posters - targeting the kinome · posters p01 analysis of ... p03 activation of a dna repair...

Posters

P01 Analysis of distinct Aurora kinase inhibitors: mitosis, apoptosis and drug

resistance Rebecca Tyler and Patrick A Eyers

P02 Functional and structural studies of the extracellular ligand binding domain of

VEGFR-2 C. Ruch, A. Prota, M. Steinmetz, G. Skiniotis, T. Walz and K. Ballmer-Hofer

P03 Activation of a DNA repair pathway by the Met RTK- a novel target for tumor

radiosensitization. Ganapathipillai S, Aebersold DM, Berthou S, Streit B, Lipson KE, Greiner RH and Zimmer Y

P04 The ErbB2 effector protein MEMO is essential for development and has a role in

tumor-induced angiogenesis and metastasis Régis Masson, Subha Susan Jacob, Patrick Kaeser, Francesca Maurer, Nancy Hynes P05 Pathogenic Mutation G2019S enhances autophosphorylation of the Leucine Rich

Repeat Kinase 2 Giorgio Rovelli, Miriam Bibel, Jutta Blank, Michael Forstner, Stefan Frentzel, Paulette

Greenidge, Bernd Kinzel, Lorenz Mayr, Matthias Mueller, Etienne Regulier, Albert Schmitz, Derya Shimshek, Thomas Troxler, Kaspar Zimmermann, Christoph Wiessner, Rainer Kuhn

P06 Mechanism of Ras and PI3K-PKB stabilization of mRNA by phosphorylation of

the posttranscriptional regulator BRF1 Don Benjamin, Martin Schmidlin, Lu Min and Christoph Moroni P07 The role of IRAK-4 kinase activity in IL-1β-mediated signaling.

Koziczak-Holbro M, Joyce C, Glück A, Tschopp C, Kinzel B, Müller M, Mathison J, Ulevitch R and Gram H.

P08 PKB modulator protein (CTMP), a mitochondrial protein with a potential role in

regulating mitochondria homeostasis Lionel A Tintignac, Susanne Schenk, Vesna Oliveri, Peter Cron, Arnaud Parcellier, Markus Durrenberger and Brian A Hemmings

P09 Modulation of phosphorylation-dependent protein kinase conformational

transitions with low molecular weight compounds Matthias Engel, Valerie Hindie, Laura A. Lopez-Garcia, Adriana Stroba, Francis

Schaeffer, Iris Adrian, Jochen Imig, Leila Idrissova, Wolfgang Nastainczyk, Stefan Zeuzem, Pedro M. Alzari, Rolf W. Hartmann, Albrecht Piiper and Ricardo M. Biondi

P10 Molecular mimicry of an active immune inhibitory receptor by the bacterial

pathogen Bartonella Patrick Guye, Hermine Schein, Thomas Rhomberg, Paul Jenö, Christoph Dehio

P11 Signalling by specific PI3-Kinase isoforms in human neuroblastoma D. Boller, T. Shalaby, M.A. Grotzer, A. Arcaro

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P12 Autocrine insulin-like growth factor-I signaling promotes growth and survival of

human acute myeloid leukemia cells via the phosphoinositol 3-kinase/Akt pathway Kathrin T. Doepfner, Olivier Spertini, and Alexandre Arcaro

P13 Akt/mTOR pathway activation and Bcl-2 family proteins modulate the sensitivity

of human small cell lung cancer cells to RAD001 (Everolimus) Marin Marinov, Algirdas Ziogas, Olivier E. Pardo, Liwen Terence Tan, Heidi A. Lane, Nicholas Lemoine, Uwe Zangemeister-Wittke, Michael J. Seckl, and Alexandre Arcaro

P14 Thrombin control of PI3K signaling underlies plasticity of hair follicle dermal cell Feutz AC, Barrandon Y, Monard D P15 MNK1 and MNK2 regulation in HER2 over-expressing breast cancer cell lines Carol A. Chrestensen, Jacquelyn K. Shuman, Andrew Eschenroeder, Mark

Worthington, Herman Gram, and Thomas W. Sturgill

P16 Src-mediated PDK1 activation requires Tyr-9 phosphorylation of PDK1 and the assistance of Hsp90

Keum-Jin Yang, Sanghee Shin, Longzhen Piao, Eulsoon Shin, Yuwen Li, Miran Roh, Kyung Ah Park, Hee Sun Byun, Minho Won, Gang Min Hur, Jeong Ho Seok, Brian A Hemmings and Jongsun Park

P17 Identification of new molecular targets for human medulloblastoma using RNA

interference screening A.S. Guerreiro, A.N.D. Elsing, T. Shalaby, M.A. Grotzer, and A. Arcaro

P18 Pentacyclic triterpenoids inhibit Iκb kinase activity and promote apoptosis in

androgen-independent prostate cancer T. Syrovets, J.E. Gschwend, B. Büchele, Y. Laumonnier, Th. Simmet

P19 HIPK2 phosphorylates p53 serine 46 in response to DNA double-strand breaks and is regulated by checkpoint kinase ATM Ilka Dauth, Jana Krüger and Thomas G. Hofmann

P20 Constitutively activated IGF-IR causes complete transformation of mammary

epithelial cells and is associated with an epithelial to mesenchymal transition via NF-kappaB-mediated induction of snail and reversed by the new IGF-IR small molecule inhibitor BMS-536924 Lee AV, Cui X, Grabiner B, Lin X, Litzenburger BC, Lewis MT, Carboni J, Gottardis M, Wong TW, Attar R, Kim HJ.

P21 PKB is activated by DNA-PK in response to DNA double-strand breaks to

regulate survival and proliferation Lana Bozulic, Banu Surucu, Ekaterina Gresko, Jianhua Feng1, Jongsun Park2 and Brian A Hemmings

P22 Defining pathways influencing paclitaxel sensitivity

Marani Michela P23 Wnt1 transactivates EGFR in human breast cancer cells

Thomas Schlange, Yutaka Matsuda and Nancy E. Hynes

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P24 TSC1-TSC2 Complex Inhibits mTORC2 in Human Cells Klaus D. Molle and Michael N. Hall

P25 Regulation of EGF receptor signaling by myotubularins

Philipp Berger, Imre Berger, Christiane Schaffitzel, Kristian Tersar, Kurt Ballmer-Hofer, and Ueli Suter

P26 The purinome, a complex mix of drug and toxicity targets

Timothy A. J Haystead and Steven Hall P27 Profiling of signaling molecules in four different human prostate carcinoma cell

lines before and after induction of apoptosis Iben H.E. Skjøth and Olaf-Georg Issinger

P28 Centrosome-associated NDR kinase regulates centrosome duplication

Alexander Hergovich, Stefan Lamla, Erich A. Nigg, and Brian A. Hemmings

P29 The role of DNA-PK in the activation of PKB by Ser473 hydrophobic motif phosphorylation Banu Surucu, Lana Bozulic, Debby Hynx and Brian A. Hemmings

P30 Increased rate of T-cell lymphoma in NDR1-deficient mice

Hauke Cornils, Mario Stegert, Stephan Dirnhofer and Brian A. Hemmings P31 Identification of Src kinase substrates regulating anchorage independency of

lung cancer cells Ryuichi Sakai and Takamasa Uekita

P32 Using mammalian and yeast systems to study MEMO, a novel ERBB2 effector

protein Maria Meira, Regis Masson, Nancy Hynes

P33 Enhancing cisplatin sensitivity in MCF-7 human breast cancer cells by

downregulation of Bcl-2 and cyclin D1 Christina Westmose and Olaf-Georg Issinger

P34 Molecular characterization of the mitotic spindle checkpoint protein Bub1

Christiane Klebig, Patrick Meraldi P35 Purification and characterization of the CK2alpha’-based holoenzyme, an

isozyme of CK2alpha: a comparative analysis Birgitte B. Olsen, Brigitte Boldyreff, Karsten Niefind and Olaf-Georg Issinger

P36 Loss of NOTCH2 positively predicts survival in distinct subgroups of human

oligodendroglioma and glioblastoma Jean-Louis Boulay, André R. Miserez, Christian Zweifel, Balasubramanian Sivasakaran, Anthony Ghaffari, Veronika Kana, Cordelia Luyken, Michael Sabel, Abdessamad Zerrouqi, Morten Wasner, Otmar Gratzl, Erwin van Meir, Markus Tolnay, Guido Reifenberger and Adrian Merlo

P37 Negative regulation of c-Src by binding to the PDZ domain of AF-6

Gerald Radziwill, Andreas Weiss, Jochen Heinrich, Prisca Boisguerin, Koji Owada, and Karin Moelling

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Olsen+BB%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Niefind+K%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Issinger+OG%22%5BAuthor%5D

P38 Identification of LNX as a novel Src-interacting protein Andreas Weiss, Gerald Radziwill, and Karin Moelling

P39 Raf Kinase

Karin Moelling P40 PP1 positively regulates the TNF/NF - κB pathway

Shinya Mitsuhashi, Hiroshi Shima, Nobuhiro Tanuma, Kunimi Kikuchi, Makoto Ubukata

P41 The human tumour suppressor LATS1 is activated by human MOB1

at the membrane Alexander Hergovich, Debora Schmitz, Brian A. Hemmings

P42 The role of Frizzled-4 and -6 in angiogenesis

Ivana Samarzija, Patrizia Sini, Thomas Schlange, Nancy Hynes P43 Combination of suboptimal concentrations of EGFR inhibitor and microtubule

stabilizer induces apoptosis of glioblastoma cells Korur S, Failly M, Lino MM, Boulay JL, Egler V and Merlo A

P44 Tenascin-C is a novel RBPJk- induced target gene for Notch2 signaling in

gliobastomas Balasubramanian Sivasankaran, Martin Degen, Anthony Ghaffari, Christian Zweifel, Mihai Ionescu, Markus Tolnay, Morten Wasner, Robert Kiss, Ruth Chiquet-Ehrismann, Adrian Merlo and Jean-Louis Boulay

P45 Tie2 kinase inhibitors inhibit angiogenesis and tumor growth

A Vaupel, P Furet, K Masuya, C Schnell, McCarthy, M Ferretti, E. Billy, L, J Brueggen, C, V Rasetti, , M Lang and G Martiny-Baron

P46 SIN1 Regulation of mammalian TOR Complexes

Nelyn Soto, Valeria Facchinetti, Carolyn Lowry, Bing Su, and Estela Jacinto P47 High-throughput crystallography of protein kinases as a tool for drug discovery

Stefan Knapp P48 Endothelin receptor type B inhibits tenascin-C-induced focal adhesion and

stress fiber disorganization Katrin Lange, Antje Dittmann, Wentao Huang, Erika Fluri and Gertraud Orend

P49 Phosphatidylinositol-3-kinase γ (PI3Kγ) in mast cell adhesion

Emilie Clément, Matthew Thomas and Matthias P. Wymann P50 WD-Repeat FYVE Protein Interacts with the Akt Kinase and Promotes

Adipogenesis Thorsten Fritzius and Karin Moelling

P51 Activation of PI3Kγ in mast cells downstream of FcεRI, Ca2+-influx and PKCβ Romy Walser, Katja Björklöf, Peter Küenzi, Muriel Laffargue, Michael Leitges, Emilio Hirsch, Matthias P. Wymann

P52 Rrd2 mediates suppression to topoisomerase I induced DNA damage in yeast

M.Carmen Díaz de la Loza and Ralf E. Wellinger

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P53 Potential use of pharmacological cyclin-dependent kinase inhibitors as anti-HIV

therapeutics Agbottah E, Nekhai S, Kashanchi F

P54 Structure of the human protein Kinase STK16 Reveals a Novel Activation Loop

Architecture Jeyanthy Eswaran, Antonio Bernad, Jose M. Ligos, Barbara Guinea, Judit É Debreczeni, Sirlester A. Parker, Rafael Najmanovich, Benjamin E. Turk and Stefan Knapp

P55 Mechanism of induction of programmed cell death by the β-GBP cytokine in

colon cancer cells Dong-yun Shi

P56 The Ret receptor tyrosine kinase mediates anchorage-independent proliferation

of breast cancer cells Anne Boulay, Madlaina Breuleux, Michael Stumm, Christine Stephan, Cathrin Brisken, Heidi A. Lane, Nancy Hynes

P57 Self-limiting feedback of E2F-1 overexpression in cells resistant to E2F-1

induced apoptosis Vincent Roh, Daniel Candinas, Stephan A. Vorburger

P58 VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding

shows attenuated signalling potential through VEGFR-2 Stéphanie Cébe Suarez, Michel Pieren, Sandie Arn, Luca Cariolato, Ute Hoffmann, Augustyn Bogucki, Corinne Manlius, Jeanette Wood, and Kurt Ballmer-Hofer

P59 The role of dimerisation in VEGFR-2 activation

Debora Dosch and Kurt Ballmer-Hofer

P60 CGP-582, an imatinib derivative selective for PDGFR and Kit: exploring the

molecular reason using the MM-PBSA computational technique A. Bortolato, C. Gambacorti-Passerini and L. Scapozza

P61 Modulation of phosphorylation-dependent protein kinase conformational

transitions with low molecular weight compounds Matthias Engel, Valerie Hindie, Laura A. Lopez-Garcia, Adriana Stroba, Francis Schaeffer, Iris Adrian, Jochen Imig, Leila Idrissova, Wolfgang Nastainczyk, Stefan Zeuzem, Pedro M. Alzari, Rolf W. Hartmann, Albrecht Piiper and Ricardo M. Biondi

P62 X-ray crystallograpfy of ERK1 protein

Elad Elkayam, Boaz Shaanan and Yossef Granot P63 The potential of novel naphtoquinones in prevention of restenosis Yaron Aviv, Sharon Etzion, Shmuel Bittner, Yossi Granot P64 Arginine-vasopressin and JAK-STAT signaling in vascular smooth muscle cells

Levy Oren and Granot Yossi P65 Glycogen synthase kinase-3 as a key component of estrogen receptor alpha

signalling pathway Jean Grisouard, Alexander Hermani And Doris Mayer

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P66 Class I PI3Ks as drug targets in melanoma Romina Marone, Sauveur-Michel Maira, Anke Klippel, Frederic Stauffer, Pascal Furet, Carlos Garcia-Echeverria and Matthias P. Wymann

P67 On the role of Protein Phosphatase 2A in cell migration

Veerle Janssens, Karen Zwaenepoel, Carine Rossé, Marleen M. Petit, Jozef Goris and Peter J. Parker

P68 On the complex structure of PP2A

Zwaenepoel Karen, Longin Sari, Jordens Jan, Goris Jozef, Janssens Veerle P69 Phosphoprofile of activated EGF receptor

Isabell Huber, Judith Koch, Frank Neumann, Julia Riedel, Gabi Schmitt, Petra Schuessler, and Heinz Hoschuetzky

P70 Validation of PhosphoBioMarkerTM specific monoclonal antibodies

Isabell Huber, Petra Schuessler, and Heinz Hoschuetzky P71 EGFR phosphoprofiling with Fluorescent Microspheres (Luminex)

Isabell Huber, Petra Schuessler, and Heinz Hoschuetzky P72 Epigallocatechin gallate (EGCG) mimics insulin’s phosphorylation of the

transcription factor FOXO1a and elicits cellular responses in the presence and absence of insulin. Siobhan Anton, Laura Melville and Graham Rena

P73 Anti-apoptotic effect of Src kinase in Xenopus oocyte extracts is mediated by Src

SH3 domain Alexander A. Tokmakov, Mariko Ikeda, Mikako Shirouzu, Shigeyuki Yokoyama

P74 Regulation of the protein kinase MELK, an interactor of NIPP1

Monique Beullens, Sadia Vancauwenbergh, Nick Morrice, Rita Derua, Hugo Ceulemans, Etienne Waelkens and Mathieu Bollen

P75 Activating alleles of JAK3 in acute megakaryoblastic leukemia

Denise K. Walters, Thomas Mercher, Ting-Lei Gu, Thomas O’Hare, Jeffrey W. Tyner, Marc Loriaux, Valerie L. Goss, Kimberly A. Lee, Christopher A. Eide, Matthew J. Wong, Eric P. Stoffregen, Laura McGreevey, Julie Nardone, Sandra A. Moore, John Crispino, Titus J. Boggon, Michael C. Heinrich, Michael W. Deininger, Roberto D. Polakiewicz, Gary Gilliland and Brian J. Druker; presented by Christopher Bunker

P76 A novel role of ERK5 in integrin-mediated breast cancer cell adhesion and

motility via FAK signaling – implications in cancer metastasis Rajinder S. Sawhney, Maria Orsino, Majed Aggi, Jennie Hauser, and Michael Brattain

P77 The Kinase Knowledge Base: computational tools for kinase drug discovery Eric Martin, Sullivan D, Jansen H, Lindvall M, Hendrickson T, Kondracki J,

Ringgenberg J, Silveria S P78 MEK kinase 1 (MEKK 1) is critical for thymus-dependent (TD) immunity, and

demonstrates the signaling specificity of mitogen-activated protein kinase kinase kinase (MAP3Ks) Ewen Gallagher, Thomas Enzler, Amy Anzelon-Mills, Dennis Otero, Tord Labuda, Min Gao, and Michael Karin

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P79 Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3 Stegert MR, Hergovich A, Tamaskovic R, Bichsel SJ, Hemmings BA

P80 Bacterial co-expression of LKB1, MO25� and STRAD� results in the formation

of the active heterotrimeric complex Dietbert Neumann, Marianne Suter, Roland Tuerk, Uwe Riek, Theo Wallimann

P81 A single point mutation in the PDZ ligand sequence of c-Src impairs epithelial

cell-sheet integrity during wound healing and promotes directional single cell migration Martin Baumgartner, Gerald Radziwill, Andreas Weiss and Karin Moelling

P82 Role of Memo a novel ErbB2 effector protein in tumor cell biology

Barbara Haenzi, Régis Masson, Subha Susan Jacob, Nancy E. Hynes P83 TRAF4 is a signalling adaptor overexpressed in human cancers

Sophie Camilleri-Broët, Catherine Sautès-Fridman and Catherine H. Régnier P84 Roles of δEF1 family, SIP1 and ZEB1, in the Regulation of TGF-β-induced

Epithelial-Mesenchymal Transdifferentiation Masao Saitoh, Takuya Shirakihara, and Kohei Miyazono

P85 JNK2 is required for Ras transformation independently of AP-1

Christina Nielsen, Jacob Thastrup, Trine Bøttzauw, Marja Jäättelä and Tuula Kallunki P86 PMA-induced PKC translocation is inhibited by new PKC C1 domain ligands

Talman Virpi, Aitio Olli, Ekokoski Elina, Boije af Gennäs Gustav, Finel Moshe, Yli-Kauhaluoma Jari, Tuominen Raimo K

P87 Integrin linked kinase is a molecular adaptor for T-cadherin-induced Akt/GSK3β-

axis signaling in endothelial cells Manjunath B Joshi, Danila Ivanov, Maria Phillipova, Paul Erne, Therese J Resink

P88 Functional and structural studies of the extracellular ligand binding domain of

VEGFR-2 C. Ruch, A. Prota, M. Steinmetz, G. Skiniotis, T. Walz and K. Ballmer-Hofer

P89 The human kinetochore protein hsMcm21R is regulated in a cell cycle dependent

manner Mukta Belwal, Sarah McClelland, Andrew McAinsh and Patrick Meraldi

P90 Axl as a target for therapeutics against invasive breast cancer: from target

validation to experimental therapy Yixiang Zhang, Yuri V. Cheburkin, Yuri P. Knyazev, Wolfgang E. F. Klinkert, György Kéri, Axel Ullrich, Peter G. Knyazev

P91 Characterization of human Rio2 function in late steps of 40S subunit biogenesis

Ivo Zemp and Ulrike Kutay

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P92 Realizing the promise of chemical genetics: from substrate identification to selective inhibition of a single essential protein kinase in human cells Stephane Larochelle, Karl A. Merrick, Marie-Emilie Terret, Lara Wohlbold, Nora M. Barboza, Chao Zhang, Kevan M. Shokat, Prasad V. Jallepalli and Robert P. Fisher

P93 The scaffold protein CNK1 in Raf-dependent signaling

Rafael D. Fritz, Algirdas Ziogas, Karin Moelling and Gerald Radziwill P94 Sequential tyrosine-phosphorylation of LAT controls life or death of developing T

lymphocytes Zara Hovhannisyan, Leah Alabanza, Dominique Roubaty, Ed Palmer and Guy Werlen

P95 Regulation of TGF-β1-induced apoptosis and epithelial-to-mesenchymal

transition by Protein Kinase A and STAT3 Yanan Yang, Xinchao Pan, Weiwei Lei, Jun Wang, Jian Shi, Fuqiang Li, and Jianguo Song

P96 Sutent/sunitinib malate - towards a response prediction signature

Michaela Bairlein, Jesper V. Olsen, György Keri, Matthias Mann, Henrik Daub and Axel Ullrich

P97 Life with a single isoform of PKB/Akt: mice lacking PKBβ/Akt2 and PKBγ/Akt3 are

viable but display impaired glucose homeostasis and growth deficiencies Bettina Dummler, Oliver Tschopp, Debby Hynx, Zhong-Zhou Yang, Stephan Dirnhofer, and Brian A. Hemmings

P98 Multiplexed monitoring of activation state markers in signal transduction pathways

Jens Tränkle, Claudio Calonder, Gina Balciunaite, Daniel Rechsteiner, and Markus Ehrat P99 Dihydroxyacetone kinases: a new family of kinases with metabolic and

transcription control functions Annapurna Srinivas, Sandra Christen, Priska Baehler, Anja Zeller, David Pridmore, Christoph Bieniossek, Ulrich Baumann and Bernhard Erni

P100 The MEN kinase cascade promotes chromatin decompaction at the end of

mitosis Varela E., Leroy, D., Shimada K., Laroche, T. and Gasser, S. M.

P101 Probing the kinome with kinase inhibitors: chemical genomics based screening,

selectivity profiling, and mode-of-action studies D. Eberhard, M. Bantscheff, Y. Abraham, S. Bastuck, G. Bergamini, V. Reader, T. Werner, U. Kruse, G. Neubauer, N. Ramsden, B. Küster, G. Drewes

P102 Characterisation of activation of the Akt/mTOR pathway in various human

malignancies reveals a wide heterogeneity between individual tumours Nelly Marmy Conus, Richard Young, Wayne Phillips, Melanie Trivett, Rick Pearson, Ian Campbell and Grant McArthur

P103 Structure of the catalytic domain of human Protein Kinase C-beta II with a

bisindolylmaleimide inhibitor Stephan Grant

P104 MAPKKK6 is a negative regulator of apoptosis

Elisabeth Ortner and Karin Moelling

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P105 Approach to molecular basis of Insulin/IGF ageing signalling on Drosophila

melanogaster Tomoatsu Ikeya and Linda Partridge

P106 A functional interaction between Sprouty proteins and Caveolin-1

Miguel A. Cabrita, Fabienne Jäggi, Sandra P. Widjaja and Gerhard Christofori P107 Phosphoproteomic indication of a MAPK kinase-mediated phosphorylation of an

antioxidant enzyme in tomato Tim Xing, Christof Rampitsch, Natalia Bykova

P108 Chimeric receptor models of CD5-signalling in chronic lymphocytic leukemia

(CLL) Michaela Patz, Aditya Veldurthy, Michael Hallek and Günter Krause

P109 TGFβ1-mediated activation of protein kinase B/Akt is important for epithelial-

mesenchymal transition in diabetic nephropathy Kattla JJ, Godson C, Brazil DP

P110 ECO-4601, a novel antitumor compound targeting the Ras-MAPK pathway

induces apoptosis H. Gourdeau, V. Zvereff, M-A. Wioland, D. Lobreau, and P. Falardeau

P111 Conditional knock down of c-Myc in the mouse mammary gland

Tina Stoelzle, Andreas Trumpp and Nancy E. Hynes P112 Lymphoid microenvironment inhibits apoptosis in B-CLL cells through activation

of PI3K/Akt pathway Medhat Shehata, Dita Demirtas, Susanne Schnabl, Martin Hilgarth, Rainer Hubmann,

Josef D. Schwarzmeier, Ulrich Jaeger P113 A cellular test sytem for assaying inhibitors of the kinase activity of B-Raf VE600

Jan E. Ehlert, Bettina Mutschler, Melanie Müller, and Michael H.G. Kubbutat P114 Increases in c-Src expression level and activity do not promote growth of human

colorectal carcinoma cells in vitro and in vivo Arkadiusz Welman, Christopher Cawthorne, Lourdes Ponce-Perez, Jane Barraclough, Sarah Danson, Stephen Murray, Jeff Cummings, Terry D. Allen and Caroline Dive

P115 Bcl10 controls TCR- and FcγR-induced actin polymerization

Daniel Rueda, Olivier Gaide, Liza Ho, Elodie Lewkowicz, Florence Niedergang, Fabien Rebeaud, Stephan Hailfinger, Montserrat Guzzardi, Béatrice Conne, Marcus Thelen, Jerome Delon, Uta Ferch, Tak W. Mak, Jürgen Ruland, Jürg Tschopp, Jürg Schwaller and Margot Thome

P116 New transition state-based inhibitor for human ornithine decarboxylase inhibits

growth of tumor cells Fang Wu and Heinz Gehring

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P117 Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice Arash M Tehrani, Soon-Kyung Hwang, Tae-Hee Kim, Chong-Su Cho, Jin Hua, Woon-Sung Nah, Jung-Taek Kwon, Jun-Sung Kim, Seung-Hee Chang, Kyung-Nam Yu, Sung-Jin Park, Dilli R Bhandari, Kee-Ho Lee, Gil-Hwan An, George R. Beck Jr., Myung-Haing Cho

P118 A novel role of AMP-activated protein kinase in ginsenoside-Rh2-induced

apoptosis in cancer cells Min-Jung Kim and Joohun Ha

P119 AMP-activated protein kinase suppresses apoptosis via feedback inhibition of c-

Jun N-terminal kinase in glucose-deprived cancer cells Hee Yun and Joohun Ha

P120 Fas (APO-1/CD95) activates AMP-activated protein kinase in DU145 human

carcinoma: a novel role of AMPK in Fas-induced apoptosis Seung-Nam Jung, Sung-Soo Kim, and Joohun Ha

P121 Anti-diabetes and anti-obesity effect of cryptotanshinone via activation of AMP-

activated protein kinase Eun Ju Kim, Sung-Soo Kim, Joohun Ha P122 Interleukin-6-induced activities of the Src family kinases Hck, Lyn and Fyn in

myeloma cells are blocked by an acidic petide A. Hausherr, R. Tavares, M. Hallek, and G.F. Krause

P123 Protein Kinase B, a survival kinase in chronic lymphocytic leukaemia of B cells

Sebastian Hofbauer, Inge Tinhofer, Richard Greil P124 PI3Kγ adaptors relay PI3Kγ activity to specific cell responses

Bohnacker T, Marone R, Calvez R, Clément E, Hirsch E, Wymann MP P125 PhosphoSite® update 2007: global analyses of the phosphoproteome

Peter Hornbeck, John Rush, Jon Kornhauser, Julie Nardone, Ela Skrzypek, And Bin Zhang

P126 Human DNA polymerase λ is phosphorylated by the cyclin-dependent kinase

Cdk2/cyclin A complex Wimmer U, Frouin I, Ferrari E, and Hübscher U P127 Chemical genetics: functional analysis of the analog sensitive allele of S.pombe

p34cdc2 (cdc2-as) and the role of cdc2 in regulating cytokinesis Sandra Dischinger, Andrea Krapp, Lifeng Xie, James R. Paulson and Viesturs Simanis

P128 Generation of new powerful and selective inhibitors of protein kinase CK2 by in

silico analysis and molecular derivatization of cinnamic and coumarinic scaffolds G. Cozza, M.A. Pagano, G. Poletto, F. Meggio, S. Moro, A. Guiotto, A. Chilin, G. Zagotto and L.A. Pinna

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P129 Cell-Based Approaches to Protein Kinase Assays Using GFP-fusion Substrates Brian Pollok, Thomas Machleidt, Matthew Robers, Kevin Vedvik, Steve Riddle and Kurt Vogel

P130 The effect of statins on K-Ras localization and cellular proliferation

H.Gbelcová, Z.Knejzlík, J.Zelenka, M.Leníček, L.Vítek, T.Ruml P131 Growth inhibition of a gefitinib resistant NSCLC cell line with the anti-EGFR

monoclonal antibody cetuximab Denis Irmer, Jana Plagge, Christiane Amendt and Andree Blaukat

P132 Identification of MAT2A as a new substrate of the protein tyrosine kinase Pyk2

by a modified KESTREL approach Friederike Krop, Vanessa Jacob, Roland Kellner, Dirk Vocke and Andree Blaukat

P133 Generation of biologically active Reg-4 and PAP-1 Fc-fusion proteins that

activate kinase cascades in cancer cells Dirk Vocke, Silvia Jezek, Iris Mannberger and Andree Blaukat

P134 Analysing the role of a novel cell-cycle-dependent modification of the yeast Chk2

homologue Rad53p Thomas Schleker, Kenji Shimada, Ragna Sack and Susan M. Gasser

P135 Association of common haplotypes of the p85α subunit of phosphoinositide 3-

kinase gene (PIK3R1) with glycemic response to 26 weeks of treatment with an insulin sensitizing PPAR-γ/α agonist in type 2 diabetic patients L. Hansen, C. T. Ekstrøm, R. Tabanara Y Palacios, M. Anant, K. Wassermann, R. R. Reinhardt

P136 Quantitative Western blot: step by step analysis

F. Heidebrecht, A. Heidebrecht and A. Bader P137 Coupled mchanism of activation of AGC kinases by linker and hydrophobic motif

phosphorylation sites Camilla Hauge, Torben L. Antal, Ulrik Doehn, Kathrine Thorup, Leila Idrissova, Ricardo M. Biondi and Morten Frödin

P138 Systems biology modelling of signalling pathways in drug discovery

S. Reinker, J. Voshol, K. Mayawala, C.R. Cho, J. van Oostrum P139 A KESTREL screen for substrates of the TORC2 effector kinase Ypk2

Alexandre Huber and Robbie Loewith P140 Targeting the EGF-receptor with EGF-derived peptides fused to a multimeric

enzyme Annette Gaida, Illip Burmester and Kristian M. Müller

P141 Identification of an EGF-receptor binding peptide for tumor targeting, based on

phage display and random fragmentation Illip Burmester, Annette Gaida, Kristian Müller

P142 Novel 2-acylaminopyridin-4-yl imidazoles: potent and metabolically stable p38

MAP kinase inhibitors Ziegler K, Laufer S

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P143 Design, synthesis and selectivity profile of a novel structural class of p38 MAP kinase inhibitors Ahrens G, Karcher S, Nieß R, Hering J, Laufer S

P144 Profile and molecular modeling of 3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-

pyrrole-2,5-dione as a highly selective VEGF-R2/3 inhibitor Christian Peifer, Gerd Dannhardt and Stefan Laufer

P145 From five to six membered rings: 3,4-diarylchinolinone as lead for novel

p38MAP-kinase inhibitors Peifer C, Abadleh M, and Laufer S

P146 Compound B1 leads to recovery from alcohol-induced fatty liver in rats.

Sanghee Shin, Keum-Jin Yang, Longzhen Piao, Eulsoon Shin, Li Yuwen, Miran No, Kyung Ah Park, Hee Sun Byun, Minho Won, Gangmin Hur, Jeong Ho Seok, Jin Man Kim, Minho Song and Jongsun Park

P147 Kinase inhibitors as antifungals: targeting Septoria tritici

Marc Morgan, Jane Endicott, Martin Noble, Jane Wibley, Andy Corran P148 Mammalian sterile 20-like kinase Mst1 is a multi-functional signal transducing in

prostate cancer Hager M, Cinar B, Oancea, E, DiVizio D and Freeman MR P149 The increased metastatic potential of human pancreatic cancer cell lines is

associated with the expression of constitutively active KRAS Stephan Gysin, Byron Hann and Martin McMahon

P150 PASKIN: link between energy metabolism and protein translation in mammals?

Juliane Tröger, Katrin Eckhardt, Emanuela Borter, Patrick Spielmann, Gieri Camenisch and Roland H. Wenger

P151 Haematopoietic progenitor cells utilise conventional PKC to suppress PKB/Akt

activity in response to c-Kit stimulation Charlotte E. Edling, Malin Pedersen, Leif Carlsson, Lars Rönnstrand, Ruth H. Palmer and Bengt Hallberg

P152 Quantitative analysis of receptor tyrosine-kinase signaling exploited by Listeria

monocytogenes Tobias Reinl, Joseph Wissing, Roman Fischer, Claudia Hundertmark, Frank Klawonn, Henrik Daub, Jürgen Wehland, Lothar Jänsch

P153 Activation by drugs of the PKCζ-LKB-AMPK signaling cascade is necessary for

transcriptional regulation of cytochromes P450 Sharon M. Blättler, Michel R. Kaufmann, Franck Rencurel and Urs A. Meyer

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Poster 01

Analysis of distinct Aurora kinase inhibitors: mitosis, apoptosis and drug resistance Rebecca Tyler and Patrick A Eyers Molecular Cancer Studies, Faculty of Life Sciences, University of Manchester, Manchester, UK The Aurora kinase inhibitors VX-680 and several quinazolines, exemplified by ZM447439, represent two distinct class of small molecule anti-cancer agent that are potent Aurora kinase inhibitors. The successful application of inhibitors as therapeutic agents requires some understanding of their specificity and mechanism of action, especially in patients that become refractory to treatment through mutation. In this study we have investigated the in vitro specificity of these drugs towards the Aurora kinases and c-Abl, the tyrosine kinase whose activation causes CML. We have analysed drug-resistance to both targets, and identify mutations in the Aurora kinases that may be important for resistance to one, but not the other, compound. We also compare and contrast the cellular effects of both drugs, highlighting different effects in cells which are likely to be a consequence of their different intracellular specificity.

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Poster 02

Functional and structural studies of the extracellular ligand binding domain of VEGFR-2 C. Ruch, A. Prota, M. Steinmetz, G. Skiniotis1, T. Walz1 and K. Ballmer-Hofer Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland and 1Harvard Medical School, Dept. of Cell Biology, Boston MA, USA Angiogenesis, the process through which new blood vessels are formed from preexisting vasculature, plays a pivotal role in normal development and in a variety of diseases such as cancer, retinopathies, atherosclerosis and preeclampsia. Vascular endothelial growth factor receptors are key regulators of angiogenesis and are activated by ligands of the VEGF family. Three VEGF-receptors have been described VEGFR-1, -2, and -3 and additional receptors whose role in angiogenesis is not yet well defined at the molecular level, neuroplin-1 and -2. VEGFRs are receptor tyrosine kinases consisting of 7 extracellular Ig-like domains, a transmembrane domain and a cytoplasmic split tyrosine kinase domain followed by a domain carrying tyrosine residues involved in receptor signalling. These receptors are activated upon binding of VEGF family growth factors, VEGF-A, -B, -C, -D, -E, -F, and PlGF (placenta growth factor). Each VEGF variant interacts with a specific set of VEGFRs and induces or stabilises receptor dimerisation. Subsequent activation of the intracellular split kinase domain initiates signalling pathways that regulate cell proliferation, differentiation, migration and vessel permeability. Understanding the mechanism of receptor activation in the context of its 3-dimensional structure will open new avenues for the design of drugs interfering with angiogenic signalling. We combine functional biochemical studies with structural information on VEGF receptors. The extracellular domain of VEGFR-2 was produced in mammalian cells, purified to homogeneity and submitted to binding studies with VEGF ligands. Electron microscopy was used to obtain low resolution structural information on receptor/ligand complexes. Our data show that the extracellular domain of VEGFR-2 is highly flexible in the absence of bound ligand while ligand-bound receptor assumes a tight and apparently stable conformation. X ray crystallography and small angle scattering are currently used to obtain detailed structural information of ligand/receptor complexes.

66

Poster 03

Activation of a DNA repair pathway by the Met RTK - a novel target for tumor radiosensitization Ganapathipillai S, Aebersold DM, Berthou S, Streit B, Lipson KE, Greiner RH and Zimmer Y Department of Radiation Oncology, Inselspital Bern; Switzerland, Department of Clinical Research, University of Berne,Switzerland; SUGEN Inc, South San Francisco, US

Objective: The aims of the present work are to reveal the molecular pathways by which Met activation leads to DNA repair and to explore the potential of the anti Met small molecule SU11274 as a radiosensitizer.

Materials and Methods: NIH 3T3 cell lines that ectopically express wild type (WT) Met and the mutated varients, M1268T and Y1248H were used. western blotting, co-immunoprecipitations, clonogenic assays, confocal microscopy and various flow cytometry methodologies were used in the present study. The Met specific small molecule inhibitor SU11274 was from SUGEN Inc, South San Francisco.

Results: Our results show that in cell lines with expression of the Met mutated variants, M1268T and Y1248H, constitutive tyrosine phosphorylation of C-Abl and Rad51 is detected and both proteins are physically associated with the mutated receptor. Neither C-Abl or Rad51 phosphorylations, nor association between Met and these proteins have been detected in cells that express Met WT, even following treatment with HGF.

To establish the link between Met, C-Abl and Rad51 we have treated the corresponding cell lines with the Met small molecule inhibitor SU11274. Exposure to 2µM of SU11274 for 16hrs led to both disruption of the interaction between the receptor, C-Abl and Rad51 and to a reduction in the phosphorylated forms of these proteins only in the cells that express the SU11274 sensitive mutant M1268T, but not in cells with the resistant SU11274 Met variant Y1248H. To further confirm the link between Met and Rad51 in the context of DNA repair and cell survival, we have studied the impact of Met inhibition by SU11274 on overall DNA damage and clonogenic survival following administration of IR. DNA damage was monitored by evaluation of cellular levels of the phosphorylated form of the histone H2AX (pH2AX). pH2AX levels were measured by flow cytometry following exposure of cells to a single dose of 10Gy. Apart from pretreatment with 2µM SU11274, cells were also treated for 1 hr with 20µM of the p53 inhibitor, pifithrin alpha, to circumvent a strong G1 arrest that is induced by SU11274 and, which we speculated to be p53 dependent. Our data show, that only cells that express the sensitive variant M1268T treated by IR, SU11274 and pifithrin alphaexhibit higher levels of pH2AX, e.g. more DNA damage, than cells that were treated only with IR and pifitrin alpha, suggesting therefore that SU11274 may inflict through Met inhibition, an attenuation of a DNA repair proccess, indicating hence that Met inhibition may have a radiosensitizing impact.

In parallel to these observations, clonogenic assays in which M1268T and Y1248H cells were pretreated with 2µM of SU11274 prior to exposure to IR, suggest that the Met inhibitor reduced the number of resistant colonies to IR, resulting in lower survival as compared to cells that received IR alone. This was only in the case of cells expressing the SU11274 sensitive mutant M1268T, but not with cells that expressing SU11274 resistant mutant Y1248H. Finally, using confocal microscopy, our work shows that pretreatment of cells with 2µM of SU11274 prior to administration of a single dose of 10Gy , reduced typical post IR Rad51 foci formation in the nucleus. This finding was again observed only with M1268T cells but not with Y1248H. These results were also confirmed by western blot analysis with nuclear and cytoplasmic fractions. These findings suggest that Met blocking by SU11274 leads to inhibition of Rad51 phosphorylation via C-Abl inhibition, which eventually causes an impairment of Rad51 nuclear translocation following induction of DNA damage.

Conclusions: Our results contribute to better understand the link between Met and DNA repair and the molecular basis of radio-resistance of tumor cells with aberrant Met activity. The work also suggest that employing of Met inhibitors such as SU11274, may sensitize the cells to cytotoxicity induced by IR. Nevertheless, successful radiosensitization depends in the case of the inhibition of Met activiaty due to point mutations, on the specific mutation, which is targeted.

67

Poster 04

The ErbB2 effector protein MEMO is essential for development and has a role in tumor-induced angiogenesis and metastasis Régis Masson, Subha Susan Jacob, Patrick Kaeser, Francesca Maurer, Nancy Hynes Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. ERBB receptor tyrosine kinases initiate signaling cascades that affect cellular processes via recruitment of phosphotyrosine binding proteins to their cytoplasmic domain. We identified MEMO1 (mediator of ErbB2 driven cell motility) as a novel ERBB2 interacting protein. MEMO is conserved through evolution and its homologues are found in all branches of life. Although MEMO function is still unknown, it was originally shown to be essential for ERBB2-induced cell motility in vitro. We are investigating both the physiological and pathological functions of MEMO.ErbB2 is crucial for embryonic development. We have designed knock-out mice for Memo and have shown that homozygous mutant mice die at midgestation. Memo -/- embryos shows obvious defects in placental vascularization and heart development. To address whether MEMO has a more general role in metastatic cancer development, we explored the role of MEMO in 4T1 cells, a highly metastatic mouse mammary tumor cell line. A shRNA vector designed to stably down-regulate MEMO expression was introduced into 4T1 cells and clones showing moderate to strong decrease in MEMO level were selected. The in vitro migratory ability of MEMO knock-down cells in response to serum was decreased in comparison to control cells. Importantly, we observed a lower rate of lung metastasis from the primary 4T1-induced mammary tumor that correlated with MEMO expression level in the knock-down cells suggesting that MEMO has an important role in the metastatic process. 1.Marone R, Hess D, Dankort D, Muller WJ, Hynes NE, Badache A. Memo mediates ErbB2-driven cell motility. Nat. Cell Biol. 2004, 6(6): 512-22. 2. Erickson SL, O’Shea KS, Ghaboosi N, Loverro L, Frantz G, Bauer M, Lu LH, Moore MW. ErbB3 is required for normal cerebellar and cardiac development: a comparison with ErbB2- and heregulin-deficient mice. Dev. 1997, 124, 1999-5001.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Hess+D%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Dankort+D%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Muller+WJ%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Hynes+NE%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_Abstract&term=%22Badache+A%22%5BAuthor%5D

Poster 05

Pathogenic Mutation G2019S enhances autophosphorylation of the Leucine Rich Repeat Kinase 2 Giorgio Rovelli1, Miriam Bibel1, Jutta Blank3, Michael Forstner3, Stefan Frentzel1, Paulette Greenidge2, Bernd Kinzel4, Lorenz Mayr4, Matthias Mueller4, Etienne Regulier1, Albert Schmitz1, Derya Shimshek1, Thomas Troxler2, Kaspar Zimmermann2, Christoph Wiessner1, Rainer Kuhn1

1Neuroscience, 2Global Discovery Chemistry, 3Discovery Technologies, 4Models of Disease Center, Novartis Institute for BioMedical Research (NIBR) Basel, Switzerland The Leucine Rich Repeat Kinase 2 (LRRK2) gene has recently emerged as a central player in the etiology of familial Parkinson’s disease (PD). Mutations in LRRK2 cause an autosomal-dominant form of parkinsonism which is clinically indistinguishable from late onset sporadic PD. The common pathological feature of LRRK2 mutations is degeneration of dopaminergic neurons in the Substantia Nigra. LRRK2 is a large multi-domain protein that contains an enzymatically active MAPKKK domain. The kinase domain closely resembles receptor-interacting protein kinases (RIPKs) and mixed lineage kinases (MLKs), which are known to be upstream of the MAP kinase pathways involved in transducing cellular stresses. LRRK2 also contains a Ras-like GTP-binding domain and several structural domains mediating protein-protein interactions, i.e., leucine rich repeats (LRR), “C-terminal of Ras” (COR) and WD-40 repeats (WD40). Genetic and clinical data have provided definitive evidences that missense mutations involving at least five distinct residues are pathogenic: I1122, R1441, Y1699, G2019 and I2020. Remarkably, the mutations affecting residues G2019 and I2020 are located in the kinase activation segment, close to the highly conserved “DYG” epitope. Since mutations at the corresponding residues in B-Raf, EGFR and MLK4 are known to be oncogenic, we tested the hypothesis whether pathogenic LRRK2 mutations cause a dysregulation in the kinase activity consistent with the ‘gain-of-toxic-function’, a characteristic shared by many dominantly inherited diseases. LRRK2 wild type and selected pathogenic mutants were transiently expressed in HEK293 cells, recombinant proteins were immunoprecipitated and kinase activity tested by autophosphorylation. Here, we show that autophosphorylation of the three pathogenic mutations G2019S, I2020T and R1414G is increased when compared to wild type LRRK2.

69

Poster 06

Mechanism of Ras and PI3K-PKB stabilization of mRNA by phosphorylation of the posttranscriptional regulator BRF1 Don Benjamin, Martin Schmidlin, Lu Min and Christoph Moroni Institute for Medical Microbiology, University of Basel Signalling pathways have been shown to stabilize mRNA transcripts bearing an AU-rich element (ARE) in the 3'UTR. Many ARE bearing genes are involved in growth and cell-cycle regulation such as proto-oncogenes, growth factors and cytokines. We show that the posttranscriptional regulator BRF1 is inactivated by PKB phosphorylation resulting in increased levels of its target transcripts. Two critical residues at Ser92 and Ser203 were identified as essential for PKB dependent regulation. Phosphorylation at these sites leads to stabilization of an ARE-RNA probe both in an in vitro decay assay and in in vivo cotransfection assays. A phospho-null BRF1 mutant carrying Ser-to-Ala substitutions at positions 92 and 203 is unresponsive to regulation under conditions where PKB activity is increased either through stimulation by insulin, or by co-transfection with constitutively active PKB. Supporting evidence indicates that BRF1 repression occurs via phosporylation dependent binding to 14-3-3 at these sites. While BRF1 activity is clearly regulated at both sites in response to the PI3K-PKB pathway, over-expression of ras is still able to over-ride constitutive activation in the phospho-null BRF1 mutant pointing to the existence of additional regulatory sites on BRF1. Derailment of BRF1 regulation in oncogenically transformed cells possessing highly active PKB or ras could result in the lifting of post-transcriptional regulation over its downstream target transcripts and may contribute to the pleiotropic changes observed in tumour cells.

70

Poster 07

The role of IRAK-4 kinase activity in IL-1β-mediated signaling Koziczak-Holbro M1, Joyce C1, Glück A1, Tschopp C1, Kinzel B1, Müller M1, Mathison J2, Ulevitch R2 and Gram H.1

1Novartis Institutes for BioMedical Research, Basel, Switzerland 2The Scripps Research Institute, La Jolla, USA Activation of interleukine-1 receptor (IL-1R) or toll like receptors (TLRs) by binding of pro-inflammatory cytokines or pathogen-associated molecular products leads to induction of cellular signaling pathways, involving a cascade of adaptor molecules, kinases and transcription factors, that control expression of inflammatory genes. IL-1R-associated kinases (IRAKs) are recruited to the receptor complex upon activation. IRAK-4 has been reported pivotal for IL-1R- and TLR-mediated signaling, but controversial data exist concerning its kinase function. In order to investigate the role of IRAK-4 kinase activity in vivo, ‘knock-in’ mice were generated by replacing the wild type (wt) IRAK-4 with a mutant gene encoding kinase inactive protein. Homozygous mutant mice (IRAK-4 KD) were viable and reproduced as wt mice. Analysis of the IL-1β-induced signaling in mouse derived embryonic fibroblasts (MEF) revealed disrupted activation of NFkB, p38 and JNK pathways. Lipopolysaccharide (LPS)-stimulated bone marrow derived macrophages (BMDM) from IRAK-4 KD or IRAK-4-/- mice also displayed defects in JNK and p38 signaling while activation of NFkB pathway was like in wt cells. In line with this finding, we observed early LPS-mediated induction of mRNAs coding for inflammatory proteins in both wt and mutated cell. In contrast to wt BMDM, expression of most LPS-induced mRNAs declined over time in mutated cells, resulting in significantly reduced production of the respective proteins. This suggests that IRAK-4 plays a role in late phases of LPS-mediated gene expression. Additionally, our studies in animal models of chronic and acute inflammation verified the role of IRAK-4 kinase activity in immune responses in vivo. All these results indicate that IRAK-4 kinase activity is indispensable in IL-1R- and TLR-mediated signaling and expression of pro-inflammatory genes.

71

Poster 08

PKB modulator protein (CTMP), a mitochondrial protein with a potential role in regulating mitochondria homeostasis Lionel A Tintignac*, Susanne Schenk*, Vesna Oliveri#, Peter Cron*, Arnaud Parcellier*, Markus Durrenberger# and Brian A Hemmings*. *Friedrich Miescher Institute for Biomedical Research, CH 4058, Basel, Switzerland; # Microscopy center Pharmazentrum, University of Basel Klingelbergstrasse 50/70 CH-4056 Basel, Switzerland Constitutive activation of PKB/Akt occurs frequently in numerous cancers, mainly through inactivation of the tumor suppressor PTEN that normally downregulates PKB activity. Our group identified the human C-terminal modulator of PKB protein (CTMP) as another negative regulator of PKB. hCTMP directly binds the hydrophobic motif of PKB at the membrane and prevents its activation. Moreover, anti-sense ablation of hCTMP1 increases the ability of PKB to activate its downstream effectors upon growth factor stimulation. Overexpression of hCTMP in tumorigenic cells expressing the viral homolog of PKB (v-Akt) restores cell cycle arrest (under low serum concentration) and inhibits tumor growth in nude mice. This data suggest that CTMP functions as a tumor suppressor. Here, we report that CTMP protein resides almost exclusively in the mitochondria. Immunolocalisation of both endogenous and exogenous hCTMP coupled to cell fractionation protocols show that hCTMP protein is mostly restricted to the mitochondria, whereas almost none is detectable in the cytosol. Full length hCTMP protein is targeted to the mitochondria through a conserved hydrophobic mitochondrial targeting signal (MTS). The MTS is encoded in the 20 first amino acid residues of hCTMP, and is followed by a highly probable MPP- (mitochondrial processing peptidase) cleavage site located at residues 34 to 38. Overexpression of various N-terminal deletion- and point- hCTMP mutants in HeLa cells shows that the hCTMP protein is synthesized as a 240 amino acid precursor, and cleaved into a 204 amino acid (missing the 36 first aa) mature protein in the mitochondria. Immunogold EM staining, as well as immunodetection of hCTMP in selectively permeabilized mitochondria shows that hCTMP is accessible at the inner membrane space (IMS) and mitochondrial matrix (MM) of HeLa cells. Inducible knockdown of hCTMP expression in HeLa cells dramatically alters the shape of the mitochondria and enhances ATP production. hCTMP knocked-down cells accumulate ‘’swelling mitochondria’’, characterized by dilated ring and lariat-like structure as opposed to the filamentous structure observed in control cells. Our experiments identified CTMP as a mitochondrial protein. The altered mitochondrial volume observed in hCTMP knockdown cells suggests that CTMP protein may function as a regulator of mitochondria homeostasis.

72

Poster 09

Modulation of phosphorylation-dependent protein kinase conformational transitions with low molecular weight compounds Matthias Engel, Valerie Hindie, Laura A. Lopez-Garcia, Adriana Stroba, Francis Schaeffer, Iris Adrian, Jochen Imig, Leila Idrissova, Wolfgang Nastainczyk, Stefan Zeuzem, Pedro M. Alzari, Rolf W. Hartmann, Albrecht Piiper and Ricardo M. Biondi University of Saarland, Department of Internal Medicine II, Homburg, Germany Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate PDK1 by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogues, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode-of-action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.

73

Poster 10

Molecular mimicry of an active immune inhibitory receptor by the bacterial pathogen Bartonella Patrick Guye1, Hermine Schein1, Thomas Rhomberg1, Paul Jenö1, Christoph Dehio1

1Biozentrum der Universität Basel, Basel Switzerland Tyrosine-phosphorylation plays a crucial role in eukaryotic signaling pathways and is therefore a prominent target for abuse by pathogens. Here we show the VirB/VirD4 Type IV secretion system of the vascular tumor-inducing pathogen Bartonella henselae to secrete a protein, termed BepE, into infected cells, where it localizes to the plasma membrane and to cell-cell contacts. BepE is tyrosine-phosphorylated by host cell kinases on its immune receptor tyrosine-based inhibitory motifs (ITIM), immune receptor tyrosine-based switch motifs (ITSM) and a Csk binding-like motif. Additionally, we show that the phosphatase SHP2 as well as the C-terminal Src kinase (Csk) associate with these tyrosine-phosphorylated motifs. Together, these data suggest that the translocated bacterial effector protein BepE is mimicking activated immune inhibitory receptors, to our knowledge a novel mechanism to subvert host cell signaling.

74

Poster 11

Signaling by specific PI3-Kinase isoforms in human neuroblastoma D Boller1, T Shalaby2, MA Grotzer2, A Arcaro1

(1) Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland; (2) Department of Oncology, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland The phosphoinositide 3-kinase (PI3K) pathway is involved in the regulation of fundamental cellular processes, including cell growth, survival and motility. The aim of this project is to study the potential of targeting the PI3K pathway in neuroblastoma. When compared to adrenal gland tissue, neuroblastoma patient samples were found to express high levels of PI3KR1 and PI3KCD mRNA, a finding also observed in cell lines such as SH-SY5Y. SH-SY5Y cells stably transfected with a vector encoding shRNA against p110δ show decreased cell viability in low, but not high serum conditions, while targeting the catalytic isoforms p110α or p110β did not have a comparable effect. Furthermore, increased caspase 3/7 activity was observed in SH-SY5Y cells with decreased levels of p110δ under low serum conditions. While cells transfected with shRNA targeting p110α, p110β or p110δ showed comparable proliferative responses to growth factor stimulation, differences in signal transduction via the Akt pathway were observed. The involvement of this pathway was further highlighted by the observation that cells expressing an activated Akt mutant did not show a reduction in proliferation upon transfection with the shRNA construct targeting p110δ. Together, our data provide novel insights into the individual functions of the class IA PI3K isoforms in human neuroblastoma.

75

Poster 12

Autocrine insulin-like growth factor-I signaling promotes growth and survival of human acute myeloid leukemia cells via the phosphoinositol 3-kinase/Akt pathway Kathrin T. Doepfner 1, Olivier Spertini 2, and Alexandre Arcaro 1

1Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland; 2Service and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudois, CH-1014 Lausanne, Switzerland Insulin-like growth factor (IGF) signaling plays an important role in various human cancers. Therefore, the role of IGF-I signaling in growth and survival of acute myeloid leukemia (AML) cells was investigated. Expression of the IGF-I receptor (IGF-IR) and its ligand IGF-I were detected in a panel of human AML blasts and cell lines. IGF-I and insulin promoted the growth of human AML blasts in vitro and activated the phosphoinositide 3-kinase (PI3K)/Akt and the extracellular signal-regulated kinase (Erk) pathways. Proliferation of a panel of AML cell lines and AML patient blasts was inhibited by a pharmacological IGF-IR kinase inhibitor or by an IGF-IR neutralizing antibody. IGF-I-stimulated growth of AML blasts was furthermore blocked by an inhibitor of the PI3K/Akt pathway. Down-regulation of the class IA PI3K isoforms p110β and p110δ by RNA interference or isoform-specific inhibitors impaired IGF-I-stimulated Akt activation, cell growth and survival in AML cells. In addition to its anti-proliferative effects, p110δ shRNA-transfected cells were sensitized to etoposide-induced apoptosis. Together our data describe a novel role for autocrine IGF-I signaling in the growth and survival of primary AML cells, which involves the class IA PI3K isoforms p110β and p11δ as signal transducers. Impairing IGF-IR/PI3K/Akt signaling in combination with chemotherapeutic agents may therefore represent a novel approach to target human AML.

76

Poster 13

Akt/mTOR pathway activation and Bcl-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001 (Everolimus) Marin Marinov 1, Algirdas Ziogas 2, Olivier E. Pardo 3,4, Liwen Terence Tan 3, Heidi A. Lane 5, Nicholas Lemoine 6, Uwe Zangemeister-Wittke 2, Michael J. Seckl 3, and Alexandre Arcaro 1,3, *

1Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich; 2Department of Pharmacology, University of Bern; 3Lung Cancer Biology Group, Imperial College Faculty of Medicine, Hammersmith Hospital, London; 4CRUK London Research Institute, London; 5Novartis Institutes for BioMedical Research Basel, Oncology, Novartis Pharma AG, Basel6; Centre for Molecular Oncology, Institute of Cancer, Queen Mary’s University of London Small-cell lung cancer (SCLC) represents 20% of all lung tumours and the overall survival is very poor. We found elevated expression of the mammalian target of rapamycin (mTOR) in SCLC cell lines. The impact of the rapamycin derivative RAD001 on SCLC cell growth and survival was investigated. RAD001 impaired basal and growth factor-stimulated SCLC cell growth in vitro. Moreover RAD001 treatment markedly enhanced the anti-proliferative effects of etoposide. H-69 cells treated with RAD001 and etoposide displayed an induction of caspase-3 activity and PARP cleavage. H-69 cells displayed high basal levels of p21WAF1 and MDM2 and combining RAD001 with etoposide resulted in a reduction in the levels of p21WAF1 and MDM2. In contrast, RAD001 alone or in combination with etoposide did not alter the levels of Bcl-2, Bcl-XL or Bax. SCLC cells with high Akt pathway activation were more sensitive to RAD001. Constitutive activation of the Akt/mTOR pathway was sufficient to sensitise resistant SCLC cells to RAD001. Furthermore down-regulation of Bcl-2 family proteins in SCLC cell lines that were less sensitive to RAD001 sensitised these cells to RAD001. Moreover, SCLC tumour growth in vivo was inhibited by RAD001. Together our data demonstrate that inhibiting mTOR signalling with RAD001 potently disrupts growth and survival in human SCLC cells.

77

Poster 14

Thrombin control of PI3K signaling underlies plasticity of hair follicile dermal cell Anne-Cathrine Feutz, Yann Barrandon and Denis Monard Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland and EPFL, Lausanne, Switzerland Hair follicile development and cyclic growth is tightly regulated by the interactions between a subset of mesenchymal cells forming the dermal papilla and follicular epithelial cells. The serine protease inhibitor nexin-1 (PN-1) shows a cyclic expression in both mesenchymal structures of hair follicle: the dermal papilla (DP) and dermal sheath (DS). Here, we show that during anagen PN-1 colocalized with thrombin and controls its activity. In follicles from PN-1 -/- mice thrombin activity is increased and the protein accumulates in DP. As cultured DP cells spontaneously express the thrombin receptor PAR-1, cells lacking PN-1 show a thrombin-dependent overgrowth in vitro. In addition, increased expression of smooth muscle cells actin protein (SMA) correlates with a thrombin-dependent gain in contractile properties. Both thrombin effects involve activation of the PI3K/Akt pathway suggesting that they are coordinated. Those two in vitro features indicate that PN-1 protects DP cells from acquiring the characteristics of “myofibroblastc” DS cells. The relevance of our in vitro data was confirmed by detecting a global overactivation of the PI3K pathway and an increased expression of SMA protein restricted to PAR-1 positive DS cells of PN-1 -/- anagen hair follicles. Morover, SMA expression was reinforced in trailing dermal sheath cells and appeared in DP during catagen indicating that DP to DS-like transition correlates with natural disappearance of PN-1 expression and upregulation of PAR-1. In PN-1 -/-, change in cell differentiation was associated with a decreased ability of DP cells to sustain growth and survival of cultured keratinocytes. Such a deficiency could be reproduced by pretreatment of wt DP cells with an excess of thrombin. Our study demonstrates that control of thrombin activity is involved in maintaining both identity and functional integrity of DP. It strongly suggests that thrombin links control of DP cells differentiation and switch in exocrine activity during follicular cycle.

78

Poster 15

MNK1 and MNK2 regulation in HER2 over-expressing breast cancer cell lines Carol A. Chrestensen1,*, Jacquelyn K. Shuman1, Andrew Eschenroeder1, Mark Worthington2, Herman Gram3, and Thomas W. Sturgill1 Department of Pharmacology1, University of Virginia, Charlottesville, Virginia, 22908; Division of Digestive Diseases2, Johns Hopkins Bayview Medical Center, Baltimore, MD 21224; and Arthritis and Bone Metabolism3, Novartis Pharma AG, CH-4002 Basel, Switzerland. Current address*- Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, GA 30144 Correspondence: Thomas W. Sturgill, Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, Charlottesville, VA USA 22908-0735. Tel: (434) 924-9191. Fax: (434) 924-5207. eMail: [email protected] Map kinase-interacting protein kinases 1 and 2 (MNK1, MNK2) function downstream of p38 and ERK MAP kinases, but there are large gaps in our knowledge of how MNKs are regulated and function. As proteins activated in the HER2/ras/raf/ERK pathway the MNKs are of potential interest in HER2 over-expressing cancers. We utilized a panel of breast cell lines (HCC1419, AU565, SKBR3, MCF7 and MC10A), three of which over-express HER2, to characterize the amounts and activation status of MNKs, and other pathway enzymes (ERKs, RSKs) in these cells. We generated a phosphospecific antibody to pT214 in the T-loop of MNKs and found that phosphorylations of both T209 and T214 in huMNK1 are required for activation. Increased phosphorylation and activity of the MNKs correlates with HER2 over-expression and a with Novartis compound CGP57380 characterized in several studies as a specific MNK inhibitor reduces colony formation in soft agar. Our work identifies the MNKs as potential therapeutic targets for breast cancer treatments.

79

Poster 16

Src-mediated PDK1 activation requires Tyr-9 phosphorylation of PDK1 and the assistance of Hsp90 Keum-Jin Yang1, Sanghee Shin1, Longzhen Piao1, Eulsoon Shin1, Yuwen Li1, Miran Roh1, Kyung Ah Park2, Hee Sun Byun2, Minho Won2, Gang Min Hur2, Jeong Ho Seok2, Brian A Hemmings3 and Jongsun Park1 1Cell Signaling Laboratory, Cancer Research Institute, 2Department of Pharmacology, College of Medicine, Chungnam National University, Taejeon, South Korea, 3Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell-signaling. PDK1 activate members of the AGC family of protein kinases including PKB, S6K, SGK and PKC, by phosphorylating serine/threonine residue in the activation loop. PDK1 has been reported to be constitutively active in resting cells and is further activated by tyrosine-phosphorylation (Tyr9 and Tyr373/376) following the treatment of cell with insulin or pervanadate. Furthermore, Tyr373/376 is important for PDK1 catalytic activity. In addition, Tyr373/376 phosphorylation of PDK1 is dependent on Tyr9 phosphorylation, suggesting that the activity of Tyr373/376 kinase is influenced by Tyr9 residue. Here, we investigate the possible involvement of SH2 domain which is known to recognize tyrosine phosphorylation of protein, in PDK1 activation process. In vitro, pervanate-stimulated PDK1 binds directly to SH2-contaning proteins, Src, Crk and GAP. Using deletion mutants of Src, we confirmed that SH2 domain of Src is responsible for the interaction with PDK1. Furthermore, the specificity of Src-SH2 domain for sites of tyrosine phosphorylation on PDK1 was verified by using two phosphopeptide binding assay and PDK1 phospho-mutants. HSP90 inhibitor destabilized the PDK1 protein level. This destabilization is partially blocked in the HEK 293 cells expressing PDK1-Y9F. Co-expression of HSP90 enhances the PDK1/Src complex formation and lead to further increase in PDK1 activity. Taken together, these data suggest that Src-induced PDK1 activation and it’s tyrosine phosphorylation might be another mechanism how Src acts as an oncogenic tyrosine kinase in various cancers together with Hsp90’s assistance. This work was supported by the SRC/ERC Program (Grant R11-2002-100-02006-0) and Basic Research Program grant (R01-2005-000-10240-0, F01-2005-000-10011-0) of MOST/KOSEF.

80

Poster 17

Identification of new molecular targets for human medulloblastoma using RNA interference screening A.S. Guerreiro1, A.N.D. Elsing1, T. Shalaby2, M.A. Grotzer2, and A. Arcaro1

1 Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, Zurich, Switzerland; 2 Neuro-Oncology Program, University Children’s Hospital, Zurich, Switzerland Rationale: Medulloblastoma, a malignant embryonal tumor of the cerebellum, is the most common cause of cancer-related death in children. As little is known about the molecular mechanisms underlying tumor growth in medulloblastoma, we sought to identify kinases that regulate cell proliferation and apoptosis using high-throughput RNA interference (RNAi) screen targeting most of the human genes encoding protein and lipid kinases in medulloblastoma cell lines. Cell proliferation and evasion of apoptosis are hallmarks of cancer, and an understanding of the regulation of these processes may lead to identification of new molecular prognostic markers and facilitate the development of targeted therapies for medulloblastoma. Methods: DAOY medulloblastoma cells were transiently transfected with 1111 shRNAs directed against 561 known and putative human kinases. Cells were incubated 24h-72h to allow target knockdown. Cell growth was assessed using the MTT assay. Apoptosis was assessed by measuring caspase-3 activity. shRNA-mediated downregulation of gene expression was confirmed by Western blot. Results: About 10% of the total number of shRNA constructs inhibited growth of DAOY cell line by more than 80%, while 25% inhibited growth by more than 70%. The shRNA constructs resulting in most efficient suppression of DAOY cell growth were grouped into categories based on the known function of their targeted genes, including cell cycle control, AGC kinases, receptor tyrosine kinases (RTK), phosphoinositide 3-kinases (PI3K), mitogen-activated protein kinase (MAPK) signaling and Src kinases. These constructs were selected and tested in a secondary screen for induction of apoptosis. Kinases targeted by the shRNA constructs that most efficiently inhibited cell growth and induced apoptosis were selected for further studies. Conclusions: RNAi targeting of specific protein and lipid kinases in a large scale approach allowed identification of key regulators of cell proliferation and apoptosis in medulloblastoma cell lines. In addition to known kinases, new survival kinases were identified. Further validation of the positive kinases and translation to clinical knowledge will hopefully bring a new understanding of medulloblastoma disease biology.

81

Poster 18

Pentacyclic triterpenoids inhibit IκB kinase activity and promote apoptosis in androgen-independent prostate cancer T. Syrovets, J.E. Gschwend, B. Büchele, Y. Laumonnier, Th. Simmet Dept. of Pharmacology of Natural Products and Clinical Pharmacology and Dept. of Urology, University of Ulm, D-89081 Ulm, Germany Signaling through NF-κB has been implicated in the malignant phenotype as well as chemoresistance of various cancers. Here we show that the chemically pure pentacyclic triterpenoids acetyl-β-boswellic acid (AβBA) and acetyl-11-keto-β-boswellic acid (AKβBA) inhibit proliferation and elicit concentration- and time-dependent cell death in chemoresistant androgen-independent PC-3 prostate cancer cells both in vitro and in vivo. Apoptosis was shown in cultured PC-3 cells by phosphatidylserine expression, appearance of the sub-G0/1 peak, mitochondrial cytochrome c release and DNA fragmentation. At the molecular level these compounds intercept the IκB kinase (IKK) activity and inhibit the constitutively activated NF-κB signaling of PC-3 cells; signaling through the interferon-stimulated response element remained unaffected suggesting specificity for IKK inhibition. This led to impaired phosphorylation of p65 and reduced nuclear translocation of NF-κB proteins as well as down-regulation of the constitutively overexpressed and NF-κB-dependent antiapoptotic proteins Bcl-2 and Bcl-xL. Additionally, expression of cyclin D1, a crucial cell cycle regulator, was reduced as well. Down-regulation of IKK by antisense oligodeoxynucleotides confirmed the essential role of IKK for the proliferation of the PC-3 cells. Both compounds tested were active in vivo, yet AKβBA proved to be far superior. Indeed, topical application of water-soluble AKβBA-γ-cyclodextrin on PC-3 tumors xenografted onto chick chorioallantoic membranes induced concentration-dependent inhibition of proliferation as well as apoptosis. Similarly, in nude mice carrying PC-3 tumors i.p. injection of AKβBA-γ-cyclodextrin inhibited the tumor growth and triggered apoptosis in the absence of detectable systemic toxicity. Thus, AKβBA and related compounds acting on IKK might provide a novel approach for the treatment of chemoresistant human tumors such as androgen-independent human prostate cancers.

82

Poster 19

HIPK2 phosphorylates p53 serine 46 in response to DNA double-strand breaks and is regulated by checkpoint kinase ATM Ilka Dauth, Jana Krüger1 and Thomas G. Hofmann

Zelluläre Seneszenz, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 1Heinrich-Pette-Institut, Hamburg, Germany The tumor suppressor p53 is known as the “guardian of the genome” because of its role in maintaining genomic stability. p53 is a transcription factor that is stabilized after different kinds of DNA damage including UV, Cisplatin and ionizing radiation (IR). p53 is involved in different cellular responses such as DNA repair, cellular senescence and induction of apoptosis. p53 activity is mainly regulated by its subcellular distribution and posttranslational modifications, in particular through phosphorylation and ubiquitination. It is established that the phosphorylation of p53 at serine 46 (p53 Ser46) is necessary for the induction of apoptosis after different kinds of DNA damage. UV induced DNA damage leads to p53 Ser46 phosphorylation by Serine/Threonine kinase HIPK2 (Homeodomain Interacting Protein Kinase 2). The kinase phosphorylating the Ser46 residue of p53 after double strand breaks (DSBs) induced by IR is currently unknown. Here we show that IR provokes HIPK2 accumulation, activation and complex formation with endogenous p53. Furthermore, IR-induced HIPK2 accumulation strictly correlates with p53 Ser46 phosphorylation. Strikingly, down regulation of HIPK2 by RNA interference specifically inhibits IR-induced p53 Ser46 phosphorylation. Furthermore, we could show that HIPK2 is regulated by the checkpoint kinase ATM (ataxia telangiectasia mutated). ATM is an important kinase involved in DNA-damage response after DSBs through phosphorylation of effector proteins such as p53 (Ser15). Interestingly, cells from A-T patients show defects in the stabilization of HIPK2 in response to IR and p53 Ser46 is not phosphorylated after IR. Pharmacological inhibition of the ATM kinase in cells leads to accumulation failure of HIPK2 after IR. In summary, our data show that HIPK2 is the IR-activated p53 Ser46 kinase and provide evidence for its regulation by ATM. Finally, our results suggest that HIPK2 might prove to be a promising target in cancer therapy.

83

Poster 20

Constitutively activate IGF-IR causes complete transformation of mammary epithelial cells and is associated with an epithelial to mesenchymal transition via NF-kappaB-mediated induction of snail and reversed by the new IGF-IR small molecule inhibitor BMS-536924 Lee AV, Cui X, Grabiner B1, Lin X1, Litzenburger BC, Lewis MT, Carboni J2, Gottardis M2, Wong TW2, Attar R2, Kim HJ. Breast Center, Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. 1Department of Molecular and Cellular Oncology, University of Texas, M.D. Anderson cancer center, Houston, Texas, 2Oncology Drug Discovery, Bristol-Myers Squibb Research Institute, Princeton, New Jersey. Type I insulin-like growth factor receptor (IGF-IR) is overexpressed in a number of cancers and contributes to tumor invasion and metastasis. We have previously reported that a constiutively active IGF-IR (CD8-IGF-IR) causes rapid mammary tumorigenesis when overexpressed in the mouse mammary gland. To elucidate the molecular mechanisms of tumor formation, we stably overexpressed CD8-IGF-IR in immortalized, but non-transformed, mammary epithelial cells (MCF-10A). MCF-10A-CD8-IGF-IR cells showed constitutive IGF-IR phosphorylation in the absence of any IGF stimulation. MCF-10A-CD8-IGF-IR showed numerous features of transformation including growth in the absence of serum, lack of contact inhibition in monolayer and foci formation, anchorage-independent growth in soft-agar, and invasion through matrigel. Interestingly, MCF-10A-CD8-IGF-IR cells were also able to grow as xenografts in immunocomprimised mice (when injected with or without matrigel) an uncommon feature following transformation of MCF-10A cells with a single oncogene. BMS-536924 was effective at blocking both IGF-I stimulated wild-type IGF-IR and also CD8-IGF-IR activity. Inhibition was maximal at 1μM, a concentration which didn’t affect epidermal growth factor (EGF)-mediated activation of EGFR signaling. Monolayer growth assays showed that BMS 536924 induced a dose dependent inhibition of proliferation with an IC50 of 0.4-0.8μM, whereas the IC50 in anchorage independent growth was nearly a log-fold lower. Flow cytometry indicated that BMS-536924 caused a G0/G1 block in the cell cycle. BMS-536924 was also able to completely reverse the CD8-IGF-IR induced invasion. Finally BMS-536924 at 100mg/kg/day caused a 70% reduction in MCF-10A-CD8-IGF-IR xenograft volume. In addition to the transformation, we also found that MCF-10A-CD8-IGF-IR cells underwent an epithelial to mesenchymal transition (EMT). EMT was revealed at the molecular level by decreased levels of epithelial markers E-cadherin, and alpha- and beta-catenin, and increased expression of mesenchymal markers fibronectin, vimentin, N-cadherin and alpha-smooth muscle actin. Consistent with this EMT, MCF-10A-CD8-IGF-IR cells showed dramatically increased migration compared to parental MCF-10A cells which were non-migratory. MCF-10A-CD8-IGF-IR cells showed low levels of E-cadherin mRNA and promoter activity, which was correlated with increased levels of the transcriptional repressor snail, which binds and inhibits the E-cadherin promoter. BMS-536924 (1μM) completely blocked CD8-IGF-IR activity and caused a decrease in snail levels, re-induction of E-cadherin levels, and a morphological reversion of the EMT. CD8-IGF-IR cells showed increased NF-kappaB activity as assessed by gel-shift assay and p65 localization in the nucleus. Pharmacologic blockade of NF-kappaB activity lowered snail levels and morphologically reversed the EMT, consistent with previous reports that NF-kappaB binds and regulates the snail promoter and is critical for EMT. This study indicates that CD8-IGF-IR activates NF-kappaB to increase snail levels, downregulate E-cadherin, and cause EMT. In addition the study reveals that the new small molecule, BMS 536924, is an effective inhibitor of IGF-IR, causing complete reversion of an IGF-IR-mediated transformation and reversing EMT.

84

Poster 21

PKB is activated by DNA-PK in response to DNA double-strand breaks to regulate survival and proliferation Lana Bozulic, Banu Surucu, Ekaterina Gresko, Jianhua Feng1, Jongsun Park2 and Brian A Hemmings Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; 1University Hospital Zürich, Center for Integrative Human Physiology, Zürich, Switzerland; 2 Cancer Research Institute, Department of Pharmacology, College of Medicine, Chungnam National University, South Korea

The three PKB isoforms share a conserved structure that includes three functional domains: an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory domain. Activation of all three isoforms is regulated via phosphorylation of the conserved residues, Thr308 in the activation loop, and Ser473 in the hydrophobic motif (for the PKBα isoform). Phosphorylation at Ser473 stabilizes

the active conformation state, and therefore is the key step in the activation of PKB that needs to be tightly controlled. These phosphorylation events involve recruitment of PKB to the plasma membrane. Once activated at the plasma membrane, phosphorylated PKB can translocate to the cytosol and the nucleus; some recent data suggest that activation can even occur in the nucleus. Recent findings from our laboratory and other groups proposed the members of the PI3K-like family of protein kinases (PIKKs): DNA-PK, mammalian target of rapamycin (mTOR) and ataxia-telangiectasia mutated (ATM) as bona fide candidates for Ser473 kinase. We found the DNA-dependent protein kinase (DNA-PK) to be the major Ser473 kinase activity purified from HEK293 cells plasma membrane fraction, and an effective Ser473 kinase in vitro. However, as a second fraction of Ser473 activity was present in our plasma membrane preparations, and cells deficient in DNA-PK still exhibited low but persistent levels of Ser473 phosphorylaton, we concluded that a second physiological kinase for Ser473 must exist. Employing a stable inducible RNAi system targeting mTOR, DNA-PK or ATM in HEK293 cells, we found that ablation of ATM by RNAi has no effect on PKB activity. mTOR knockdown results in loss of PKB Ser473 phosphorylation and failure to phosphorylate downstream targets GSK3 and FKHRL1, suggesting that mTOR is the major Ser473 kinase in growing cells with little or no contribution from DNA-PK. In contrast, when cells are starved, or starved and subsequently stimulated with insulin, both mTOR and DNA-PK contribute to the activation of PKB, which is reflected by PKB downstream signaling. Finally, using γ-irradiation, we found that DNA-PK is the major kinase responsible for PKB activation in response to DNA double-strand breaks. Downregulation of DNA-PK by RNA interference in HUVEC cells results in an impaired response of PKB to γ-irradiation. Pretreatment of HUVEC cells with NU7026, a highly specific small molecule inhibitor of DNA-PK, ablates Ser473 phosphorylation upon DNA-damaging treatment. Furthermore, immunoprecipitated PKB from cells treated with the inhibitor shows significantly reduced in vitro kinase activity. DNA-PK co-immunoprecipitates with PKB in irradiated cells, but not in control cells or NU7026 treated cells. Consequently, mouse embryonic fibroblasts (MEF) deficient in DNA-PK fail to activate PKB in response to γ-irradiation, but not to insulin stimulation. DNA-PK knock-out MEFs are hypersensitive to radiation induced apoptosis. Ablation of total PKB by siRNA mediated knockdown results in increased apoptosis and altered cell cycle following γ-irradiation. Interestingly, the PKBα but not PKB β/γ knockout MEFs resemble the DNA-PK deficiency phenotype, and show about 65% apoptosis following irradiation treatment as opposed to 25% observed in WT or PKB β/γ knockout cells. We conclude that DNA-PK specifically activates PKB by hydrophobic motif Ser473 phosphorylation in response to DNA DSBs. Active PKB then provides a survival signal for the cell, influencing anti-apoptotic and cell cycle parameters. Furthermore, this action may be resticted to the PKBα isoform.

85

Poster 22

Defining pathways influencing paclitaxel sensitivity Marani Michela Cancer Research UK, 44 Lincoln's Inn Field, London, WC2A 3PX, UK De novo and acquired chemotherapy drug resistance is a major factor determining treatment outcome in cancer patients. New insights into the mechanism of action of anticancer drugs are essential to improve their efficacy and to help identify patients with chemo-sensitive disease. This study describes an RNA interference-based strategy aimed at discovering genes involved in resistance of human cancer cells to the widely used chemotherapeutic agent paclitaxel (Taxol). For this purpose we have challenged colon, breast and lung human cancer cell lines with siRNAs targeting all known human kinases together with the microtubule stabilizing agent Taxol. Recent data strongly suggest that the efficacy of Taxol in killing cancer cells depends on the integrity of the mitotic checkpoint. While silencing of known mediators of the spindle assembly checkpoint did indeed mediate drug resistance in all cancer cell lines under study, we have also characterised novel human protein kinases whose effect on mitosis determines the sensitivity of cancer cells to the microtubule-disrupting chemotherapy. The discovery of cell signalling pathways leading to drug resistance may provide novel targets for increasing treatment efficacy.

86

Poster 23

Wnt1 transactivates EGFR in human breast cancer cells Thomas Schlange, Yutaka Matsuda and Nancy E. Hynes Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Background: Wnt growth factors induce a number of signaling pathways that play context dependent roles in the development of several types of cancer. While mutations in the “destruction“ complex for beta-catenin and in the N-terminus of beta-catenin itself are causative for the majority of colorectal cancers, de-regulation of the Wnt pathway seems to occur in an autocrine fashion in breast cancer. Previous work from our lab linked Wnt signaling to the activation of EGFR receptor tyrosine kinase in non-transformed mouse mammary epithelial cells. Activation of EGFR in human breast cancers is discussed as a possible mechanism for development of resistance to targeted treatment against estrogen receptor (ER)-postive and ErbB2 over-expressing cancer cells in the clinic. Materials and Methods: Therefore, we analyzed the effect of Wnt mediated EGFR transactivation in human breast cancer cell lines treated with 4-hydroxytamoxifen (4-HT) or trastuzumab/4D5 in vitro. sFRP1 was used as a naturally occurring inhibitor of Wnt signaling in conditioned medium and stably transfected breast cancer cells to analyze the effect of inhibition of Wnt signaling in vitro and in vivo (tumor xenografts and tail vein injection. Results: We provide evidence for a cross-talk of a non-canonical Wnt signal with EGFR that is mediated in a similar fashion as transactivation of this receptor tyrosine kinase (RTK) by different GPCR ligands. The mechanism depends on the activity of heterotrimeric G proteins, PLC, Src kinase and metalloprotease activity, but is apparently independent of beta-catenin. Expression data reveal that human breast cancer cell lines express several Wnt ligands, which may account for the autocrine activation of different signaling pathways downstream of the Wnt ligands. Furthermore, we show that inhibition of autocrine Wnt signaling by the secreted inhibitor sFRP1 reduces the growth rate of human breast cancer cells in vitro and in vivo. Conclusions: Data presented imply that targeting the Wnt pathway in breast cancer may slow the growth of tumor cells and may provide a new therapeutic tool to prevent development of resistance against established treatments. Funded by the Swiss Cancer League (Onco Suisse 01445-12-2003) and the Novartis Research Foundation. Civenni, G., Holbro, T., and Hynes, N.E. (2003). Wnt1 and Wnt5a induce cyclin D1 expression through ErbB1 transactivation in HC11 mammary epithelial cells. EMBO Rep. 4, 166-171.

87

Poster 24

TSC1-TSC2 complex inhibits mTORC2 in human cells Klaus D. Molle and Michael N. Hall Department of Biochemistry, Biozentrum, University of Basel The growth controlling mammalian Target of Rapamycin (mTOR) is a conserved Ser/Thr kinase found in two structurally and functionally distinct complexes, mTORC1 and mTORC2. The tumor suppressor TSC1-TSC2 complex inhibits mTORC1 by acting on the small GTPase Rheb, but the role of TSC1-TSC2 and Rheb in the regulation of mTORC2 is unclear. Here we examined the role of TSC1-TSC2 in the regulation of mTORC2 in human embryonic kidney 293 cells. Induced knockdown of TSC1 and TSC2 (TSC1/2) stimulated mTORC2-dependent actin cytoskeleton organization and Paxillin phosphorylation. Furthermore, TSC1/2 siRNA increased mTORC2-dependent Ser473 phosphorylation of plasma membrane bound, myristoylated Akt/PKB. This suggests that loss of Akt/PKB Ser473 phosphorylation in TSC mutant cells, as reported previously, is due to inhibition of Akt/PKB localization rather than inhibition of mTORC2 activity. Amino acids and overexpression of Rheb failed to stimulate mTORC2 signaling. Thus, TSC1-TSC2 also inhibits mTORC2, but possibly independently of Rheb. Our results suggest that mTORC2 hyperactivation may contribute to the pathophysiology of diseases such as cancer and Tuberous Sclerosis Complex.

88

Poster 25

Regulation of EGF receptor signaling by myotubularins Philipp Berger1,3, Imre Berger2, Christiane Schaffitzel2, Kristian Tersar1, Kurt Ballmer-Hofer3, and Ueli Suter1

1 Institute of Cell Biology, ETH Zurich, Switzerland 2 Institute of Molecular Biology and Biophysics, ETH Zurich, Switzerland 3 Paul Scherrer Institute, Villigen, Switzerland Myotubularins (MTMRs) form one of the largest and most conserved families of dual-specific protein tyrosine phosphatase-like enzymes. Mutations in three genes of the myotubularin gene family have been associated with human diseases. Charcot-Marie-Tooth disease, a peripheral neuropathy, is caused by mutations in MTMR2 or MTMR13/sbf2 and mutations in myotubularin, the founding member of this family, lead to X-linked myotubular myopathy. We previously showed that the phosphoinositides PI-3-P and PI-3,5-P2 are the major enzymatic substrates of MTMR2, which removes the phosphate at the D3 position. Interestingly, not all MTMRs are active phosphatases. Six family members, including MTMR13/sbf2, bear substitutions in catalytically essential amino acids of the phosphatase domain, rendering them inactive. Here we show that MTMR13/sbf2 interacts with MTMR2 and that it modulates the function of MTMR2 either by increasing the catalytic activity or by acting as a substrate trap. Stimulation of the EGF receptor by EGF leads to the immediate production of PI-3,4,5-P3, a delayed increase of PI-3,5-P2 levels, and the degradation of the EGF receptor within 3 hours. Overexpression of PI-3,5-P2 dephosphorylating MTMR2 in these cells blocks the degradation of the EGF receptor. Surprisingly, this effect of MTMR2 is reversed by simultaneous overexpression of MTMR13/sbf2, which contains a PI-3,4,5-P3-binding pleckstrin homology domain. These results suggest that PI-3,4,5-P3 and PI-3,5-P2 signaling are linked together and that active and inactive myotubularins regulate signaling by regulating receptor levels.

89

Poster 26

The purinome, a complex mix of drug and toxicity targets Timothy A. J Haystead* and Steven Hall# Departments of Pharmacology and Cancer Biology*, Duke University, NC 27710 and #Serenex Inc., Foster Street, Durham NC, USA. Much attention has focused on the development of protein kinases as drug targets to treat a variety of human diseases from diabetes, cancer, hypertension to arthritis. To date there is a least one example of a drug targeting a protein kinase that has successfully treated a human cancer, Gleevec. Several other protein kinase inhibitors are in clinical development. However, protein kinases are in fact part of a larger collection of some 2000 distinct proteins expressed by the genome (the purinome) that like the protein kinases also bind purines, either to be utilized as substrates or as co-factors in the form of NAD, NADP and co-enzyme A. The solution structures of many representative gene family members within the purinome show these proteins bind purines in a similar orientations to that observed in all protein kinases. Several non-protein kinase purine utilizing proteins are established drug targets such as HMG CoA reductase, dihydrofolate reductase, phosphodiesterase or HSP90. Searches of OMID identifies many purine utilizing enzymes that are associated with inborn errors in metabolism. Inhibition of any one of which by a drug could lead to an undesirable side effect. The purinome is therefore somewhat of a drug discovery mixed blessing. It is a rich source of therapeutic targets, but also contains a large collection of diverse proteins whose inhibition could result in an adverse outcome. Drug discovery within the purinome should therefore encompass strategies that enable broad assessment of selectivity across the entire purinome at the earliest stages of the discovery process. Herein we describe the technology of proteome mining and how this was utilized to capture the purinome to discover highly selective novel inhibitors of the signal transduction chaperone protein HSP90.

90

Poster 27

Profiling of signaling molecules in four different human prostate carcinoma cell lines before and after induction of apoptosis Iben H.E. Skjøth and Olaf-Georg Issinger University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark We have treated four prostate tumor cell lines, DU-145, PC-3, LNCaP and 22RV1 with various concentrations of cisplatin in order to check for influence on viability and for onset of apoptosis induction. At a cisplatin concentration of 20 µM, 22RV1 and DU-145 cells showed ~22% and 18% and PC-3 and LNCaP cells showed ~4% and 10% dead cells, respectively. When checking for apoptosis induction, the differences among the cell lines became even more evident. DU-145 and 22RV1 cells showed apoptosis induction at 5 µM and 2 µM cisplatin, whereas in the case of LNCaP and PC-3 cells comparable apoptosis induction was observed at 100 µM cisplatin; hence, the difference between the two groups of cell lines with respect to apoptosis induction is 20- and 50-fold, respectively. We used 37 antibodies to screen the expression levels of key signalling molecules and their phosphorylation status where appropriate. DU-145 and PC-3 cells are androgen-receptor negative and harbour non-functional p53, whereas LNCaP and 22RV1 cells are androgen-receptor positive and harbour wild-type p53. The results of the profiling of DU-145 and PC-3 support the notion that an intact PTEN/AKT pathway (as found in DU-145 and 22RV1 cells) and the presence of active p38 are responsible for the high sensitivity to apoptosis induction and that neither the androgen-receptor nor the p53 status is of primary importance for the differences observed with respect to apoptosis induction.

91

Poster 28

Centrosome-associated NDR kinase regulates centrosome duplication Alexander Hergovich1, Stefan Lamla2, Erich A. Nigg2, and Brian A. Hemmings1

1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; 2Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried bei München, Germany Human NDR kinases are upregulated in certain cancer types, yet their functions still remain undefined. Here, we report the first known function of a mammalian NDR kinase by demonstrating that human NDR directly contributes to centrosome duplication. A subpopulation of endogenous NDR localizes to centrosomes in a cell-cycle dependent manner. Overexpression of NDR resulted in centrosome overduplication in a kinase-activity dependent manner, while expression of kinase-dead NDR or depletion of NDR by small interfering RNA (siRNA) negatively affected centrosome duplication. By targeting NDR to the centrosome, we also show that the centrosomal pool of NDR is sufficient to generate supernumerary centrosomes. Furthermore, our data indicate that NDR-driven centrosome duplication requires Cdk2 activity and that Cdk2-induced centrosome amplification is affected when NDR activity is reduced. Overall, considering that centrosome overduplication is linked to cellular transformation, our observations may also provide a molecular link between mammalian NDR kinases and cancer.

92

Poster 29

The role of DNA-PK in the activation of PKB by Ser473 hydrophobic motif phosphorylation Banu Surucu, Lana Bozulic, Debby Hynx and Brian A. Hemmings Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr308 and Ser473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 kinase (Ser473K), respectively. Although PDK1 has been well characterized, the identification of the Ser473K remains controversial. A major PKB Ser473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK is a member of PIKK family that includes ATM, ATR, mTOR and is known to be activated by DNA damage. Significantly, both mTOR and ATM have also been reported to regulate Ser473 phosphorylation. Our aim is to delineate the role of DNA-PK in PKB activation in response to different stimuli including insulin and DNA damage. As processing of multiple inputs may be used by cells to regulate the activation of PKB, the phosphorylation of the Ser473 could be stimulus, signaling pathway and/or cell type specific. Therefore, our studies make use of both genetically modified cell lines and in vivo mutant mice models lacking DNA-PK. To evaluate the involvement of DNA-PK in regulation of PKB under physiological conditions in response to insulin stimulation, we treated DNA-PK WT and KO mice with insulin, which is a well-established stimulus for PKB activation. Our results revealed that PKB Ser473 as well as S6K Thr389 phosphorylation is not compromised in several tissues tested. Moreover, preliminary results from insulin and glucose tolerance tests do not suggest insulin resistance and glucose intolerance in DNA-PK knock-out mice.

As the role of DNA-PK in response to DNA damage is well established, we investigated PKB activation by DNA double strand breaks. DNA-PK KO mice and cell lines deficient in DNA-PK are hypersensitive to ionizing radiation and to radiomimetric drugs, as can be observed in PKB� KO mice. Accordingly, we analyzed PKB activation following γ-irradiation or doxorubicin (topoisomerase II inhibitor that introduces DNA double strand breaks) treatment in DNA-PK WT and KO MEFs. We observed that PKB phosphorylation on Ser473 and Thr308 is induced in WT MEFs but this response was impaired in KO MEFs upon the induction of DNA damage. Our results indicate that PKB activation is promoted in response to DNA damage and DNA-PK is required for phosphorylation of the key regulatory site (Ser473) in the hydrophobic motif in this setting. Further in vivo analysis of PKB regulation in response to γ-irradiation is currently under investigation. Taken together, these results indicate that DNA-PK is not the physiological Ser473 kinase in response to insulin stimulation. Rather, DNA-PK is involved in regulation of PKB upon DNA damage induction indicating the stimulus specific regulation of PKB activation.

93

Poster 31

Identification of Src kinase substrates regulating anchorage independency of lung cancer cells Ryuichi Sakai and Takamasa Uekita Growth Factor Division, National Cancer Center Research Institute, Japan Solid cancer cells frequently show resistance to apoptosis induced by loss of attachment to extra cellular matrix, which is also called as anchorage independency. Several cellular changes acquired during the initiation and progression of cancer cells might contribute to anchorage independency. We noticed that soft agar colonies shown in several lung cancer cell lines are blocked by the Src-family inhibitor PP2 or by the siRNAs for Src family kinases. It was further revealed that several tyrosine-phosphorylated proteins characteristic to a subset of lung cancer cell lines are associating with Fyn kinase at the suspension culture condition. These phosphotyrosine-containing proteins are purified using two-step affinity purification from the suspension culture of an adenocarcinoma cell line A549 and several prominent phospho-tyrosine containing proteins are identified by mass spectrometry analysis. Two of the major Fyn-associating phosphotyrosine-containing proteins at the molecular weight of 135kD and 80kD in the suspension culture turned out to be a single protein CDCP1. The transmembrane protein CDCP1 is reported to be overexpressed in several cancer cell lines and its phosphorylation condition is regulated by factors including integrin stimulation. Partial scission of 135kD CDCP1 by serine proteases produces the 80kD product. Phosphotyrosine-mediated association of CDCP1 with c-Src has recently been reported although the biological role of this molecule in cancer is still unclear. It was confirmed that CDCP1 is associating with Fyn in vitro in A549 cells, and both expression and phosphorylation of CDCP1 in these cells are significantly elevated during the suspension culture. Knock-down of CDCP1 by RNAi in A549 cells caused apoptosis in suspension condition but not in adherent condition, while no significant effect was observed on the cell proliferation. At the same time, the ability to form colonies on soft agar is significantly reduced by knock-down of CDCP1. It was suggested CDCP1 is a molecule regulating anchorage independency under the control of Src family kinases in a subset of lung cancer cells and therefore possible candidate for the target to block unregulated growth of lung cancer.

95

Poster 32

Using mammalian and yeast systems to study MEMO, a novel ERBB2 effector protein Maria Meira, Regis Masson, Nancy Hynes Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. The Neu/ErbB2 receptor tyrosine kinase has been shown to play an important role in cancer cell motility and metastases formation. This receptor is often overexpressed in human tumors of diverse origins including breast and ovarian. Our studies focused on signaling molecules that interact with autophosphorylated tyrosine residues of the cytoplasmic tail of ErbB2 receptor. We have discovered that two autophosphorylation sites, Tyr 1201(YC) and Tyr1227(YD) were fully able to restore the migratory phenotype and promote cell motility to breast carcinoma cells upon Heregulin (HRG) stimulation, whereas the receptor lacking the 5 major autophosphorylation sites was impaired in stimulating migration. A novel protein has been identified and named Memo for Mediator of ErbB2-driven cell Motility. Memo interacts with the phosphorylated Tyr1227(YD) and is essential for HRG induced motility (1). Based upon the fact that Memo knock-down (KD) does not block the ability of cells expressing the Tyr 1201 add-back mutant to migrate, we hypothesized that two parallel pathways stimulate migration in the context of a wild type ErbB2 receptor. We have found that PLCγ1 binds to Tyr 1201, and KD of PLCγ1 blocks HRG-induced cell migration. Furthermore, PLCγ1 KD cells, like Memo KD cells, show decreased microtubule outgrowth and strengthened actin stress fibers in response to HRG treatment. We are now focusing on the mechanisms underlying the effects of Memo and PLCγ1 on migration and initiated a genetic approach to accomplish this. Memo is a conserved protein and has an orthologue with 40% sequence identity in S. Cerevisiae. Strains lacking the Memo gene (YJR008W) are viable. Accordingly, we have performed a synthetic lethal screen (in collaboration with the group of Dr. Matthias Peter in Zurich) by mating a Memo deleted strain to an ordered array of 4700 yeast strains containing non essential gene deletions. This has led to the identification of a few genes that give a synthetic phenotype. 1. Marone, R. et al. 2004. Nature Cell Biol. 6: 515 - 522

96

Poster 33

Enhancing cisplatin sensitivity in MCF-7 human breast cancer cells by downregulation of Bcl-2 and cyclin D1 Christina Westmose and Olaf-Georg Issinger Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark MCF-7 human breast cancer cells are relative resistant to cisplatin treatment compared to other breast cancer cell lines. In order to identify possible targets for sensitizing the breast cancer cells to cisplatin treatment protein expression levels and the phosphorylation status of 27 different signaling proteins were examined. MCF-7 cells expressed high levels of anti-apoptotic Bcl-2 protein relative to more cisplatin sensitive breast cancer cells. After cisplatin treatment a decrease in cyclin D1 was seen in all the breast cancer cells studied. Therefore, Bcl-2 and cyclin D1 were chosen as putative targets for increasing cell death and growth arrest induced by cisplatin and thereby enhancing the drug sensitivity in MCF-7. RNA interference, using Bcl-2- and cyclin D1- siRNAs sensitized MCF-7 cells to cisplatin treatment and by simultaneous knockdown of both Bcl-2 and cyclin D1 further sensitization was seen. This shows the potential of targeting both apoptotic- and cell cycle-regulating pathways to enhance the effect of chemotherapy.

97

Poster 34

Molecular characterization of the mitotic spindle checkpoint protein Bub1 Christiane Klebig, Patrick Meraldi Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland Bub1, a conserved protein kinase acting at kinetochores, performs two crucial functions during mitosis. It is essential for the spindle checkpoint and is required for correct kinetochore-microtubule attachment. It has been shown that in Bub1 depleted cells an inactive spindle checkpoint and chromosome alignment defects lead to chromosome missegregation resulting in aneuploidy and genetic instability, a hallmark of cancer cells. Interestingly, a correlation between Bub1 mutations and carcinogenesis has already been described underlining the importance of a functioning Bub1 in normal cells. It is not yet clear how Bub1 contributes at the molecular level to its different functions and whether an impairment of the functions is strong enough to cause the development of a malignant phenotype. To elucidate the specific roles of Bub1 in the mitotic spindle checkpoint control, the establishment of correct kinetochore-microtubule attachment and how an impairment of its functions may lead to carcinogenesis, we are screening Bub1 mutants for separation of function and carcinogenicity in human cells. These different Bub1 mutants lack conserved domains or carry point-mutations found in human cancers. We are currently introducing these mutants by RNAi complementation in HeLa cells and will analyze the stable cell lines by cell biological techniques and live cell imaging. We will demonstrate whether the different mutations lead to an impairment in the spindle checkpoint control and/or kinetochore-microtubule attachment defects. Moreover, the effect of several mutants regarding the development of a malignant phenotype by using untransformed RPE-hTERT cells will be investigated. In a second step, we aim to use those tools to better understand at a molecular level how the spindle checkpoint functions and how failure of the checkpoint machinery might lead to cancer development.

98

Poster 35

Purification and characterization of the CK2alpha’-based holoenzyme, an isozyme of CK2alpha: a comparative analysis

Birgitte B. Olsen1, Brigitte Boldyreff2, Karsten Niefind3 and Olaf-Georg Issinger1

1Institute for Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. 2KinaseDetect Aps, Forskerparken 10C, Odense, Denmark. 3Institute for Biochemistry, University Köln, Germany.

Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.

99

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Olsen+BB%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Niefind+K%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&itool=pubmed_AbstractPlus&term=%22Issinger+OG%22%5BAuthor%5D

Poster 36

Loss of NOTCH2 positively predicts survival in distinct subgroups of human oligodendroglioma and glioblastoma Jean-Louis Boulay 1, André R. Miserez 2 , Christian Zweifel 1,3, Balasubramanian Sivasakaran 1, Anthony Ghaffari 1,3, Veronika Kana 1, Cordelia Luyken 4, Michael Sabel 4, Abdessamad Zerrouqi 5, Morten Wasner 3, Otmar Gratzl 3, Erwin van Meir 5, Markus Tolnay 6, Guido Reifenberger 4 and Adrian Merlo 1,3. 1 Laboratory of Molecular Neuro-Oncology, Department of Research, University Hospital, CH-4031 Basel, Switzerland; 2 Department of Internal Medicine, University Hospital, CH-4101 Bruderholz, and Cardiovascular Research Group, Diagene, CH-4153 Reinach, Switzerland; 3 Neurosurgical Clinic, University Hospital, CH-4031 Basel, Switzerland; 4 Departments of Neuropathology, and Neurosurgery, Heinrich-Heine-University, D-40225 Düsseldorf, Germany; 5 Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, Emory University, Atlanta, GA 30322, USA; 6 Institute of Pathology, University Hospital, CH-4031 Basel, Switzerland Histological classification and WHO grading represent the gold standard to estimate prognosis and guide therapy of glial brain tumors. Mean survival time of glioma patients varies from about 10 months in glioblastoma (GBM) to 10 years in oligodendroglioma (OG) grade II. Genetically, OG differs from GBM by the frequent loss of heterozygosity (LOH) of chromosome 1p that is associated with favorable prognosis and response to radio- and chemotherapy in anaplastic OG. Whether loss of 1p is also a prognostic marker in GBM is not known. We therefore constructed a somatic deletion map on chromosome 1p in 144 malignant brain tumors to relate distinct haplotypes to patient survival. We found that loss of 1p centromeric marker D1S2696 within the NOTCH2 gene is associated with favorable prognosis in both OG and GBM and detected NOTCH2 homozygous deletions in OG. We further developed a NOTCH2-based genetic assay that distinguishes GBM from OG with 1p loss and sharply predicts 24-months survival of glioma patients. Our data strongly suggest NOTCH2 to be causally linked to prognosis in both glioma subtypes.

100

Poster 37

Negative regulation of c-Src by binding to the PDZ domain of AF-6 Gerald Radziwill1, Andreas Weiss1, Jochen Heinrich1, Prisca Boisguerin2, Koji Owada3, and Karin Moelling1

1 Institute of Medical Virology, University of Zurich, 8006 Zurich, Switzerland; 2

Institute of Medical Immunology, Charité, 10115 Berlin, Germany; 3 Department of Molecular Bioregulation, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan The protooncogene c-Src encodes a tightly regulated non-receptor tyrosine kinase. c-Src plays a key role in cell adhesion, cell morphology and motility as well as in cell proliferation and survival. Elevated c-Src protein expression level and increased kinase activity have been found in several human cancers. Deletion of the C-terminal sequence results in a constitutively activated c-Src mutant detected in some human colon carcinoma. Replacement of the C-terminal sequence is characteristic for v-Src, the viral homologue of c-Src encoded by the avian Rous Sarcoma Virus (RSV), which causes tumours in chickens. In this study we describe the very C-terminus of c-Src as ligand for a PDZ domain protein. PDZ domain proteins are multidomain proteins and function as scaffolds to organize cell adhesion complexes, to cluster transmembrane proteins, and to align proteins of a signalling cascade, e.g. shown for Src/Raf-1 signalling (1). In many cases PDZ proteins mediate anti-proliferative effects. We demonstrate by mutational analysis that the C-terminal amino acid Leu of c-Src is essential for the binding to a PDZ domain. As candidate PDZ protein we identified AF-6, a junctional adhesion protein, involved in formation and maintenance of cell junctions (2) and in negative regulation of signalling (3). Here, we show that the PDZ domain of AF-6 strongly restricts the number of c-Src substrates, while knockdown of AF-6 has the opposite effect. The AF-6 PDZ domain interferes with phosphorylation of c-Src at Tyr 527 by the C-terminal Src kinase, CSK, and reduces autophosphorylation at Tyr 416 resulting in a moderately activated c-Src protein. The interaction of c-Src with a PDZ domain protein, e.g. AF-6, may spatially restrict c-Src to subcellular regions, e.g. cell-cell contacts. We identified a new type of negative regulation of c-Src through binding to a PDZ domain protein. 1. Ziogas A., Moelling K., and Radziwill G. J Biol Chem. 280: 24205-24211; 2005. 2. Lorger M. and Moelling K. J Cell Sci. 119: 3385-3398; 2006. 3. Radziwill G., Erdmann R., Margelisch U., Moelling K. Mol Cell Biol. 23: 4663-4672;

2003.

101

Poster 38

Identification of LNX as a novel Src-interacting protein Andreas Weiss, Gerald Radziwill, and Karin Moelling Institute of Medical Virology, Gloriastr. 30, CH-8006 Zurich, Switzerland Overexpression or increased activity of the proto-oncogene c-src is frequently detected in human malignancies. The non-receptor tyrosine kinase c-Src is important for many cellular processes such as receptor-mediated signaling, cell adhesion, motility, cell proliferation and survival. However, the precise functions of c-Src in normal tissues and in cancer remain unclear. Recently, we described the very C-terminus of c-Src, GENL (Gly-Glu-Asn-Leu), as a class III ligand for the PDZ domain protein AF-6 (see abstract Radziwill et al.). PDZ domain proteins are multidomain proteins and function as scaffolds to organize cell adhesion complexes, to cluster transmembrane proteins, and serve as points of integration or divergence of signaling cascades. In many cases PDZ proteins contribute to the maintenance of a non-proliferating state. To further study the role of PDZ ligand-mediated interactions of c-Src we performed a screen for PDZ domains that interact with c-Src. One candidate we identified to interact with c-Src is a PDZ domain of the Ligand-of-Numb protein X (LNX). LNX is a scaffold and a RING type E3 ubiquitin ligase, which contains, in addition to its RING domain, an NPXY motif and four PDZ domains. Here, we report the interaction of c-Src with LNX. By using a mutant where the C-terminal Leu of c-Src is mutated to Ala, a mutation that was shown to abolish binding of the ligand to a PDZ domain, we demonstrate that the c-Src/LNX interaction is at least in part PDZ-dependent. Furthermore, we show that LNX is a substrate for Src-mediated phosphorylation. Expression of LNX leads to the ubiquitination of its target proteins. Co-expression of c-Src and LNX, however, interferes with LNX-mediated ubiquitination. Our findings suggest an interplay between c-Src and LNX which might have an impact on the function of LNX as a scaffold or E3 ubiquitin ligase, or on the spatial regulation of c-Src activity as described for AF-6 before. 1. Radziwill G, Erdmann R, Margelisch U., Moelling K., Mol Cell Biol. 23: 4663-

4672; 2003 2. Lorger M. and Moelling K., J Cell Sci. 119: 3385-3398; 2006

102

Poster 39

Raf kinase Karin Moelling Institute of Medical Virology, University of Zurich, Switzerland The Raf kinase is an important signal transducer. It has isoforms, homologues, and structural features and biological properties, characteristic of many kinases. Based on known Raf-mediated signal transduction pathways one can draw simple stick models about signaling. These comprise positive or negative feedbacks, cross-talk, signal integration, bidirectional signaling, lateral signaling, transient or constituent signaling, inside-out, outside-in signaling, oscillations, etc. Electrical circuits can help to simplify apparently complex situations. 20 knowledge-based models will be presented and the question raised about prediction of further unknown ones. Some suggestions will be made. A mathematical description is also presented.

103

Poster 40

PP1 positively regulates the TNF/NF-κB pathway Shinya Mitsuhashi1, 2, Hiroshi Shima1, 3, Nobuhiro Tanuma1, Kunimi Kikuchi1, Makoto Ubukata2

1Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan 2Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan 3 Division of Cancer Chemotherapy, Research Institute, Miyagi Cancer Center, Natori, 981-1293, Japan Engagement of TNFR1 by TNFα activates three important signaling pathways: NF-κB, JNK, and caspases. The extent to which each pathway is activated determines the overall effect of TNFα on cell function such as apoptosis, inflammation, the immune response, and cell growth and differentiation. It is proposed that TNFα activates IKK and JNK, promoting IKK-induced phosphorylation and degradation of IκB and subsequent nuclear translocation of NF-κB. We were interested in defining differential contributions of two protein phosphatases, PP1 and PP2A, to TNFα-induced signaling. Activities of PP2A have been previously investigated using the specific inhibitor okadaic acid (OA). Here we used tautomycetin (TC), a highly specific PP1 inhibitor, to compare those effects. We show that TC specifically inhibits activation of NF-κB among the three TNFα effectors (NF-κB, JNK and caspase). Specifically, TC-treatment suppressed activation of IKK by attenuating T-loop phosphorylation of IKKα and IKKβ, resulting in inactivation of the NF-κB pathway. Co-immunoprecipitation experiment showed that catalytic subunit of PP1 physically interacted with IKK complex. Ours is the first demonstration that PP1 positively regulates the TNFα-induced NF-κB pathway at the level of IKK activation. Although PP2A negatively regulated TNFα-induced IκB phosphorylation and JNK activation as previously reported, we show that it functions positively in IKK activation. This study suggests that TC could be used therapeutically to suppress the NF-κB pathway.

104

Poster 41

The human tumour suppressor LATS1 is activated by human MOB1 at the membrane Alexander Hergovich, Debora Schmitz, Brian A. Hemmings Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Mammalian LATS kinases are implicated in regulating cell cycle progression and apoptosis. As members of the highly conserved NDR family, they belong to the AGC group of kinases. LATS1 deficient mice develop ovarian stromal cell tumours and soft tissue sarcomas. Promoter inactivation and missense mutations affecting LATS1 have been reported in human sarcomas, ovarian carcinomas, and breast cancer. These data suggest that dysregulated LATS1 expression contributes to tumour formation in mammals. Consistent with these observations, the warts/lats gene is a tumour suppressor in invertebrates. The signaling pathway involving the upstream kinase Hippo, Salvador, Lats/Warts, and Yorkie proteins is essential for proliferation control in D. melanogaster. Strikingly, Lats/Warts tumour suppressor activity depends on the direct interaction with the coactivator Dmob (Drosphila Mps1-One binder). Both Hippo and MST1/2 – the upstream kinase of mammalian LATS – belong to the Ste20-like kinase family. Recent data reports human LATS1 to interact with human MOB1 (hMOB1), but the activation of LATS1 was not addressed. Here, we identify a highly conserved hMOB1-binding motif within LATS1’s primary structure. While coexpression of LATS1 with hMOB1 did not elevate LATS1 kinase activity in mammalian cells, membrane-targeting of hMOB1 resulted in a significant increase of LATS1 activity. This stimulation was dependent on the activation segment – Ser909 – and the hydrophobic motif phosphorylation site – Thr1079 – and occurred a few minutes after membrane association. Based on these results we propose an in vivo mechanism of LATS1 activation through rapid recruitment to the plasma membrane by hMOB1 followed by multi-site phosphorylation. Our results provide key insights into the molecular regulation of the LATS tumour suppressor in mammalian cells.

105

Poster 42

The role of Frizzled-4 and -6 in angiogenesis Ivana Samarzija, Patrizia Sini, Thomas Schlange, Nancy Hynes Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland The Wnt family of secreted signaling molecules and the downstream components of Wnt signal transduction are highly conserved among animal species. Wnt molecules bind to and activate cell surface receptors encoded by the Frizzled (Fzd) gene family leading to phosphorylation of an intracellular intermediate Dishevelled (Dvl). Several Wnt-mediated independent intracellular signaling pathways have been characterized. These include the canonical pathway, which results in stabilization and nuclear translocation of β-catenin, the planar cell polarity (PCP) pathway and the Ca2+ pathway. Accumulating evidence suggests that the particular pathway activated depends on multiple factors, including the cell identity as well as the specific Wnt:Fzd pair. Several groups have identified a role for signaling through the Wnt pathway in both normal as well as situations of pathological angiogenesis, such as neovascularization of tumours. Wnt ligands as well as their receptors, are expressed by endothelial and vascular smooth muscle cells. β-catenin, a key downstream component of the Wnt signaling pathway, is stabilized in the neovascular endothelium and numerous genes essential for angiogenesis have been demonstrated to be targets of the Wnt signaling pathway. Despite this, detailed characterization of the role that activation of specific Fzd receptors plays in vascular development is lacking. We have initiated studies to determine the identity of the Fzd receptors expressed on endothelial cells and how these receptors directly regulate endothelial cell biology. Analysis of the gene expression profile of various endothelial cell lines indicated that Fzd-4 and Fzd-6 are the only members of this family expressed in both human and mouse endothelial cells. We examined the effect of recombinant Wnt-3a and Wnt-5a on Dvl phoshorylation, apoptosis and survival of Human Umbilical Vein Endothelial Cells (HUVEC). We observed that, although both the ligands induced Dvl phosphorylation, only Wnt-3a led to an increase proliferation, but not survival, of HUVEC. The Wnt- induced response was blocked by co-addition of a soluble form of Fzd-4. The effect on endothelial cell biology was not VEGF-(FGF/EGF) signaling dependent. Indeed, specific inhibitors for VEGFR, EGFR, and FGFR did not affect Wnt-induced HUVEC proliferation. Currently, we are carrying out studies to further delineate the signaling cascades and how canonical and non-canonical Wnt signaling pathway may regulate the biological response in endothelial cells. In addition, we are addressing the role of Fzd-4 and Fzd-6 in the in vivo development of normal as well as pathology associated vasculature employing Fzd-4 and Fzd-6-null mice.

106

Poster 43

Combination of sub-optimal concentrations of EGFR inhibitor and microtubule stabilizer induces apoptosis of glioblastoma cells Korur1 S, Failly1 M, Lino1 MM, Boulay1 JL, Egler1 V and Merlo1,2 A 1Laboratory of Molecular Neurooncology, 2Departments of Research and Surgery, University Hospitals, Schanzenstrasse 46, CH-4031 Basel, Switzerland. Glioblastoma multiforme (GBM) is the most frequent brain tumor, with a median survival of ten months. In transgenic mice, glioblastoma (GBM) cannot develop upon single, but only on simultaneous activation of the EGFR signaling mediators RAS and AKT. However, complete blockade of EGFR activation does not result in apoptosis in human GBM cells, suggesting additional cross-talk between downstream pathways. Based on these findings we investigated combination therapies using protein kinase inhibitors (PKI) against EGFR, PDGFR and mTOR and assessed GBM cell survival. Combined blocking of EGFR, PDGFR and/or mTOR activity with PKIs at clinically relevant dose levels did not induce GBM cell apoptosis. In contrast, blocking EGFR with AEE788 in combination with sublethal concentrations of the microtubule stabilizer patupilone induced apoptosis and reduced cell proliferation in GBM cells, accompanied by reduced AKT and ERK activity. Moreover, simultaneous inactivation of the EGFR downstream targets MEK and PI3K by U0126 and wortmannin triggered rapid tumor cell death. These data underline the critical role of the PI3K/AKT and the RAS/RAF/MEK/ERK signaling cascades in the cell-intrinsic survival program of sensitive GBM cell lines. We conclude that drug combinations, which down-regulate both ERK and PKB/AKT activity, may prove effective in overcoming cell resistance in a subgroup of GBM.

107

Poster 44

Tenascin-C is a novel RBPJk- induced target gene for Notch2 signaling in gliobastomas Balasubramanian Sivasankaran1†, Martin Degen2†, Anthony Ghaffari1,3, Christian Zweifel1,3, Mihai Ionescu1, Markus Tolnay4, Morten Wasner3, Robert Kiss5, Ruth Chiquet-Ehrismann2, Adrian Merlo1,3* & Jean-Louis Boulay1* 1Laboratory of Molecular Neuro-Oncology, University Hospitals, CH-4031 Basel, Switzerland 2Friedrich Miescher Institute, Novartis Research Foundation, CH-4058 Basel, Switzerland 3Neurosurgical Clinic, University Hospitals, CH-4031 Basel, Switzerland 4Institute of Pathology, University Hospitals, CH-4031 Basel, Switzerland 5Laboratory of Toxicology, Institute of Pharmacy, Free University, B-1070 Bruxelles, Belgium Glioblastomas (GBM) are very aggressive human cancers. We found that GBM frequently harbor amplifications at the Notch2 locus and express high levels of Notch2 protein. High Notch2 levels coincided with the presence of tenascin-C, an established prognostic marker for GBM. Transfection of the Notch2 intracellular domain activated tenascin-C gene transcription in an RBPJk-dependent manner and increased endogenous tenascin-C production in human fibroblasts and glioma cells. In a tenascin-C-negative glioma cell line, we identified a mutation in the sequence encoding the conserved RBPJk-interacting RAM domain of Notch2 (L1711M) that abolished the RBPJk-dependent induction of the tenascin-C promoter. The molecular link between Notch2 and Tenascin-C defines a novel pathway in gliomagenesis.

108

Poster 45

Tie2 kinase inhibitors inhibit angiogenesis and tumor growth A Vaupel, P Furet, K Masuya, C Schnell, McCarthy, M Ferretti, E. Billy, L, J Brueggen, C, V Rasetti, , M Lang and G Martiny-Baron Novartis Institutes for Biomedical Research NIBR, Basel Oncology, WKL125.1.16 Klybeckstrasse, 4002 Basel, Switzerland Inhibition of angiogenesis is an attractive approach to inhibit tumor growth and metastasis. VEGF and its receptors have been identified and validated clinically as important therapeutic targets to inhibit tumor angiogenesis and prolong the survival of cancer patients. Tie2 is another receptor tyrosine kinase which is specifically expressed on endothelial cells and crucial for embryonic vascular development. Inhibition of Tie2 function by the use of soluble Tie2 extracellular domain has been shown to inhibit tumor angiogenesis and growth. Furthermore, kinase activating mutations have been associated with hereditary venous malformation and formation of haemangiomas. We further validated the target by the use of siRNA, demonstrating that down regulation of Tie2 protein inhibits VEGF-driven angiogenesis in vivo. For all these reasons we aimed for the development of specific inhibitors of the Tie2 kinase. We identified bisaryl-ureas as potent inhibitors of the Tie2 kinase in vitro and in cellular assays. After oral application, micromolar concentrations of the compounds are achieved in plasma and tissues and Tie2 phosphorylation was inhibited in lung tissue. Furthermore, representatives of this compound class inhibited VEGF driven angiogenesis in vivo and tumor growth of orthotopically grown B16 melanomas. Thus, we have further validated Tie2 as an attractive target to inhibit tumor angiogenesis and demonstrated it’s drugability. Inhibitors of Tie2 might be also beneficial for other diseases, like retinopathies, psoriasis and inflammatory diseases, where increased angiogenesis plays an important role for the progression of the disease.

109

Poster 46

SIN1 regulation of mammalian TOR complexes Nelyn Soto1, Valeria Facchinetti2, Carolyn Lowry1, Bing Su2, and Estela Jacinto1

1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey; 2Section of Immunology, Yale University School of Medicine, New Haven, Connecticut

The mammalian target of rapamycin (mTOR) forms two distinct protein complexes (TOR Complexes or TORC1 and TORC2) to regulate cell growth in response to nutrients and growth factors/hormones. While mTOR and mLST8 are found in both complexes, raptor is TORC1- and rictor is TORC2-specific. TORC1 is rapamycin-sensitive and regulates protein synthesis. TORC2, on the other hand, is rapamycin-insensitive and controls actin reorganization. TORC2 was recently reported to be the elusive “PDK2” that phosphorylates one of two key residues in Akt/PKB, Ser473 in the hydrophobic motif. We have identified a critical component of TORC2, mammalian SIN1 (Stress activated protein kinase-interacting protein 1). SIN1 is required for TORC2 integrity and function. Using SIN1 knockout cells, we show that SIN1 is a central regulator of TORC2 for Akt Ser473 phosphorylation. In the absence of SIN1, phosphorylation of the TORC1 effector S6K at Thr389 is elevated, indicating that SIN1 may also negatively regulate TORC1. Thus, SIN1 regulates TORC2 directly and may regulate TORC1 indirectly.

110

Poster 47

High-throughput crystallography of protein kinases as a tool for drug discovery Stefan Knapp Structural Genomics Consortium1, Oxford University, UK The Structural Genomics Consortium (SGC) is a charitable organization working to substantially increase the number of protein structures of therapeutic interest available in the public domain. A key goal is to provide 3-D structures of proteins that can then be used as a basis for drug discovery. At the time of writing this abstract, structure determination efforts in the kinase target area in Oxford led to the determination of 14 novel human high resolution protein kinases structures as well as a large number of inhibitor complexes. Many of the novel kinase structures were determined from kinase subfamilies for which no structural information was available previously, including the NEK family member NEK2, MPSK1 (STK16), cdc2 like kinases CLK1 and 3 and the MAPK3 family member ASK1. This diverse set of protein kinases revealed a number of new kinase structural motifs which gave novel insights into kinase function and regulation and which may be explored for the development of novel strategies for the design of specific inhibitors. Screening of the studied kinase targets against an inhibitor library of well known kinase inhibitors currently used for the development of novel drugs as well as novel kinase targeted scaffolds, resulted in the identification of new inhibitors and suggested a number of new applications for current drug candidates. In addition, determination of a large number of kinase co-crystal structures with these inhibitors established an interesting resource for the development of more potent inhibitors.

1 The Structural Genomics Consortium is a registered charity (no. 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, the Swedish Governmental Agency for Innovation Systems, the Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research, and the Karolinska Institute.

111

Poster 48

Endothelin receptor type B inhibits tenascin-C-induced focal adhesion and stress fiber disorganization Katrin Lange, Antje Dittmann, Wentao Huang, Erika Fluri and Gertraud Orend Institute of Biochemistry and Genetics, Center for Biomedicine, Department of Clinical and Biological Sciences, DKBW, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland The extracellular matrix molecule tenascin-C is highly expressed in most solid tumors. It appears to inhibit the tumor proliferation-attenuating effect of fibronectin by blocking the integrin α5β1 coreceptor function of syndecan-4. This causes cell rounding and stimulates tumor cell proliferation (Huang et al., 2001, Cancer Res 61, 8586). We have shown that tropomyosin-1 (TM1), an actin stress fiber stabilizing tumor suppressor-like molecule, is a critical target of tenascin-C and that tenascin-C induces endothelin receptor type A (EDNRA) expression (Ruiz et al., 2004, Cancer Res 64, 7377). Here, we investigated whether EDNR signaling influences tenascin-C-induced cell rounding. We observed that tenascin-C collaborates with EDNRA signaling in blocking TM1 and RhoA expression and FAK activation in a MEK-dependent manner. In contrast to EDNRA, EDNRB signaling blocked the effect of tenascin-C and caused cell spreading in the presence of tenascin-C. EDNRB signaling restored FAK phosphorylation, as well as RhoA and TM1 expression and was dependent on EGFR and PI3K. Our data suggest that tenascin-C action in the tumor microenvironment can be enhanced by EDNRA and blocked by EDNRB signaling. Tenascin-C may enhance migration of cancer cells through activation of EDNRA signaling and subsequent disruption of actin stress fibers. This information might be useful for prediction of tumor malignancy and for cancer therapy.

112

Poster 49

Phosphatidylinositol-3-kinase γ (PI3Kγ) in mast cell adhesion Emilie Clément, Matthew Thomas* and Matthias P. Wymann Institute of Biochemistry and Genetics, Basel, Switzerland * Novartis for Biomedical Research, UK Mast cells (MCs) are primary effector cells in allergy and inflammation. We have recently documented that class IB PI3K (PI3Kγ) plays a major role in MC hyper-activation: antigen/IgE stimulation triggers autocrine/paracrine stimulation of bone marrow-derived MCs (BMMCs) through G protein-coupled receptors (GPCRs), e.g. the adenosine the A3 adenosine receptor (A3AR). Moreover, adhesion of MCs is important for the recruitment of progenitors to tissues, and modulation of differentiation, survival and the activation of mature MCs. Activated BMMC adhere to extracellular matrix proteins like fibronectin (FN) and express various integrins such as α5β1, αVβ3 and αIIbβ3. Challenged subcutaneously with IgE injections, wild type mice increased local numbers of MCs, while PI3Kγ null mice did not respond. Activation of cell surface receptors like c-kit and FcεRI enhance BMMC adhesion. Wild type and PI3Kγ-deficient BMMC adhere to FN upon PMA and SCF stimulation, whereas adenosine and IB-MECA (a specific A3AR agonist) induce adhesion to FN in a PI3Kγ-dependent fashion. Here, adhesion to FN is α5β1 integrin-mediated. Due to these results, we think that PI3Kγ might take an important role in recruitment of BMMC progenitors into tissues. As expression levels of integrins are equal in both wild type and PI3Kγ-deficient cells, we are investigating how PI3Kγ regulates integrins avidity and migration.

113

Poster 50

WD-repeat FYVE protein interacts with the Akt kinase and promotes adipogenesis Thorsten Fritzius and Karin Moelling Institute of Medical Virology, University of Zurich, Switzerland The protein kinase Akt is an important regulator of many processes, including metabolism, proliferation and differentiation. Recently, several studies have suggested a role of Akt in preadipocyte differentiation. We have identified a protein, consisting of seven WD-repeats, presumably forming a β-propeller, and a FYVE domain, ProF, which interacts with the kinases Akt and protein kinase Cζ, PKCζ. The interaction between ProF and the kinases is increased upon hormonal stimulation of the cell [1]. We identified ProF as a positive regulator of preadipocyte differentiation. This is demonstrated by knockdown of ProF using RNA interference, which led to decreased adipogenesis, as shown by reduced lipid accumulation and impaired expression of the late differentiation markers PPARγ and C/EBPα, which indirectly led to decreased uptake of glucose into adipocytes, while overexpression of ProF caused the opposite. We demonstrate ProF as positive regulator of preadipocyte differentiation and anticipate a role of ProF as potential mediator for the phosphorylation of Akt substrates by the kinase. 1. Fritzius, T., Burkard, G., Haas, E., Heinrich, J., Schweneker, M., Bosse,

M., Zimmermann, S., Frey, A.D., Caelers, A., Bachmann, A.S. and Moelling, K. (2006) A WD-FYVE protein binds to the kinases Akt and PKC zeta/lambda. Biochem J, 399, 9-20.

114

Poster 51

Activation of PI3Kγ in mast cells downstream of FcεRI, Ca2+-influx and PKCβ Romy Walser1, Katja Björklöf1, Peter Küenzi1, Muriel Laffargue1, Michael Leitges2, Emilio Hirsch3, Matthias P. Wymann1

1Inst. Biochem. and Genet., Dept. Clin. & Biol. Sciences, University of Basel, Switzerland; 2Max-Planck-Inst. Exp. Endocrinol., Hannover, Germany; 3Dept. Genet., Biol. & Biochem., University of Torino, Italy Allergy and chronic inflammation is driven by mast cell-derived inflammatory mediators. IgE and allergen trigger mast cell activation through the high affinity receptor for IgE (FcεRI), which is coupled to protein tyrosine kinase activation culminating in Ca2+ mobilisation. Full-scale mast cell activation requires an autocrine/paracrine hyperactivation loop involving G protein-coupled receptor (GPCR) signalling and class IB PI3Kγ. Here, we demonstrate that PI3Kγ is also activated by another pathway in mast cells which depends on extracellular Ca2+-influx. Stimulators of Ca2+-mobilisation, such as thapsigargin and calcium-ionophores, cause degranulation in wild type bone marrow-derived mast cells (BMMCs), but fail to do so in PI3Kγ–/– BMMCs. Furthermore, only wild type BMMCs show PKB/Akt phosphorylation in response to Ca2+-influx and phorbol myristate acetate (PMA), while PI3Kγ–/– BMMCs do not. Ca2+-mediated activation of PI3Kγ can be interrupted by pharmacological and genetic targeting of protein kinase Cβ (PKCβ), but not PKCα or PKCγ. Co-immunoprecipitation studies demonstrated, that PI3Kγ constitutively associates with activated, but not with inactive forms of PKCβ. Althogether, our results delineate a Ca2+- and PKCβ-dependent, GPCR and Ras-independent pathway to PI3Kγ, which is relevant to FcεRI action, and thus might provide new ways to manage allergic disease.

115

Poster 52

Rrd2 mediates suppression to topoisomerase I induced DNA damage in yeast M.Carmen Díaz de la Loza and Ralf E. Wellinger Universidad de Sevilla-Cabimer, Avd. Américo Vespucio S/N, 41092 Sevilla, Spain. E-mail: [email protected] DNA topoisomerases are needed to avoid torsional stress mediated by DNA dependent processes such as transcription, replication or recombination. Topoisomerase I creates a transient single-strand break in the DNA backbone, that upon changing the supercoiling of the DNA, is ligated by the same enzyme. Yeast topoisomerase I mutant alleles are known which lead to the formation of a complex consisting of the enzyme covalently attached to the 3´ end of the DNA nick. Interestingly, such mutant alleles are lethal. Most likely, during S-phase, replication forks encountering the DNA nick fall apart leading to the formation of double-strand breaks. If not repaired, these double-strand breaks are mortal. We used a mutant topoisomerase I allele and an overexpression library to screen for supressors in yeast cells. Interestingly, overexpression of the RRD2 gene (the counterpart of the human PTPA gene) conferred resistance to topoisomerase I damage. Rrd2p is part of the Tap42-phosphatase complex that has been shown to interact with the PP2A phosphatase-protein complex. PP2A has recently been shown to be important for checkpoint control after double-strand break formation. The PP2A complex appears to dephosphorylate checkpoint proteins as well as H2AX histones in human cells. We find that RRD2 overexpression did not affect the topoisomerase I mRNA levels. Lethality was restored in a rad52 background indicating that topoisomerase I damage suppression depends on homologous recombination. Overexpression of the RRD2 did not suppress other DNA damages induced by either camptothecine, hydroxyurea, MMS, or UV irradiation. Studies are on the way to investigate by which mechanism RRD2 overexpression confers a selective resistance to topoisomerase I damage.

116

Poster 53

Potential use of pharmacological cyclin-dependent kinase inhibitors as anti-HIV therapeutics Agbottah E, Nekhai S, Kashanchi F. Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine, Washington, DC 20037, USA. Email: [email protected] Cyclin-dependent kinases (cdks) are key regulators of the cell cycle and RNA polymerase II transcription. Several pharmacological cdk inhibitors (PCIs) are currently in clinical trials as potential cancer therapeutics since cdk hyperactivation is detected in the majority of neoplasias. Within the last few years, the anti-viral effects of PCIs have also been observed against various viruses, including human immunodeficiency virus (HIV), herpes simplex virus, and murine leukemia virus. There are currently 80 million individuals in the world infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality. Current HIV-1 inhibitors target the fusion of the virus to the cell and two viral proteins, reverse transcriptase and protease. We have previously published data indicating that other non-viral targets (i.e., non-essential cellular genes) could be utilized to block HIV-1 replication. We specifically asked whether we could target cellular kinases needed for HIV-1 transcription including cyclin E/cdk2, cyclin A/cdk2 and cyclin T/cdk9. Our results indicate that to increase therapeutic index for infected cells, all three kinases could be targeted in either cell lines or primary infected PBMCs. Low concentration of cdk inhibitors are not toxic or static in uninfected control cells. Finally, this microbicide approach against HIV-1 is currently being tested in our mouse model using human stem cells. Therefore, targeting cellular enzymes necessary for HIV-1 transcription, which are not needed for cell survival, is a compelling strategy to inhibit wild type and mutant HIV-1 strains.

117

Poster 54

Structure of the human protein kinase STK16 reveals a novel activation loop architecture Jeyanthy Eswaran1, Antonio Bernad2, Jose M. Ligos2, Barbara Guinea2, Judit É. Debreczeni1, Sirlester A. Parker3, Rafael Najmanovich4, Benjamin E. Turk3 and Stefan Knapp1 1University of Oxford, Structural Genomics Consortium, Botnar Research

Centre, Oxford OX3 7LD, UK 2Departamento de Inmunología y Oncología; Centro Nacional de

Biotecnología, C.S.I.C., Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain

3Yale University School of Medicine, Department of Pharmacology, New Haven, CT 06520, USA

4European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, CB10 1SD, UK

The activation segments of protein kinases comprise conserved sequence and structural elements, including the DFG motif which forms a magnesium binding site, a short β-sheet, the activation loop and the P1-loop. The activation loop, together with the P1 loop, form part of the substrate recognition site and regulate kinase activity. We have identified a new architecture of an activation segment in the human kinase STK16 that consists of an antiparallel β-sheet structure as well as a large α-helical insertion at the C-terminus of the activation segment. Sequence comparisons together with secondary structure predictions suggested that similar activation segments are present in other STK16 family members as well as in members of the MAST kinase family. The sequence specificity of STK16 was determined using peptide library screens, revealing a consensus sequence of P/V/I-φ-H/Y-T*-N/G. Furthermore, we identified the GTPase DRG1 as a STK16 interaction partner and specific substrate. The presented data reveal a novel kinase structural motif and suggest a role of STK16 regulating DRG1, a GTPase involved in regulation of cellular growth and homeostasis.

118

Poster 55

Mechanism of induction of programmed cell death by the β-GBP cytokine in colon cancer cells Dong-yun Shi Pharmaceutical Sciences Research Division, School of Biomedical and Health Sciences, King’s College London Beta-galactoside binding protein (β-GBP) is a negative cell cycle regulator and an antiproliferative cytokine. My studies are aimed at defining the molecular mechanisms whereby β-GBP induces apoptosis in colon cancer cells. I have selected three cell lines: SW480, derived from a primary tumour of the colon, SW620 a metastatic derivative of SW480 and LoVo a very aggressive drug resistant cell line. I have focused on the Ras-ERK pathway and the PI3 kinase/Akt pathway which control cell proliferation and cell survival. My investigations on the effect of β-GBP on cell proliferation and on apoptosis are based on growth curves and on assessment of apoptotic events using TMRE, Annexin, Caspase-3 and TUNEL. They have shown in a similar pattern: early growth arrest followed by apoptosis after 2/3 days. I have examined the differential expression of Ras and ERK activation and of PI3 kinase activity/Akt activation in the above two conditions. For PI3 kinase activity I have set up a new method based on the isolation of the PI3 kinase enzyme by immunoprecipitation of its regulatory subunit (p85) and the assessment of the catalytic activity of the p110 subunit in a kinase assay, followed by quantitation of phosphatidylinositol-(3,4,5)-P3 (PIP3) in an ELISA. My results are not consistent at present with a significant involvement of ERK. By contrast, I have found that PI3K is downregulated by β-GBP in all cases and I am therefore investigating the effect of β-GBP downstream of PI3 kinase, focusing on cyclins, E2F-1 transcription factor and Akt function.

119

Poster 56

The Ret receptor tyrosine kinase mediates anchorage-independent proliferation of breast cancer cells Anne Boulay1, Madlaina Breuleux2, Michael Stumm2, Christine Stephan 2, Cathrin Brisken3, Heidi A. Lane2, Nancy Hynes1.

1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. 2 Novartis Institute for Biomedical Research, Oncology, Basel, Switzerland. 3 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland. Ret - the receptor tyrosine kinase for glial-derived neurotrophic factor (GDNF) family of growth factors - is essential for the development of the sympathetic, parasympathetic and enteric nervous system and the kidney. Ret is a paradigm of a single gene that causes different types of human cancers when targeted by different genetic alterations. Different germ line gain of function point mutations in Ret cause three related dominantly inherited cancer syndromes affecting neuroendocrine tissues: multiple endocrine neoplasia type 2A (MEN2A) and type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Moreover, sporadic or radiation-induced papillary thyroid carcinomas (PTC) are characterized by Ret activation following rearrangements of the tyrosine kinase domain with various partners, resulting in ligand-independent dimerization and constitutive activation of the chimeric proteins. However, there is little evidence as to Ret expression and function in non-neuroendocrine tumor cells. We observed that Ret is expressed in MCF7 and T47D human breast tumor cell lines. Stimulation of breast tumor cells with GDNF (and the recombinant soluble co-receptor GFRα1 for T47D) resulted in increased activation of Erk and JNK pathways. This was prevented by a pharmacologic inhibitor and siRNAs directed against Ret, indicating that Ret mediates GDNF effects in breast cancer cells. Interestingly, GDNF does not modulate proliferation of adherent cells; however, it promotes proliferation in the absence of anchorage to the substratum in a Ret-dependent manner. Our data support therefore, that Ret regulates the invasive phenotype of human breast cancer cells. Importantly, we also observed Ret expression in human breast tumor biopsies, indicating that Ret has potential as a target for therapeutic intervention in human breast cancers.

120

Poster 57

Self-limiting feedback of E2F-1 overexpression in cells resistant to E2F-1 induced apoptosis Vincent Roh, Daniel Candinas, Stephan A. Vorburger Department of Visceral and Transplantation Surgery, Clinical Research, Inselspital Bern, Switzerland E2F-1 is a transcription factor regulating cell proliferation. It is also known to efficiently induce a broad variety of tumor cells to undergo apoptosis. Interestingly, some cell lines, like the breast cancer cells MCF-7, are resistant to E2F-1 induced apoptosis. In this study, we show that E2F-1 expressed from a replication deficient adenovirus under control of the human telomerase reverse transcriptase (hTERT) promoter can reciprocally downregulate the hTERT core promoter that drives its own expression. MCF-7 cells were infected with adenoviral vectors that carried the E2F-1 transgene either under control of hTERT or under control of the unspecific cytomegalovirus (CMV) promoter. Western-blot analysis for transgene expression showed, indeed, an early decline of E2F-1 in the AdhTERT-E2F treated cells compared to AdCMV-E2F treated cells. These findings let us speculate, that in cells that are not sensitive to E2F-1 overexpression, the transgene suppresses its expression in a negative feed-back cycle, whereas in sensible cells, E2F-1 overexpression induced apoptosis before it can reciprocally suppress the hTERT promoter. The concept that transgene expression could be limited to cells that are sensitive to the therapeutic effect of the transgene only is tempting. The difference of the targeted (promoter driven) expression of a therapeutic gene in tumor cells and the limitation of the therapeutic gene expression to tumor cells that are sensitive to its therapeutic effect only lies in the additional reduction of unwanted transgene toxicity. It has the, at least theoretical, advantage that unexpected effects of the gene transduced are avoided. In the case of gene therapy with the transcription factor E2F-1 one major concern is the potential to induce proliferation and tumor growth in those cells that resist the apoptotic effect of E2F-1. These findings suggest that, in that case, the use of a hTERT regulated E2F-1 vector could be helpful to quickly shut down the expression of E2F-1 if the transduced cell does not undergo apoptosis.

121

Poster 58

VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signalling potential through VEGFR-2 Stéphanie Cébe Suarez1, Michel Pieren1, Sandie Arn1, Luca Cariolato1, Ute Hoffmann1, Augustyn Bogucki2, Corinne Manlius3, Jeanette Wood3, and Kurt Ballmer-Hofer1. 1Paul Scherrer Institut, Laboratory of Biomolecular Research, Molecular Cell Biology, Villigen-PSI, Switzerland; 2Friedrich Miescher Institute, Basel, Switzerland, 3Novartis Pharma Ltd, CH-4057 Basel, Switzerland.

The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vascular endothelial growth factors (VEGFs). VEGF family proteins are produced in multiple isoforms with distinct biological properties and bind to three types of VEGF receptors. A VEGF-A splice variant, VEGF-A165b, has recently been isolated from kidney epithelial cells. This variant is identical to VEGF-A165 except for the last six amino acids encoded by an alternative exon. VEGF-A165b and VEGF-A165 bind VEGF receptors 1 and 2 with similar affinity. VEGF-A165b elicits drastically reduced activity in angiogenesis assays and even counteracts signalling by VEGF-A165. VEGF-A165b weakly bound to heparan sulfate and did not interact with neuropilin-1, a coreceptor for VEGF receptor 2. To determine the molecular basis for altered signalling by VEGF-A165b we measured VEGF receptor 2 and ERK kinase activity in endothelial cells in culture. VEGF-A165 induced strong and sustained activation of VEGF receptor 2 and ERK-1 and -2, while activation by VEGF-A165b was only weak and transient. Taken together these data show that VEGF-A165b has attenuated signalling potential through VEGF receptor 2 defining this new member of the VEGF family as a partial receptor agonist.

122

Poster 59

The role of dimerisation in VEGFR-2 activation Debora Dosch1 and Kurt Ballmer-Hofer1

1Molecular Cell Biology, Biomolecular Research at the Paul Scherrer Institut, 5232-Villigen-PSI, Switzerland Vascular endothelial growth factors, VEGFs, are among the most important biomolecules regulating blood and lymphatic vessel homeostasis. They bind to three variants of receptor tyrosine kinases, VEGFR-1, -2, or -3, expressed on the surface of vascular endothelial cells. Ligand binding results in receptor dimerisation followed by transphosphorylation and activation of the kinase domain. Receptor dimerisation is a key event in receptor activation and defects at this level lead to unregulated signalling and disease. Studies performed with related receptors such as the PDGF, Neu or EGF receptors have shown that receptor activation requires specific orientation of the kinase domains relative to each other. Proper orientation is, at least in part, regulated by the transmembrane domain of the receptor. In order to elucidate the role of dimerisation in VEGFR-2 activation, we studied the activity of membrane-bound receptor kinase dimers in vitro and in live cells. We established a screening system with which a series of constructs differing in the rotational angle at which the kinase domains associate with each other could be generated. Our data indicate that only constructs with distinct orientations of the VEGFR-2 dimers show high constitutive tyrosine kinase activity suggesting that dimerisation is necessary, but not sufficient for receptor activation.

123

Poster 60

CGP-582, an imatinib derivative selective for PDGFR and Kit: exploring the molecular reason using the MM-PBSA computational technique A. Bortolato1,2 , C. Gambacorti-Passerini3 and L. Scapozza2

1 Molecular Modeling Section, Department of Pharmaceutical Sciences,

University of Padua, via Marzolo 5, 35131 Padova, Italy 2 Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences,

University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, 1211 Genève, Switzerland

3 Department of Clinical Medicine, S. Gerardo Hospital-University of Milano-Bicocca, Monza, Italy

Protein kinases play a crucial role in the control of cascade signal pathways and their over expression or over activation results in several disease. Specific targeting of these proteins allows a selective and efficient treatment of the pathologies to them correlated, as demonstrate by Imatinib (also known as STI-571). This is a potent inhibitor of c-Abl, Bcr-Abl, c-Kit and PDGFR approved as drug for the treatment of the Chronic Myeloid Leukemia and Gastrointestinal Stromal Tumors. The molecule CGP-582 is an Imatinib analogue with the presence of a nitrogen group in benzene ring near the methyl piperazine. This small modification results in an important activity loss only versus Abl, but not against c-Kit and PDGFR. This modification in the target selectivity, confirmed on a cellular level, suggests new possible promising therapeutic applications. To understand the molecular reasons of the efficiency loss of Abl inhibition, the difference of free energy of binding between STI-571 and CGP-582 was evaluated with success using the molecular mechanics Poisson-Bolzmann surface area (MM-PBSA) computational technique. This method, involving both force field and solvation terms important for the binding, has been employed successfully in several biophysical study to predict free energy changes.

124

Poster 61

Modulation of phosphorylation-dependent protein kinase conformational transitions with low molecular weight compounds Matthias Engel, Valerie Hindie, Laura A. Lopez-Garcia, Adriana Stroba, Francis Schaeffer, Iris Adrian, Jochen Imig, Leila Idrissova, Wolfgang Nastainczyk, Stefan Zeuzem, Pedro M. Alzari, Rolf W. Hartmann, Albrecht Piiper and Ricardo M. Biondi University of Saarland, Innere Med. II, Homburg, Germany Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate PDK1 by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogues, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode-of-action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.

125

Poster 62

X-ray crystallograpfy of ERK1 protein Elad Elkayam, Boaz Shaanan and Yossef Granot. Life Sciences department, Ben Gurion University of the Negev, Beer Sheva, Israel; [email protected] Protein kinases, are enzymes that catalyze the transfer of phosphate group from ATP to OH group of Serine/Threonine/Tyrosine residues of their protein substrates. They play a crucial role in many of these processes ultimately regulate cell cycle progression. One of the most important families of kinases is the Mitogen-activated protein kinase (MAPK) that has a major role in response to extracellular stimuli. The MAPK can be divided into three major families: ERK, p38 and JNK, all consist of proline directed serine/threonine kinases that tightly control proliferation, stress response, differentiation, apoptosis and cell growth. Here, we are suggesting the use of X-ray crystal structure-based design of small molecules that will hopefully inhibit one of the major pathways, the ERK pathway, which transmits signals from growth factors leading to proliferation and differentiation in various cell types. In our work we cloned, purified and crystallized one member of the ERK family – ERK1. We believe that ERK1 crystal structure will be the basis for the design of small molecules inhibitors that will hopefully serve as drug candidates.

126

Poster 63

The potential of novel naphtoquinones in prevention of restenosis Yaron Aviv, Sharon Etzion, Shmuel Bittner, Yossi Granot Department of Life Science, Ben Gurion University of the Negev, Be’er Sheva Israel, [email protected] Restenosis is a direct result of vessel injury, local inflammation, and remodeling following balloon angioplasty and coronary stenting resulting in luminal narrowing. This process involves the release of growth factors and activation of the mitogen activated protein kinases (MAPKs) that stimulate vascular smooth muscle cells (VSMCs) to migrate and proliferate. MAPKs family consists of three major members: Extracelluar signal-regulated kinases (ERKs), which are associated with cell growth and survival, C-Jun N-terminal kinases (JNKs) and p38 that regulate cell death. In this work, we produced and scanned synthetic naphtoquinones in an attempt to reduce VSMCs proliferation and migration without affecting the survival of cardiomyocytes and endothelial cells. Two of more than 200 compounds that were synthesized demonstrated promising differential behaviors among the different cell types. Pharmacological concentrations of the compound TW96 were found to induce VSMCs death probably by increasing ROS level, induction of p38 activity and decrease of mitochondrial potential. Interestingly, this compound had a minor effect on cardiomyocytes survival and on p38 activation. Additionally, TW96 activated the survival pathway of ERK1/2 in cardiomyocytes. TW85 exclusively decreased VSMCs survival, without affecting p38 and ERK1/2 activity or ROS formation. In addition, TW85 exhibited encouraging effect on cardiomyocytes survival possibly by increasing ERK1/2 activation. Remarkably, the survival of the endothelial cells was not affected by TW85 or TW96. Here we reveal prototype naphtoquinones that distinctively decrease VSMCs survival. These observations may lead to the development of potential drugs that will prevent restenosis.

127

mailto:[email protected]

Poster 64

Arginine-vasopressin and JAK-STAT signaling in vascular smooth muscle cells Levy Oren and Granot Yossi Department of Life Sciences, Ben Gurion University of the Negev, Be'er Sheeva, Israel [email protected] JAK (Janus activated kinase)-STAT (signal transducers and activators of transcription) signaling is a major signal transduction pathway in mammalian cells. Different growth factors and cytokines were reported as activators of the JAK-STAT pathway in various cell types. Interestingly, arginine-vasopressin (AVP) was never reported as an inducer of the JAK-STAT pathway. In the present study, we show for the first time that AVP stimulation of vascular smooth muscle cells (VSMCs), which are the cells composing the vascular wall, induces STAT3 tyrosine and serine phosphorylation, followed by nuclear translocation of the phosphorylated STAT3. In addition, we found that AVP induced JAK2 tyrosine phosphorylation. Taken together, these results demonstrate that AVP activates the JAK-STAT pathway in VSMCs. Furthermore, our results indicate that AVP-induced STAT3 tyrosine phosphorylation requires both JAK2 and c-Src tyrosine kinases. The present study also implicates that extracellular signal regulated kinase (ERK1/2),which are serine/threonine kinases, are the mediators of STAT3 serine phosphorylation upon AVP stimulation. We further suggest that AVP-induced STAT3 serine phosphorylation negatively modulates STAT3 tyrosine phosphorylation. Finally, our results implicate a novel role for the JAK-STAT pathway, mediating AVP-induced VSMCs hypertrophy.

128


Poster 65

Glycogen Synthase Kinase-3 as a key component of estrogen receptor alpha signaling pathway Jean Grisouard, Alexander Hermani And Doris Mayer German Cancer Research Centre (DKFZ), Research Group Hormones and Signal Transduction, Heidelberg, Germany Background: GSK3beta is involved in the control of gene expression via the regulation of transcription factors, including estrogen receptor alpha (ERalpha). Recently, we discovered involvement of GSK-3beta in estrogen-independent and estrogen-dependent activation of ERalpha, respectively [1] and [2]. While phosphorylation of ERalpha appears to be crucial for its activation, the impact of GSK-3 on the estrogen-dependent regulation of ERalpha function and activity remains to be clarified. Methods: Phosphorylation of ERalpha by GSK-3beta was analysed by in vitro kinase assays. Thereafter, the effects of GSK-3 inhibitors on ERalpha phosphorylation and activation were analysed in breast cancer cells. Further experiments using siRNA technology in combination with western blot and luciferase reporter assays were performed to investigate the effects of GSK-3 regarding ERalpha signalling pathway. Results: In vitro kinase assays first depicted that GSK-3beta phosphorylated ERalpha at Ser-118. Moreover, the addition of a GSK-3 inhibitor (LiCl) on MELN cells (MCF-7 cells stably transfected with estrogen ERE-luciferase reporter plasmid) in culture stimulated with 17beta-estradiol (E2) led to a decrease in Ser-118 phosphorylation and to an inhibition of ERalpha-controlled luciferase expression. In agreement with the previous observations, silencing of GSK-3 by siRNA transfection resulted in decreases of E2-induced ERalpha phosphorylation at Ser-118 and E2-induced luciferase expression. Our results also suggested that GSK-3/ERalpha complex may play a crucial role in ERalpha protein stabilisation and turnover. Conclusion: We suggest that GSK-3 plays an important role in the estrogen-dependent regulation of ERalpha function and activity. [1] De Servi B., A. Hermani, S. Medunjanin, D. Mayer. (2005) Oncogene, 24: 4946-55. [2] Medunjanin S., A. Hermani, B. De Servi, J. Grisouard, G. Rincke and D. Mayer. (2005) J. Biol. Chem., 280: 33006-14.

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Poster 66

Class I PI3Ks as drug targets in melanoma Romina Marone (1), Sauveur-Michel Maira (2), Anke Klippel (3), Frederic Stauffer (2), Pascal Furet (2), Carlos Garcia-Echeverria (2) and Matthias P. Wymann (1) (1) Institute of Biochemistry and Genetics, DKBW, Centre of Biomedicine, Basel, Switzerland, (2) Novartis Oncology, Basel, Switzerland, (3) Atugen, Berlin, Germany Phosphoinositide 3-kinases (PI3Ks) play an important role in cell growth, survival and cell motility.Here we investigate the importance of the four PI3K class I isoforms in melanoma progression in a collection of melanoma lines and melanocytes covering various disease stages. The catalytic PI3K subunits p110alpha, beta and delta are expressed in melanocytes, but expression of p110delta was lost in some melanoma cell lines. Interestingly, p110gamma is the only class I PI3K isoform not expressed in melanocytes but detectable in around 15% of metastatic cells. Melanoma cells, already at early tumor stages, often show constitutive activation of MAPK and PKB, indicating that these pathways might be key targets in melanoma therapy. Treatment of cells with broadband PI3K inhibitors, such as wortmannin or LY294002, led to little reduction in cell proliferation independent on the aggressiveness of the tumor cell lines. A new generation of potent PI3K inhibitors, NG1 and NG2, on the other hand, induced complete growth arrest which correlates with no thymidin incorporation, reduced levels of cyclin D1 protein and a strong increase in the expression of the cell cycle inhibitor p27KIP1. Targeted elimination of specific PI3K class I isoforms using GeneBloc technology or shRNA expression demonstrated cell line specific dependencies on PI3K isoforms regarding phosphorylation of PKB and cell death. These results provide a first step towards identification of patient-specific requirements concerning PI3K targeting in tumor growth and migration, and might provide a starting point for anti-PI3K therapy with minimal side effects.

130

Poster 67

On the role of Protein Phosphatase 2A in cell migration Veerle Janssens1,2, Karen Zwaenepoel2, Carine Rossé1, Marleen M. Petit3, Jozef Goris2, and Peter J. Parker1

1 Cancer Research UK, London Research Institute, Protein Phosphorylation Laboratory, London, UK 2 Division of Biochemistry, Dept. of Molecular Cell Biology, Faculty of Medicine, KULeuven, Belgium

3 Laboratory of Molecular Oncology, Dept. of Human Genetics, Faculty of Medicine, KULeuven, Belgium

Protein Phosphatase 2A (PP2A) represents a major family of cellular serine/threonine phosphatases that have many important and diverse functions, and display complex regulation (1). PP2A enzymes typically exist as trimers consisting of a catalytic C, a structural A and a regulatory B-type subunit. Regulation can occur through the interaction of the catalytic subunit – via the A subunit – with these regulatory subunits, which can act as targeting and substrate specifying entities. To obtain more insight into the physiological functions of the B”/PR130 subunit we generated a PR130-specific antibody. Direct immunofluorescence indicated that PR130 can be found at various subcellular locations, including the cell protrusions and lamellipodia of migrating cells. RNAi-mediated knock-down of PR130 in HT1080 cells resulted in decreased cell migration in a wound healing assay. Rescue experiments with plasmids encoding RNAi-resistant versions of PR130 (both wild-type and several mutants) revealed the specificity of this effect. To obtain further insight into a possible mechanism, we tried to identify PR130-interacting proteins in a yeast two-hybrid screen with the PR130-specific N-terminus as bait. In parallel the PR130-specific antibodies were used in immunoprecipitations and the co-immunoprecipitating proteins identified via mass spectroscopy. Both approaches led to the identification of candidate cellular PR130-binding partners, possibly involved in cell migration. (1) Janssens, V., Goris, J., Van Hoof, C. (2005) PP2A, the expected tumor suppressor. Curr. Opin. Genet. Dev. 579, 3392-3396

131

Poster 68

On the complex structure of PP2A Zwaenepoel Karen, Longin Sari, Jordens Jan, Goris Jozef, Janssens Veerle Division Biochemistry, Departement Molecular Cell Biology, Faculty of Medicine KULeuven, Belgium Recently, the structures of the dimeric PP2A core (1) as well as of PTPA (2,3) were solved. Since some amino acids could be determined in PTPA that interact with PTPA (3) and since it was found that PTPA can act as a peptidyl-prolyl isomerase and that we were able to indicate Pro 190 in PP2AC as the target for a cis/trans isomerisation reaction (4), we can propose now a plausible structure of the PP2AD-PTPA complex that can cope with these features. This model should be helpful for further dissecting the mechanism(s) involved in the transition from the inactive to the active PP2A conformation. 1. Xing Y., Xu Y., Chen Y., Jeffrey P., Chao Y., Lin Z., Li Z., Strack S., Stock J., Shi Y. (2006). Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins. Cell, 127, 341-353 2. Leulliot N., Vicentini G., Jordens J., Quevillon-Cheruel, Schiltz M., Barford D., Van Tilbeurgh H., Goris J. (2006). Crystal Structure of the PP2A Phosphatase Activator : Implications for its PP2A- Specific PPIase Activity. Mol.Cell, 23, 413-424 3. Chao Y., Xing Y, Chen Y., Xu Y., Lin Z., Li Z., Jeffrey P., Stock J., Shi Y. (2006). Structure and Mechanism of the Phosphotyrosyl Phosphatase Activator. Mol. Cell, 23, 535-546 4. Jordens J., Janssens V., Longin S., Stevens I., Martens E., Bultynck G., Engelborghs Y., Lescrinier E., Waelkens E., Goris J., Van Hoof C. (2006). The Protein Phosphatase 2A Phosphatase Activator is a Novel Peptidyl-Prolyl Cis/trans-isomerase. J. Biol. Chem., 281, 6349-6357

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Poster 69

Phosphoprofile of activated EGF receptor

Isabell Huber1, Judith Koch1, Frank Neumann2, Julia Riedel1, Gabi Schmitt1, Petra Schuessler1, and Heinz Hoschuetzky1

1NanoTools Antikoerpertechnik GmbH & Co KG, Tscheulinstr. 21, D-79331 Teningen, Germany; 2BioAxxess, Upper Pendock, WR13 6JK, U.K. The signaling capacity of activated EGFR/ErbB receptors is not only defined by autophosphorylation of specific tyrosine residues in the intracellular domain. Phosphorylation of additional tyrosine, serine and threonine residues by kinases such as src, MAPK, PKC, PKD or CamKinase II determine signaling strength and duration, attenuation of kinase activity, receptor recycling or degradation of activated EGF/erbB receptors. This phosphoprofile gives important information on the signaling capacity of the activated receptor tyrosine kinases. NanoTools has developed a comprehensive range of phosphorylation site specific monoclonal antibodies that monitor the phosphoprofiles of the EGF/ErbB receptor tyrosine kinases and downstream targets such as MAPK, Akt, scaffold proteins and transcription factors.

133

Poster 70

Validation of phosphoBioMarkerTM specific monoclonal antibodies

Isabell Huber, Petra Schuessler, and Heinz Hoschuetzky NanoTools Antikoerpertechnik GmbH & Co. KG, Tscheulinstr. 21, D-9331 Teningen, Germany Phosphorylation site - specific monoclonal antibodies are important tools for monitoring the activation status of signaling proteins such as kinases or transcription factors. The specificity of the antibodies is validated by immunoblot analysis of whole cell lysates and by peptide competition experiments. There are important quality criteria for phosphoBioMarker - specific monoclonal antibodies: 1. The antibodies must detect the endogenous target protein in complex

protein mixtures such as whole cell lysates; 2. Target protein specificity must be controlled by immunblot analysis of cell

lysates with defined target protein expression patterns (e.g. differentiation between EGF and the highly related erbB receptors);

3. The phosphospecificity of the antibodies must be retained after pervanadate - induced hyperphosphorylation of cellular proteins (high signal to noise ratio);

4. Phosphorylation site specificity must be proven by peptide competition experiments with the corresponding peptides and with phosphopeptides of high sequence homology. These criteria are of central importance if phosphorylation site - specific monoclonal antibodies are used for monitoring the activation status of signaling proteins in liquid phase assays such as ELISA, intracellular staining, or fluorescent bead technologies. In contrast to immunoblot analysis, the experimental readout of liquid phase assays can not be correlated with the Molecular Weight of the target protein. We describe the validation of monoclonal antibodies specific for different phosphorylation sites of the EGF Receptor that do not crossreact with the highly homologous erbB Receptors.

134

Poster 71

EGFR phosphoprofiling with fluorescent microspheres (Luminex)

Isabell Huber, Petra Schuessler, and Heinz Hoschuetzky NanoTools Antikoerpertechnik GmbH & Co. KG, Tscheulinstr. 21, D-9331 Teningen, Germany The phosphoprofile of the activated EGF Receptor gives important information about the signaling capacity. Individual tyrosine phosphorylation sites correspond to activation of MAPK, Akt/PKB, or STAT signaling pathways. Other phosphorylation sites control receptor degradation, attenuation of kinase activity, endocytosis and recycling. NanoTools has developed a comprehensive range of phosphorylation site specific monoclonal antibodies to individual tyrosine, serine and threonine phosphorylation sites of the activated EGF Receptor. These antibodies have been validated for protein and phosphorylation site specificity: the antibodies are EGFR specific and do not crossreact with highly related phosphorylation sites of the homologous erbB Receptors. Thus, these monoclonal antibodies are valuable tools for analysing the phosphorylation status and the signaling capacity of the EGF Receptor in complex protein mixtures such as whole cell lysates. We describe an assay platform that allows EGFR phosphoprofiling with fluorescent microspheres. The data have been validated with immunoblots.

135

Poster 72

Epigallocatechin gallate (EGCG) mimics insulin’s phosphorylation of the transcription factor FOXO1a and elicits cellular responses in the presence and absence of insulin. Siobhan Anton, Laura Melville and Graham Rena Neurosciences Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK. DD1 9SY The green tea flavonoid epigallocatechin gallate (EGCG) is one of several compounds that have been reported to have insulin-like glucose-lowering properties in mammals. EGCG is understood to act at least in part by repression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase but the transcription factors that are targeted by EGCG are unknown. We show here that EGCG induces phosphorylation of insulin-sensitive residues on the transcription factor FOXO1a. Like insulin, EGCG induced FOXO phosphorylation is abolished by the PtdIns 3-kinase inhibitor LY294002 but not by PD98059 (an inhibitor of mitogen-activated protein kinase cascade) or by rapamycin (an inhibitor of signalling to p70 S6 kinase). EGCG differs from insulin and IGF-1 however, in that its induction of FOXO phosphorylation is sensitive to scavengers of reactive oxygen species (ROS). These results indicate that EGCG exerts its insulin mimetic effects at least in part by phosphorylation of the FOXOs through a mechanism that is similar but not identical to insulin and IGF-1 induced FOXO phosphorylation. Our results suggest that agents acting in the manner of EGCG may be useful antidiabetic agents.

136

Poster 73

Anti-apoptotic effect of Src kinase in Xenopus oocyte extracts is mediated by Src SH3 domain Alexander A. Tokmakov1, Mariko Ikeda1, Mikako Shirouzu1, Shigeyuki Yokoyama1,2,3

1Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama, 230-0045 Japan, 2RIKEN Harima Institute at Spring-8, 3University of Tokyo, Japan. E-mail: [email protected] The extracts prepared from eggs of the African clawed frog, Xenopus laevis can recapitulate a range of apoptotic events, including release of cytochrom C, caspase activation, and nuclear fragmentation (Newmeyer et al., 1994). In the present study we investigated the effect of Src kinase and its domains on the apoptosis in the cell-free extracts of Xenopus oocytes and eggs. We found that egg extracts and oocyte extracts had different susceptibilities to undergo apoptosis. The metaphase-arrested egg extracts were resistant to apoptosis and they could not be regulated by Src kinase. On the other hand, the interphase-arrested oocyte extracts were highly apoptotic. They released cytochrom C and displayed elevated levels of caspase activity after several hours-long incubation at room temperature. Caspase activation, but not cytochrom C release, in these extracts could be prevented by over-expression of either constitutively active or kinase negative Src mutants. The specific inhibitor of Src activity, PP2, had no effect on the apoptosis in this system. These data suggest that Src catalytic activity is not involved in eliciting the anti-apoptotic effects of Src kinase in the extracts. Next, we investigated the effects of Src SH2 and SH3 domains on the apoptosis in the extracts. Similar to the full-length Src protein over-expression, the addition of purified recombinant Src SH3 domain to the oocyte extracts inhibited caspase activation without affecting cytochrom C release. On the other hand, Src SH2 domain had no effect on the in vitro apoptosis. Thus, we concluded that the protective effect of Src kinase in the apoptotic Xenopus oocyte extracts is mediated by Src SH3 domain. Earlier, the MAPK pathway active in the metaphase-arrested extracts was demonstrated to protect them from apoptosis (Tashker et al., 2002). Oncoproteins can induce both cell proliferation and apoptosis, depending on cellular context. v-Src oncoprotein has been shown to activate several kinases, which can inhibit apoptosis, including ERK and PI3-K. In the present study we measured the caspase 3/7 activation and cytochrom C release in apoptotic cell-free Xenopus oocyte and egg extracts, overexpressing constitutively active (v-Src) and kinase negative (Src KN) mutants of Src kinase. No or little effect of Src overexpression on cytochrom C release was detected. On the other hand, metaphase arrested egg extracts were resistant to apoptosis and they could not be regulated by Src kinase. Our data suggest that the inhibition of apoptosis in Xenopus oocyte extracts by Src kinase is not mediated by activation of the MAPK pathway.

137


Poster 74

Regulation of the protein kinase MELK, an interactor of NIPP1 Monique Beullens, Sadia Vancauwenbergh, Nick Morrice*, Rita Derua, Hugo Ceulemans, Etienne Waelkens and Mathieu Bollen Afdeling Biochemie, Faculteit Geneeskunde, KULeuven, B-3000 Leuven, Belgium; *MRC Phosphorylation Unit, School of Life Sciences, University of Dundee, DD1 5EH, Scotland, UK NIPP1 is a regulatory subunit of a major nuclear species of protein phosphatase-1 (PP1), but is also implicated in spliceosome assembly and transcriptional repression. Previously it was shown that the phosphothreonine-binding ForkHead-Associated (FHA) domain of NIPP1 interacts with the splicing factors CDC5L and SAP155. The FHA domain also interacts with the C-terminal, TP-rich domain of Ser/Thr kinase MELK (Maternal Embryonic Leucine zipper Kinase) in a phosphorylation-dependent manner (1). MELK belongs to the family of AMP-stimulated protein kinases, but is the only member that is not regulated by LKB1. MELK is poorly characterized and has been implicated in cell cycle control, pre-mRNA splicing and stem-cell renewal. Since MELK expressed in mammalian cells is inactive, we have explored what determines its activity. We found that the C-terminal domain of MELK acted as a non-competitive inhibitor of the kinase activity. MELK is heavily phosphorylated on Ser, Thr as well as Tyr residues and we have mapped 16 autophosphorylation sites. Mutagenesis showed that autophosphorylation of Thr167 and Ser171 in the activation loop is essential for activity. Also, MELK was only active in the presence of reducing agents, suggesting that its activity is blocked by modification of cysteinyl-residues. Finally, we show that MELK binds Ca2+ and that the catalytic activity of MELK is inhibited by physiological Ca2+ concentrations. These data pave the way for a study on the function of MELK and the interaction with NIPP1 (2). 1. Vulsteke, V., Beullens, M., Boudrez, A., Keppens, S., Van Eynde, A., Rider,

M.H., Stalmans, W., and Bollen, M. (2004) J. Biol. Chem. 279, 8642-8647 2. Beullens, M., Vancauwenbergh, S., Morrice, N., Derua, R., Ceulemans, H.,

Waelkens, E., and Bollen, M. (2005) J. Biol. Chem. 280, 40003-40011

138

Poster 75

Activating alleles of JAK3 in acute megakaryoblastic leukemia Denise K. Walters (1,2,9), Thomas Mercher (3,9), Ting-Lei Gu (4,9), Thomas O’Hare (1,2), Jeffrey W. Tyner (2), Marc Loriaux (5), Valerie L. Goss (4), Kimberly A. Lee (4), Christopher A. Eide (2), Matthew J. Wong (2), Eric P. Stoffregen (2), Laura McGreevey (6), Julie Nardone (4), Sandra A. Moore (3), John Crispino (7), Titus J. Boggon (8), Michael C. Heinrich (2,6), Michael W. Deininger (2), Roberto D. Polakiewicz (4), D. Gary Gilliland (3), and Brian J. Druker (1,2); presented by Christopher Bunker (4) 1. Howard Hughes Medical Institute, Portland, Oregon 97239 2. Department of Hematology and Medical Oncology, Oregon Health &

Science University, Portland, Oregon 97239 3. Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard

Medical School, Boston, Massachusetts 02115 4. Cell Signaling Technology Inc., 3 Trask Lane, Danvers, Massachusetts

01923 5. Department of Pathology, Oregon Health & Science University, Portland,

Oregon 97239 6. Portland VA Medical Center, Portland, Oregon 97239 7. Ben May Institute for Cancer Research, University of Chicago, 924 East

57th Street, Chicago, Illinois 60637 8. Department of Pharmacology, Yale University School of Medicine, 333

Cedar Street, SHM B-302, New Haven, Connecticut 06520 9. These authors contributed equally to this work. Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3A572V, JAK3V722I, and JAK3P132T each transform Ba/F3 cells to factor-independent growth, and JAK3A572V confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.

139

Poster 76

A novel role of ERK5 in integrin-mediated breast cancer cell adhesion and motility via FAK signaling – implications in cancer metastasis Rajinder S. Sawhney, Maria Orsino, Majed Aggi, Jennie Hauser, and Michael Brattain Roswell Park Cancer Institute, Department of Pharmacology & Therapeutics, Buffalo, NY, USA Our recent studies examined the role of focal adhesion kinase (FAK) in cell motility and provided evidence that a novel signaling pathway, integrin α2/FAK/ERK1/2/μ-calpain, is important for cell motility in colon cancer cells (Sawhney et al. 2006, J Biol Chem. 281, 8497-8510). We now demonstrate a mechanism by which FAK activates the downstream ERK5 pathway (also known as Big MAPK1/BMK1), regulating cell adhesion and motility. The ERK5/MEK5 cascade has been much less studied than the ERK1/2 pathway. Aberrant regulation of the ERK5 pathway has been implicated in various clinical features of human cancers and heart diseases, thus making this cascade highly attractive for the development of new therapeutic strategies. The ERK5 pathway is known to be regulated by mitogens and cell stress agents. The role of integrin signaling in the activation of the ERK5 pathway in cancer is not understood. Vitronectin (VN) receptors (integrins) play an important role in a number of biological processes like cancer metastasis, angiogenesis, and bone resorption. In metastatic breast cancer patients, higher expression levels of vitronectin receptors are reported. In the current study, we used highly malignant and metastatic breast cancer MDA-MB-231 cells. First we examined the kinetics of ERK5 phosphorylation when MDA-MB-231 cells were plated on VN. We observed enhanced phosphorylation of ERK5 in attached cells as compared to cell suspensions at various time points (up to 2 hours). These results show that ERK5 is activated by integrin (VN receptors) signaling. Addition of function blocking anti-αVβ3, αVβ5, and αVβ6 antibodies to MDA-MB-231 cells significantly inhibited the adhesion of cells to VN; anti- αVβ5 was the most effective (~50% inhibition). Interestingly, we found that kinetics of ERK5 phosphorylation on VN corresponded to kinetics of FAK activation. To determine any relationship between FAK and ERK5 phosphorylation, further experiments showed coimmunoprecipitation of integrins αVβ3 or αVβ5 with ERK5 and FAK. To gain better insight into the role of ERK5, FAK, and VN receptors in cell adhesion and motility, we performed gain- or loss- of function experiments using specific mutants of ERK5. There are no specific small molecule inhibitors of ERK5 known. Ectopic expression of DN ERK5 AEF decreased ERK5 activation, cell adhesion and haptotactic motility on VN. Additionally, DN FAK (Y397F) expression attenuated ERK5 phosphorylation, cell adhesion, and motility. These results show that ERK5 plays a critical role in cell adhesion and motility via FAK signaling. The motility (micromotion) experiments were performed using real time quantitative Electric Cell-substrate Impedance Sensing (ECIS) technique. This study documents the novel finding that in MDA-MB-231 cells ERK5/MEK5 is a target of FAK. Our experiments unveil a novel mechanism by which vitronectin receptors and FAK could promote breast cancer metastasis via ERK5 activation.

140

Poster 77

The Kinase Knowledge Base: computational tools for kinase drug discovery Eric Martin; Sullivan,D; Jansen,H; Lindvall,M; Hendrickson,T; Kondracki,J; Ringgenberg,J; Silveria,S Novartis (NIBR), 4530 Horton St., Emeryville, CA 94608, USA Most inhibitors to all protein kinases bind to the ATP binding region. These binding regions are similar, containing subtle differences in a limited number of generic features: the “hinge”, the “gate keeper”, the hydrophobic “selectivity pocket”, the “DFG motif”, the “catalytic lysine”, the “glycine-rich loop”, etc. This lets us capture our experience from previous kinase programs, and apply it to new kinase problems. Additionally, crystallography has revealed that inhibitors induce large perturbations to these features within the active site of a given kinase. Combining structural data from diverse family members can help us make predictions despite this notorious flexibility. The “Kinase Knowledge Base” is a collection of new computational methods that apply knowledge from previous kinase programs to improve our ability to predict biology and chemistry better than all-purpose modeling tools that treat each new kinase target as an idiosyncratic new protein. This poster describes the methods and predictive power of 4 such tools:

• “Chemo-Genometric” 2-D profile-based activity predictors for virtual screening;

• “AUTOSHIM” target-specific 3-D predictive docking using an X-ray structure;

• “Surrogate Docking” for 3-D predictive docking without an X-ray structure;

• “Chemo-Kinomic” kinase “space” for “drugable” kinase profile design.

141

Poster 78

MEK kinase 1 (MEKK 1) is critical for thymus-dependent (TD) immunity, and demonstrates the signaling specificity of mitogen-activated protein kinase kinase kinase (MAP3Ks) Ewen Gallagher1,2, Thomas Enzler2, Amy Anzelon-Mills3, Dennis Otero3, Tord Labuda2, Min Gao4, and Michael Karin2

1Department of Immunology and Molecular Pathology, Royal Free and University College Medical School, Windeyer Building, 46 Cleveland Street, London, W1T 4JF, UK; 2Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA, 92093-0723; 3Program of Inflammatory Disease Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, USA, 92037; 4Pharmacopeia Drug Discovery, 3000 East Park Blvd, Cranbury, NJ, USA, 08512. MEKK1 is a MAP3K that activates the c-Jun N-terminal kinase (JNK) MAP kinase (MAPK) pathway. To understand its biological role we generated a Mekk1 kinase-deficient (Mekk1ΔKD) mouse model and studied the role of the kinase in the immune system. Following TD immune system challenge we observe reduced germinal center formation in the spleens of the Mekk1ΔKD mice, reduced splenic enlargement, and decreased levels of TD-specific immunoglobulin (Ig). By contrast the immune response following thymus-independent antigen immunizations is normal in Mekk1ΔKD mice. To understand the TD phenotypes present in the Mekk1ΔKD mouse we analyzed its cellular basis and mechanism. Mekk1ΔKD CD4+ or CD8+ T cells hyperproliferate following T cell receptor (TCR) engagement, and Mekk1ΔKD CD4+ effector T cells have enhanced production of T helper (Th) 2 cytokines following TCR priming or Th differentiation assays. Underpinning these phenotypes is the regulation of JunB turnover by the HECT E3 ligase Itch following TCR engagement. Of critical importance to Itch regulation is MEKK1-dependent JNK1 phosphorylation of the Itch proline rich region, leading to conformational changes in the E3 ligase. This novel mechanism defines an alternate mode of JNK signaling to the well-described activation of AP-1-mediated gene transcription by JNK-dependent phosphorylation of c-Jun on residues Ser63 and Ser73. Utilizing the μMT B cell-deficient mouse model in conjunction with bone marrow reconstitution or adoptive transfer of Mekk1ΔKD B cells we identified impaired B lymphocyte function in Mekk1ΔKD mice following KLH immunization. Mekk1ΔKD B cells hypoproliferate in vivo following TD antigen immunizations or in vitro following CD40 receptor engagement. Biochemical analyses reveal that JNK signaling is defective in Mekk1ΔKD B cells following CD40 receptor engagement or stimulation with BAFF. Consequently there is a deficiency in AP-1-dependent gene activation. Notably, the genes: cyclin D2, cxcr4, IL-6 and aid were found to be defectively induced in Mekk1ΔKD B cells following CD40 engagement. MEKK1 is recruited to the CD40 receptor by TRAF2, and undergoes both phosphorylation of its T loop and ubiquitylation as it becomes activated. Recruitment of the kinase to TRAF2 requires the MEKK1 N-terminal RING domain. Our results define the important role for MEKK1 in regulating B and T lymphocytes during TD immune responses, and this illustrates the biological specificity in signaling conferred by MAP3Ks.

142

Poster 79

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3 Stegert MR*, Hergovich A, Tamaskovic R, Bichsel SJ, Hemmings BA Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; *Present address: Genomics Institute of the Novartis Research Foundation, La Jolla, USA NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.

143

Poster 80

Bacterial co-expression of LKB1, MO25α and STRADα results in the formation of the active heterotrimeric complex Dietbert Neumann, Marianne Suter, Roland Tuerk, Uwe Riek, Theo Wallimann ETHZ, Institute of Cell Biology, Schafmattstr. 18, HPM D23, 8093 Zürich, Switzerland The tumor suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 protein is a Ser/Thr protein kinase and is associated with STRAD and MO25 in vivo. Here, we describe an expression and purification system for production of functional LKB1-MO25α-STRADα complex in E. coli. A tricistronic plasmid encoding MO25α STRADα and hexahistidine-tagged LKB1 for transcription of a single tricistronic messenger was constructed from monocistronic vectors using a fully modular cloning approach. After bacterial expression and lysis, LKB1, MO25α and STRADα were highly enriched from the bacterial lysates by sequential IMAC and Heparin chromatography as shown by Western blotting using specific antibodies. In size exclusion chromatography MO25α and STRADα exactly co-elute with LKB1 with an apparent molecular weight of 160 kDa. The specific activity of the size exclusion peak fraction, was 250 U/mg at approximately 25% purity using the synthetic peptide substrate, LKBtide. LKB1 and STRADα both strongly autophosphorylate in vitro, as shown by autoradiography. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the α-subunit at the Thr172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, and (ii) by LKB1-dependent incorporation of radioactive phosphate into the α-subunit of kinase dead AMPK heterotrimer. Sufficient quantities of the functional LKB1 complex should greatly facilitate studies on molecular structure, function and regulation.

144

Poster 81

A single point mutation in the PDZ ligand sequence of c-Src impairs epithelial cell-sheet integrity during wound healing and promotes directional single cell migration Martin Baumgartner, Gerald Radziwill, Andreas Weiss and Karin Moelling Institute of Medical Virology, University of Zürich, Switzerland The c-Src kinase regulates cell behavior in response to adhesion to the substratum, to cell-cell contacts and to growth factors by integrating extracellular cues into intracellular signaling networks. Radziwill and co-workers of the lab have identified the C-terminus of c-Src as ligand for a PDZ protein and demonstrated that it functions as a fine-tuning regulatory switch for c-Src activation trough PDZ domain binding. Invasion assays using c-Src with a point mutation in the PDZ ligand sequence (SrcL/A) indicated the contribution of PDZ domain binding in preventing c-Src from promoting pro-metastatic signaling. Src is well documented to regulate the motile behavior in different cellular systems and is implicated in regulating the metastatic potential of cells in synergy with the focal adhesion kinase FAK. In this study we have investigated the biological significance of the PDZ ligand sequence of c-Src in the regulation of cell migration in the human breast epithelial cell line MCF-10A. We found that SrcL/A but not SrcWT expression in MCF-10A cells impairs EGF-stimulated cell-sheet migration in wounding assays. Cells expressing SrcL/A have impaired cell-cell contacts in cell monolayers and fail to migrate in parallel trajectories during wound healing. Their Golgi apparatus was not correctly oriented towards the wound and the Src substrate FAK was miss-localized during wound healing, indicating that an intact PDZ ligand sequence of c-Src is required for proper polarization of MCF-10A cells towards the wound. In contrast, single cells expressing SrcL/A and migrating on reconstituted MCF-10A extracellular matrix in the absence of growth factors display velocity and directionality comparable to EGF-stimulated control cells. Concomitantly, SrcL/A increases phosphorylation of cortactin and FAK and causes their accumulation at the leading edge. In the absence of EGF stimulation, Src phosphorylated on Tyrosine 416, indicative for Src activation, was markedly increased in membrane and cytoskeleton fractions of MCF-10A cells expressing SrcL/A. Prolonged but low expression levels of SrcL/A prompted MCF-10A cells to undergo morphological transformation, hallmarked by flat morphology and increased activation of c-Jun N-terminal kinase and ERK. We propose that binding of PDZ domains to c-Src in epithelial cells is a novel safeguard mechanism to restrict Src functions, to maintain cell-sheet integrity, to decrease growth factor-independent adhesion turnover and to prevent morphological transformation towards an invasive phenotype.

145

Poster 82

Role of Memo a novel ErbB2 effector protein in tumor cell biology Barbara Haenzi, Régis Masson, Subha Susan Jacob, Nancy E. Hynes Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66 CH-4058 Basel, Switzerland. ErbB2, a member of the ErbB family of receptor tyrosine kinases is overexpressed in 20-30% of breast cancers due to gene amplification. High levels of ErbB2 expression correlate with poor patient prognosis and with tumor aggressiveness. Our group identified a new molecule that interacts with an autophosphorylation site on the cytoplasmic tail of ErbB2 and other receptor tyrosine kinases. The molecule was named Memo, standing for ‘Mediator for ErbB2-driven cell MOtility’ and has been shown to be essential for heregulin induced tumor cell migration (R. Marone et al. 2004). One goal is to study the role of Memo in metastasis, a process that requires migration. For these studies we used the highly metastatic 4T1 mouse mammary carcinoma cells, in which Memo expression was stably knocked down (KD) via short hairpin RNA. Clones showing moderate to strong decrease in MEMO level were selected. Recent results show that tumors generated by in vivo injection of memo KD cancer cells grow more slowly than tumors arising from control cells. Preliminary results support that this could be due to a delay in angiogenesis. Furthermore the tumors from Memo KD cells show a lower rate of lung metastasis in comparison to control tumors. This suggests that Memo plays a role in angiogenesis and in the metastatic process. To further address the role of Memo in cancer cells we will investigate pathways affected upon Memo KD in vitro.

146

Poster 83

TRAF4 is a signalling adaptor overexpressed in human cancers Sophie Camilleri-Broët, Catherine Sautès-Fridman and Catherine H. Régnier INSERM Unité 255, Centre de Recherches Biomédicales des Cordeliers, Université Paris VI, Université Paris-Descartes Faculté de Médecine, AP-HP Hôtel-Dieu, Paris, France Tumor necrosis factor receptor (TNFR) associated factors (TRAFs) belong to a family of closely related adaptor proteins involved in the intracellular signalling cascade activated by TNFR and Toll-like receptor (TLR) families. TRAF4 has been reported to mediate signalling by different TNFRs and activate mitogen-activated protein kinase (MAPK) pathways but precise in vivo evidence of such a role is still missing. TRAF4 was initially identified as a gene amplified and overexpressed in breast carcinomas. To evaluate TRAF4 overexpression in other cancer types, immunohistochemistry (IHC) analysis was performed on tissue micro-arrays (TMA ; 20 different tumor types). This analysis has shown TRAF4 protein overexpression in an average of 43% of all tumors tested (n=623), including 60% of lung adenocarcinomas (n=137). Paired primary tumors and lymph node metastases from different origins displayed mostly the same pattern of TRAF4 expression which suggests that TRAF4 up-regulation is more likely gained at an early stage of tumorigenesis rather than at later stages during cancer progression. To investigate changes in TRAF4 gene copy number, multi-tumor TMA (n=125) were also analysed by fluorescence in situ hybridization (FISH). Our investigation by IHC and FISH on the same tumor samples clearly showed that TRAF4 gene amplification is correlated with increased TRAF4 translation. Co-amplification analysis of the TRAF4 and ERBB2 genes in primary lung adenocarcinomas (n=31) showed that TRAF4 gene amplification can occur independently of ERBB2 gain in this disease. Aside from well-known oncogenes such as MYC, RAS or ERBB2, very few genes have been found amplified and overexpressed in cancers from different origins. TRAF4 is located at 17q11.2 in a region of amplification devoid of known oncogenes and represents a novel marker of human carcinomas with the signature of a candidate oncogene. The consequences of TRAF4 overexpression in cancer cells and more particularly its role in the MAPK signalling pathway is currently under investigation.

147

Poster 84

Roles of δEF1 family, SIP1 and ZEB1, in the Regulation of TGF-β-induced Epithelial-Mesenchymal Transdifferentiation Masao Saitoh, Takuya Shirakihara, and Kohei Miyazono Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Epithelial-mesenchymal transdifferentiation (EMT) is a critical morphogenic event which occurs during embryonic development and during the progression of various epithelial tumors. EMT can be induced by transforming growth factor (TGF)-β in mouse NMuMG mammary epithelial cells. Here, we demonstrate central roles of transcription factors including E47, Snail, SIP1 and ZEB1 in TGF-β-induced EMT. Epithelial cells ectopically expressing SIP1 or ZEB1 suppressed E-cadherin expression but not affected several mesenchymal markers such as fibronectin and N-cadherin, whereas Snail did not appear to enable the cells to induce EMT. In addition, overexpression of E47 also suppressed E-cadherin expression through induction of SIP1 and ZEB1 genes. In contrast, cells transfected with siRNAs against both SIP1 and ZEB1 inhibited TGF-β- and E47-induced E-cadherin suppression without any effects on mesencymal markers, and partially inhibited motility induced by TGF-β. Taken together these results suggest that δEF1 family transcription factors, SIP1 and ZEB1, are essential for TGF-β-induced EMT, especially regulation of E-cadherin.

148

Poster 85

JNK2 is required for Ras transformation independently of AP-1 Christina Nielsen, Jacob Thastrup, Trine Bøttzauw, Marja Jäättelä and Tuula Kallunki Apoptosis Department and Centre for Genotoxic Stress, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark Active Ras oncogene is expressed in approximately 30% of human cancers. Yet, very little is known about the molecular mechanisms responsible for its transforming potential. Here, we demonstrate that H-Ras-mediated transformation requires the isoform 2 of the c-Jun-N-terminal kinase (JNK). H-Ras-transduced JNK2 deficient (JNK2-/-) murine embryonic fibroblasts (MEFs) were severely inhibited in colony formation and growth in soft agar in vitro as well as in tumor formation in immunodeficient mice as compared to corresponding JNK1-/- and wild type (wt) MEFs. Accordingly, also the RNA interference-based depletion of JNK2 form wt MEFs resulted in defective Ras transformation. The extra barrier against H-Ras transformation in JNK2-/- MEFs was not due to their inability to inactivate p53 signaling, since all JNK2 deficient MEF lines had lost p19Arf. Furthermore, expression of the the E6 protein of the human papilloma virus failed to overcome the transformation defect. It could, however, be overcome by co-expression of H-Ras with the Simian virus 40 large T-antigen (SV40-LT) or c-Myc. Surprisingly, the H-Ras-transduced JNK2 deficient MEFs exhibited higher activity of activator protein-1 (AP-1) and higher levels of c-Jun expression than H-Ras-transduced JNK1 deficient or wild type cells indicating that the key target of JNK2 during the Ras transformation is divergent from AP-1. These results clearly demonstrate that a single kinase, JNK2, can control Ras transformation and thus point out a vulnerable control point that may prove important for the tumor development in general.

149

Poster 86

PMA-induced PKC translocation is inhibited by new PKC C1 domain ligands Talman Virpi1; Aitio Olli2; Ekokoski Elina1; Boije af Gennäs Gustav2; Finel Moshe3; Yli-Kauhaluoma Jari2; Tuominen Raimo K1

1 Division of Pharmacology and Toxicology, 2 Division of Pharmaceutical Chemistry, 3 Viikki Drug Discovery and Development Technology Center, Faculty of Pharmacy, PO Box 56, 00014 University of Helsinki, Finland Protein kinase C (PKC) isoenzymes are important regulators of cell proliferation and malignant transformation. In this study we investigated the effects of a series of synthesized small-molecule PKC ligands on phorbol ester binding activity and translocation of PKC. We also tested the cytotoxicity of the compounds in HeLa (human cervical cancer) cells and SMC (rabbit aorta smooth muscle) cells using MTT and LDH assays. We used the X-ray structure of PKC delta C1b domain with bound phorbol ester as a template for molecular modelling to design hydrophobic ligands that compete with phorbol esters for binding and thus modulate PKC activity. The best ligands competed with phorbol ester binding to PKC with IC50 values less than 20 µM. Translocation studies were done in living HeLa cells transfected with PKC - green fluorescent protein (GFP) constructs. The cells were pretreated with the ligands and stimulated with phorbol 12-myristate 13-acetate (PMA). The translocation of PKC-GFP was visualized with confocal microscopy and quantified from confocal microscopic images captured during the experiments. Five out of ten compounds tested inhibited PMA-induced translocation of both PKCα and PKCδ in micromolar concentrations. In cytotoxicity tests some of the active ligands caused cell growth arrest and non-necrotic cell death in HeLa but not in SMC cells. This work was supported by EU (Pro-Kinase Research project no. 503467).

150

Poster 87

Integrin linked kinase is a molecular adaptor for T-cadherin-induced Akt/GSK3β-axis signaling in endothelial cells Manjunath B Joshi, Danila Ivanov, Maria Phillipova, Paul Erne*, Therese J Resink Cardiovascular Laboratories, Department of Research, Basel University Hospital, Basel, Switzerland. * Division of Cardiology, Luzern Kantonspital, Luzern, Switzerland T-cadherin, an atypical glycosylphosphatidylinositol (GPI) anchored member of cadherin superfamily, is upregulated during pathological conditions of atherosclerosis, restenosis and tumor angiogenesis. In vitro T-cad induces endothelial cell (EC) survival, proliferation, migration and angiogenesis. We have shown that adenoviral mediated T-cad overexpression in EC (T-cad+-EC) protects against oxidative stress induced apoptosis via PI3K/Akt-axis signaling. In the present study we attempt to delineate signaling pathways involved in T-cad induced proliferation. Compared with empty vector infected EC (E-EC), and under normal culture conditions, T-cad+-EC exhibited a hyperphosphorylation of pser473-Akt and pser9-GSK3β� this effect was PI3K-dependent. Immunoblotting and immunocytochemistry demonstrated increased levels and concomitant nuclear accumulation of active β-catenin (ABC) in T-cad+-EC. Silencing of T-cad in EC using RNA interference reduced levels of pser473-Akt, pser9-GSK3β and ABC confirming specificity of T-cad effects. The functionality of ABC in T-cad+-EC was demonstrated by increased (vs. E-EC) β-catenin driven Tcf/Lef transcription factor reporter (BAT-Lux) activity and constitutive induction of cyclin D1. To determine the role of GSK3β in T-cad-induced elevation of ABC, T-cad+-EC and E-EC were cotransfected with GSK3β Kinase Mutant (KM), non-phosphorylatable GSK3β Constitutively Active (S9A) and GSK3β Wild Type (WT). Contransfection with S9A abrogated T-cad effects on ABC accumulation. Additionally, cotransfection with S9A also abrogated stimulatory effects of T-cad on EC survival. Thus phosphorylation/inhibition of GSK3β is a pivotal downstream signal response elicited by T-cad. We considered Integrin Linked Kinase (ILK) as a potential molecular adaptor for GPI-anchored T-cad and upstream regulator of T-cad-induced Akt/GSK� axis signaling in EC. ILK pull-down and in vitro kinase assay revealed increased ILK activity in T-cad+-EC. siRNA-mediated silencing of ILK abrogated stimulatory effects of T-cad on pser473-Akt, pser9-GSK3β and ABC. Western analysis of anti-ILK immuno-complex showed the presence of T-cad and integrin β3, a known binding partner of ILK. Confocal microscopy proved the colocalization of ILK and T-cad in EC; this was most prominent at the leading edge of migrating cells. We conclude that in EC T-cad transmits its outside-in signals via ILK.

151

Poster 88

Functional and structural studies of the extracellular ligand binding domain of VEGFR-2 C. Ruch, A. Prota, M. Steinmetz, G. Skiniotis1, T. Walz1 and K. Ballmer-Hofer Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland and 1Harvard Medical School, Dept. of Cell Biology, Boston MA, USA Angiogenesis, the process through which new blood vessels are formed from preexisting vasculature, plays a pivotal role in normal development and in a variety of diseases such as cancer, retinopathies, atherosclerosis and preeclampsia. Vascular endothelial growth factor receptors are key regulators of angiogenesis and are activated by ligands of the VEGF family. Three VEGF-receptors have been described VEGFR-1, -2, and -3 and additional receptors whose role in angiogenesis is not yet well defined at the molecular level, neuroplin-1 and -2. VEGFRs are receptor tyrosine kinases consisting of 7 extracellular Ig-like domains, a transmembrane domain and a cytoplasmic split tyrosine kinase domain followed by a domain carrying tyrosine residues involved in receptor signalling. These receptors are activated upon binding of VEGF family growth factors, VEGF-A, -B, -C, -D, -E, -F, and PlGF (placenta growth factor). Each VEGF variant interacts with a specific set of VEGFRs and induces or stabilises receptor dimerisation. Subsequent activation of the intracellular split kinase domain initiates signalling pathways that regulate cell proliferation, differentiation, migration and vessel permeability. Understanding the mechanism of receptor activation in the context of its 3-dimensional structure will open new avenues for the design of drugs interfering with angiogenic signalling. We combine functional biochemical studies with structural information on VEGF receptors. The extracellular domain of VEGFR-2 was produced in mammalian cells, purified to homogeneity and submitted to binding studies with VEGF ligands. Electron microscopy was used to obtain low resolution structural information on receptor/ligand complexes. Our data show that the extracellular domain of VEGFR-2 is highly flexible in the absence of bound ligand while ligand-bound receptor assumes a tight and apparently stable conformation. X ray crystallography and small angle scattering are currently used to obtain detailed structural information of ligand/receptor complexes.

152

Poster 89

The human kinetochore protein hsMcm21R is regulated in a cell cycle dependent manner Mukta Belwal, Sarah McClelland, Andrew McAinsh and Patrick Meraldi ETHZ, Institute for Biochemistry, HPM E 14, Schafmattstrasse 18, 8093 Zürich, Switzerland. Chromosome-microtubule attachment is mediated by kinetochores, multiprotein complexes assembled on centromeric DNA. Kinetochores act as platform for MT attachment and regulate the forces that power chromosome movement during mitosis. Kinetochores also monitor the state of microtubule-chromosome attachment and activate the spindle checkpoint in the presence of incorrectly attached chromosomes. Previous studies from our group led to the identification of ten novel human kinetochore proteins. We are currently investigating the functions of these proteins. My project aims at understanding the regulation of these proteins. We found that one of these, hsMcm21R, is regulated in a cell cycle dependent manner. SiRNA-mediated depletion and live-cell imaging demonstrated the role of hsMcm21R in mediating correct microtubule-kinetochore attachments. Defective attachments in hsMcm21R depleted cells interfered with spindle assembly and resulted in the formation of monopolar spindles in HeLa cells. Cell cycle synchronization followed by western blot analysis for hsMcm21R showed differential phosphorylation as cells went through the cell cycle. While the protein was unphosphorylated during interphase, most of it was phosphorylated during mitosis. The protein also appeared to get degraded as cells exited mitosis and reaccumulate as they prepared to enter the next round of mitosis. Sequence analysis of hsMcm21R showed presence of putative phosphorylation sites strongly conserved from yeast to mammals and a putative Destruction-Box motif conserved in all metazoans. By mass spec analysis of hsMcm21R isolated from HeLa cells, we will confirm if the endogenous protein is phosphorylated on these residues. We also aim to investigate the function of these sites both in vivo by RNAi complementation and in vitro by degradation and kinase assays. It would be particularly interesting to see how these regulatory events affect the function of hsMcm21R. Preliminary results from degradation assays using Xenopus laevis extracts to determine if hsMcm21R could be a substrate of APC/C suggested that it might not be a substrate. To verify the result, we plan to do the assay with endogenous hsMcm21R isolated from Hela cells together with the proteins it interacts with and also with X. laevis homolog of hsMcm21R. Another possibility we consider is it being a substrate for cullins. Many kinetochore proteins are recruited to or displaced from kinetochores just at the mitotic onset, suggesting that a functional complex is assembled only as the cell enters mitosis. The distinctive temporal pattern of kinetochore localization by constituent proteins suggests that kinetochore assembly follows a rigorously ordered pathway. In addition, the sequential appearance of certain kinases on kinetochores may also contribute to temporally regulated kinetochore assembly. Thus, biochemical characterization of candidate proteins and their post-translational modification should reveal the molecular assembly pathways mechanisms that temporally regulate their binding to the nascent kinetochore.

153

Poster 90

Axl as a target for therapeutics against invasive breast cancer: from target validation to experimental therapy Yixiang Zhang1, Yuri V. Cheburkin1, Yuri P. Knyazev1, Wolfgang E. F. Klinkert2, György Kéri3, Axel Ullrich1, Peter G. Knyazev1

1Max Planck Institute of Biochemistry, Department of Molecular Biology, Martinsried, Germany; 2Max Planck Institute of Neurobiology, Department of Neuroimmunology, Martinsried, Germany; 3Vichem Chemie Research Ltd., Budapest, Hungary Several protein kinases have been found to be involved in tumorigenesis and progression of breast cancer, and are being actively pursued as targets for therapeutic intervention. In this study, we have identified receptor tyrosine kinase Axl as a critical mediator of motility and invasivity of breast cancer. Axl was expressed in highly invasive breast cancer cells, but weakly or not expressed in breast cancer cells of low invasivity. Ectopic overexpression of Axl was sufficient to confer highly invasive behaviour on weakly invasive breast cancer cells. Experimental inhibition of Axl signalling by a dominant-negative Axl mutant, an antibody against the extra-cellular domain of Axl or shRNA knockdown of Axl inhibited motility and invasivity of highly invasive breast cancer cells. Moreover, we identified 3-quinolinecarbonitrile compounds that displayed potent inhibitory activity against Axl, and showed strong interference with motility and invasivity of highly invasive breast cancer cells. Our findings, from validating the role of receptor tyrosine kinase Axl in motility and invasivity of breast cancer cells, to experimental anti-Axl small molecule inhibitors, implicate Axl as a promising target for the therapy of invasive breast cancer.

154

Poster 91

Characterization of human Rio2 function in late steps of 40S subunit biogenesis Ivo Zemp and Ulrike Kutay Institute of Biochemistry, ETH Zurich, Switzerland The multi-step assembly process of eukaryotic ribosomes not only involves rRNAs and ribosomal proteins, but also more than 150 additional proteins. These trans-acting factors have predicted activities such as RNA helicases, RNA processing enzymes, GTPases and kinases, but mostly their functions are poorly characterized. A trans-acting protein found in pre-40S particles in yeast cells is the serine/threonine kinase Rio2p. Recombinant Rio2p shows in vitro kinase activity, and Rio2p mutants have defects in late steps of 40S subunit biogenesis (Geerlings et al, J Biol Chem (2003) 278:22537; Schafer et al, EMBO J (2003) 22:1370; Vanrobays et al, Mol Cell Biol (2003) 23:2083). Recently, the human homolog of Rio2, hRio2, was implicated in cytoplasmic processing of 18S-E pre-rRNA to the mature 18S rRNA (Rouquette et al, EMBO J (2005) 24:2862). We have cloned hRio2 from HeLa cell cDNA. hRio2 is a cytoplasmic protein at steady-state as shown by the localization of an EGFP fusion protein and immuno-detection of endogenous hRio2 in HeLa cells. Treatment of the cells with the CRM1-inhibitor leptomycin B rapidly shifts hRio2 localization to the nucleus, suggesting that hRio2 shuttles between the nucleus and the cytoplasm. Furthermore, purified hRio2 binds directly to hCRM1 in the presence of RanGTP in vitro, indicating a possible role for hRio2 as an adaptor protein for nuclear export of 40S ribosomal subunits. In sedimentation assays, hRio2 binds to purified 40S but not 60S subunits, and hRio2 co-sediments with 40S subunits on sucrose gradients. Like its yeast homolog, hRio2 undergoes autophosphorylation and is able to phosphorylate heterologous substrates in an in vitro kinase assay. We have isolated hRio2-containing ribosomal particles from HeLa cell extract by immuno-precipitation. We will present the results of the analysis of their protein content as well as of our attempt to identify hRio2 substrates within these complexes.

155

Poster 92

Realizing the promise of chemical genetics: from substrate identification to selective inhibition of a single essential protein kinase in human cells Stephane Larochelle1, Karl A. Merrick1,2, Marie-Emilie Terret1, Lara Wohlbold1, Nora M. Barboza1, Chao Zhang3, Kevan M. Shokat3, Prasad V. Jallepalli1 and Robert P. Fisher1 1Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. 2Cornell University Graduate School of Medical Sciences. 3Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA. Because of their rapid mode of action, small molecules can provide a high degree of temporal control over protein function. Therefore, the ability to target a specific protein in vivo with an inhibitory small molecule remains a major goal of chemical biology. Mutations of a conserved residue within the ATP binding pocket of protein kinases allows binding of bulky ATP analogs that have low affinity for the wild-type enzymes. The Cdk7/cyclin H/Mat1 complex is part of the core machineries of both cell-cycle and transcriptional control, serving as the CDK-activating kinase (CAK) and as the kinase component of the Pol II general transcription factor (TF) IIH. The mutation, in human Cdk7, of Phe91 to Gly renders the kinase analog-selective — able to use bulky ATP analog substrates — and sensitive — susceptible to inhibition by non-hydrolyzable analogs (AS). We have identified eight of ~12-15 protein substrates in complex cellular extracts by phosphorylation with recombinant Cdk7as and radiolabeled N6-(benzyl)-ATP. The mutant enzyme was inhibited by the non-hydrolyzable analog 1-napthylmethyl-PP1 (1-NMPP1) with an IC50 ~17 nM, whereas the wild-type kinase was unaffected by 1-NMPP1 concentrations as high as 1.5 µM. The apparent Km

ATP of Cdk7as is ~550 µM — ~sixfold higher than that of wild-type Cdk7, but below the likely intracellular ATP concentration — and the mutation did not affect enzyme turnover, suggesting that the AS enzyme would retain function in vivo. Taking advantage of powerful gene targeting technologies, based on rAAV vectors, we have engineered a human colon carcinoma cell line (HCT116) in which the wild type Cdk7 gene is replaced with the mutant version (Cdk7as). The homozygous Cdk7 as/as cells were sensitive to killing by 1-NMPP1 with an IC50 ~100 nM. In contrast, wild-type HCT116 cells were resistant to 10 µM 1-NMPP1. From this we conclude that the catalytic activity of Cdk7 is required for viability, and can be shut off selectively in vivo by a bulky ATP analog in the Cdk7 as/as cell line.

In theory, an AS version of any protein kinase can be engineered and expressed from its endogenous promoter by rAAV-directed gene replacement. We combined these strategies to investigate Cdk7, an attractive anti-cancer drug target because of its dual functions in controlling gene expression and cell proliferation. Selective inhibition of Cdk7 in G1 cells prevents activation of Cdk2 and delays S phase. Cdk7 inhibition during G2 blocks Cdk1 activation and mitosis. In vivo, and in whole cell extracts from mutant cells, inhibition of Cdk7 prevents Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation are mutually dependent. Therefore, by a novel combination of chemical genetics and homologous gene replacement in somatic cells, we have defined the biochemical functions of a mammalian cell-cycle regulator at two execution points. Upon prolonged exposure, asynchronous Cdk7 as/as cells were sensitive to killing by lower doses that did not cause obvious cell cycle arrests, possibly due to derangement of gene expression. The chemical-genetic approach we have taken ensures that the cytotoxicity is Cdk7-specific, i.e. not due to off-target effects of the analog, and should therefore allow us to evaluate CAK/TFIIH as a possible therapeutic target in a human cancer cell line. The cdk7as phenotype, moreover, could provide metrics — signatures of cell-cycle and transcriptional disruption —by which to evaluate potentially selective inhibitors of wild-type Cdk7 isolated through screens of chemical libraries.

156

Poster 93

The scaffold protein CNK1 in Raf-dependent signaling Rafael D. Fritz1, Algirdas Ziogas1,2, Karin Moelling1 and Gerald Radziwill1

1Institute of Medical Virology, University of Zurich, 8006 Zurich, Switzerland 2present adress: Department of Pharmacology, University of Bern, Switzerland The multidomain protein CNK1 (connector enhancer of kinase suppressor of Ras 1) interacts with kinases involved in different cellular processes such as proliferation, differentiation and apoptosis. We identified CNK1 as a scaffold protein in the Raf/MEK/ERK pathway. According to its scaffold function CNK1 mediates Src-dependent Raf-1 activation. This function occurred in a dose-dependent manner typical for scaffold proteins. Downregulation of endogenous CNK1 by short hairpin RNA interfered with Src-dependent activation of ERK. Thus, CNK1 is a scaffold coupling Src- and Raf-dependent signaling pathways (Ziogas et al. J Biol Chem. 280, 24205-24211, 2005). In addition, we identified CNK1 as a binding partner of the angiotensin II type 2 (AT2) receptor. The AT2 receptor is a seven transmembrane protein that plays a dual role by stimulating or inhibiting the Raf/MEK/ERK pathway depending on the cellular context. On the one hand the AT2 receptor promotes differentiation of neuronal cells by activation of the Rap1/B-Raf/MEK/ERK pathway. On the other hand the AT2 receptor represses growth factor-induced MAPK cascade activation via several protein phosphatases. We found two domains of CNK1, the SAM and the CRIC domain, to interact with the AT2 receptor. In an overexpression system we could show that this interaction is specific for the ubiquitously expressed CNK1 but not for the neuronal isoform CNK2. We characterized this interaction by analysing mutant CNK1 proteins and found mutants with increased or decreased binding affinity towards the AT2 receptor depending on the introduced mutation. The biological significance of the interaction between CNK1 and the AT2 receptor was further supported by coprecipitation of both proteins from murine heart extracts. We suggest that CNK1 may play a role in the AT2 receptor-mediated signaling pathways, either in the MAPK cascade activating branch or in the repressive branch via protein phosphatase activation.

157

Poster 94

Sequential tyrosine-phosphorylation of LAT controls life or death of developing T lymphocytes Zara Hovhannisyan, Leah Alabanza, Dominique Roubaty1, Ed Palmer1 and Guy Werlen Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, 08854, NJ, USA. 1Department of Research, University Hospital Basel. Switzerland; E-mail: [email protected] In T cell development, positive selection allows thymocytes expressing appropriate self-tolerant T cell antigen receptors (TCR) to survive and differentiate, while to avoid autoimmunity, negative selection removes overtly self-reactive thymocytes from the repertoire. Using a transgenic mouse model expressing a unique TCR, we have demonstrated that the activation kinetics of the MAPkinase, ERK decides over life and death of developing T lymphocytes. Positive selection ligands induce a slow and sustained ERK activation, while negative selection ligands trigger a fast, but transient activation of ERK. Both ligands activate the JNK and p38 MAPKs with similar kinetics (Werlen, G., et al., 2000, Nature 406; 422-426). We have furthered these studies and found that the distinct ERK kinetics result from the formation of different TCR-signalosomes, in which the transmembrane scaffold protein, LAT plays a crucial role. Negative selection ligands induce a fast but transient Tyrosine-phosphorylation of LAT, while differentiation ligands trigger a delayed and weak phosphorylation of that scaffold protein. We have generated antisera specifically recognizing individually each of the 9 phosphorylated Tyrosine residues of LAT and analyzed their phosphorylation kinetics upon TCR engagement. We found that positive and negative selection ligands induce distinct sequential phosphorylation of LAT and consequently control the kinetics of ERK activation by modulating the recruitment of specific signaling components to LAT and the TCR-signalosomes. Both ligands induce the recruitment of the phospholipase, PLCγ, which impinges on the ERK pathway via the Ras activator, RasGrp. Additionally, negative selection ligands trigger a fast and transient recruitment of the adaptor Grb2 and the Ras activator, SOS, resulting in a faster activation of ERK as opposed to stimulation with ligands inducing positive selection only. Interestingly, the tyrosine kinase Zap-70 exclusively phosphorylates LAT in response to differentiation ligands, while Zap-70 and an additional tyrosine kinase phosphorylate LAT in response to negative selection ligands. Our results demonstrate that LAT is a molecular clock, which by recruiting distinct signaling components to its sequentially phosphorylated Tyrosine-residues controls life or death of developing T lymphocytes.

158

Poster 95

Regulation of TGF-β1-induced apoptosis and epithelial-to-mesenchymal transition by protein kinase A and STAT3 Yanan Yang, Xinchao Pan, Weiwei Lei, Jun Wang, Jian Shi, Fuqiang Li, and Jianguo Song Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 320 Yue-Yang Road, Shanghai 200031, China Apoptosis and epithelial-to-mesenchymal transdifferentiation or transition (EMT) are crucial for normal development and body homeostasis. The alterations of these events are closely related to some pathological processes, such as tumor formation and metastasis, fibrotic diseases of liver and kidney, and abnormal development of embryos. The mechanism that underlies the simultaneously occurring apoptosis and EMT induced by transforming growth factor-β (TGF-β) has not been well studied. In this report, we investigated the potential mechanism that underlies TGF-β1-induced apoptosis and EMT. TGF-β1-induced apoptosis and EMT were associated with the activation of protein kinase A (PKA) and signal transducer and activator of transcription-3 (STAT3). Inhibition of PKA by specific PKA inhibitor H89 or by PKA inhibitor peptide (PKI) blocked STAT3 activation and suppressed TGF-β1-induced apoptosis and EMT. Furthermore, overexpression of a phosphorylation-deficient form of STAT3, but not wild-type STAT3, produced an inhibitory effect on TGF-β1-induced apoptosis and EMT. The results indicate that PKA is an upstream regulator for TGF-β1-induced STAT3 activation and plays an important role in TGF-β1-mediated apoptosis and EMT. These studies provided a new insight into the signaling mechanism underlying the apoptosis and EMT, which could be of importance in understanding some related physiological and pathological processes.

159

Poster 96

Sutent/sunitinib malate - towards a response prediction signature Michaela Bairlein1, Jesper V. Olsen2, György Keri3, Matthias Mann2, Henrik Daub1 and Axel Ullrich1

1Department of Molecular Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany; 2 Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Martinsried, Germany; 3 Vichem Chemie Ltd., Budapest, Hungary SUTENT (Sunitinib Maleate) is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities known to inhibit receptor tyrosine kinases like platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT and FLT3 receptor. So far, SUTENT showed promising data in preclinical and clinical trials for a variety of different cancer types and was approved in 2006 for the treatment of Gastrointestinal Stromal Tumors (GIST) and metastatic Renal Cell Carcinoma (mRCC). The aim of this study was to understand the underlying molecular mechanisms of SUTENT action in order to obtain a molecular signature of tumor sensitivity as a basis for response prediction and further applications in new tumor indications. SUTENT treatment of a broad range of cell lines from different tumor types showed anti-proliferative, anti-migratory, anti-invasive and apoptosis inducing effects. To investigate endogenous molecular sites of action of SUTENT and its physiological effects over a wide spectrum of tumor cell lines, we used optimized chemical proteomics and functional cellular assays. By immobilizing a suitable SUTENT analogue and performing affinity chromatography to capture cellular drug targets in combination with high performance mass spectrometry on a hybrid linear ion trap (LTQ)-Orbitrap we sensitively identified known kinases like PDGFRα and β as well as several previously unknown targets of SUTENT including members of the receptor tyrosine and cytosolic tyrosine kinase families as well as serine/threonine kinases involved in cancer progression. Based on the identified kinases, RNA interference techniques will be used in an attempt to connect individual kinase targets with in vitro effects of SUTENT and thereby reveal new targets for therapeutic intervention in different tumor types.

160

Poster 97

Life with a single isoform of PKB/Akt: mice lacking PKBβ/Akt2 and PKBγ/Akt3 are viable but display impaired glucose homeostasis and growth deficiencies Bettina Dummler, Oliver Tschopp, Debby Hynx, Zhong-Zhou Yang, Stephan Dirnhofer, and Brian A. Hemmings Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Protein kinase B (PKB), also known as Akt, is a key regulator of metabolism, proliferation, and differentiation. The three members of the PKB family, PKBα/Akt1, PKBβ/Akt2, and PKBγ/Akt3, are encoded by distinct genes but share similar structural organization. To address the question of isoform-redundancy and isoform-specificity of the PKB family of protein kinases in vivo, we generated mice deficient in both PKBβ and PKBγ; in these mice only the PKBα isoform remains to perform essential PKB functions. Surprisingly, we found that PKBβ-/- PKBγ-/- and even PKBα+/-PKBβ-/-PKBγ-/- mice developed normally and survived with minimal dysfunctions despite a dramatic reduction of total PKB levels in all tissues. A single functional allele of PKBα appears to be sufficient for successful embryonic development and postnatal survival. This is in sharp contrast to the previously described lethal phenotypes of PKBα-/-PKBβ-/- mice and PKBα-/-PKBγ-/- mice. However, PKBβ-/-PKBγ-/- mice were glucose and insulin intolerant and exhibited a ~25% reduction in body weights compared to wild type mice. In addition, we found a substantial reduction in relative size and weight of brain and testis in PKBβ-/-PKBγ-/- mice, demonstrating an in vivo role for both PKBβ and PKBγ in the determination of whole animal size and individual organ sizes.

161

Poster 98

Multiplexed monitoring of activation state markers in signal transduction pathways Jens Tränkle, Claudio Calonder, Gina Balciunaite, Daniel Rechsteiner, and Markus Ehrat Zeptosens - A Division of Bayer (Schweiz) AG; Benkenstrasse 254, 4108 Witterswil, Switzerland; www.zeptosens.com To increase the efficacy of kinase inhibitor treatments and to eliminate tumors before they become resistant, multi-target drugs or drug combinations that affect several pathways involved in cancer diseases are investigated [1,2]. The desire to accomplish high throughput analysis of signaling events in cells and to significantly reduce costs associated with such measurements implicates the processing of a high number of differently treated cell cultures in small sample volumes with a highly efficient procedure. This leads to the need for an ultra sensitive detection system which can handle thousands of samples and provides from each of them sufficient information to gain conclusive information on pathway activation or inhibition. For this purpose the determination of 100 proteins involved in signaling processes might be required with the same precision as typically obtained in single analyte detection. In a typical Zeptosens CeLyA (Cell Lysate Array) protein profiling study different cell lines are treated with and without drug candidates at different concentrations; the cells are harvested at different time points and immediately lysed. An aliquot of the cell lysate - corresponding to the content of less than 10 cells - is spotted onto the ZeptoMARK chip. The cell lysate arrays are then incubated with antibodies specific to either total or activated marker proteins. Ratios of protein and activation levels are quantified and correlated with the cell treatment and the incubation time. Very low sample and reagent consumption for array preparation and assay execution permits to perform the analysis of a large number of samples and thus the establishment of transient profiles with high resolution. Using similar protocols protein marker ratios in tissue samples of cancerous vs. non-cancerous specimen can be analyzed as well. The ultra high sensitivity ZeptoREADER system based on Zeptosens' planar waveguide technology allows to monitor changes in protein expression/activation of as little as 15% in less than 10 cell contents on a single spot by fluorescence measurement. We will present data that demonstrate the power of the CeLyA technology for the analysis of activation state markers in cells. The sensitivity of the technology allows the detection of as little as 1000 protein molecules per spot. Studies analyzing transient protein phosphorylation, differential phosphorylation profiles upon compound treatment and the parallel characterization of drug efficacy on several cellular targets at the same time will be presented on this poster. [1] The second wave in kinase cancer drugs, Nature Biotechnology, 24(2),127-130, (2006) [2] Drugs hitting multiple targets, Nature, 437, 942-943, (2005)

162

Poster 99

Dihydroxyacetone kinases: a new family of kinases with metabolic and transcription control functions Annapurna Srinivas1, Sandra Christen1, Priska Baehler1, Anja Zeller1, David Pridmore2, Christoph Bieniossek1, Ulrich Baumann1 and Bernhard Erni1

1Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland 2Nestlé Research Center, CH-1000 Lausanne 26, Switzerland Dihydroxyacetone (Dha) kinases are a novel family of kinases with signaling and metabolic functions. The putatively PEP-dependent Lactococcus lactis Dha kinase operon consists of three structural genes viz DhaK, DhaL and DhaM and a glycerol facilitator GlpF. Here we report the crystal structures of the transcriptional activator DhaS and the apo- and Dha-liganded coactivator DhaQ and characterize their function. DhaS belongs to the family of TetR repressors although, unlike all known members of this family, it is a transcriptional activator. DhaQ is a paralog of the Dha binding Dha kinase (DhaK) subunit and has the Dha kinase fold. DhaQ and DhaS form a stable complex that in the presence of Dha and activate transcription of the Lactococcus lactis dha operon. Dha covalently binds to DhaQ through a hemiaminal bond with a histidine and thereby induces a conformational change, which is propagated to the surface via a cantilever-like structure. DhaS binding protects an inverted repeat sequence.

163

Poster 100

The MEN kinase cascade promotes chromatin decompaction at the end of mitosis Varela E., Leroy, D., Shimada K., Laroche, T. and Gasser, S. M. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 36, Basel Switzerland The goal of successful cell division is to ensure that each daughter receives a complete copy of the genome. The task requires precise mechanisms of control to orchestrate in time and space the dynamic changes in chromatin and in cellular structure during mitosis. Once chromosomes have successfully segregated to each daughter, cells must re-establish the interphase nuclear organization, to provide the environment for controlled gene regulation. The major change in chromatin structure after segregation of the genome is chromosome decompaction and repositioning in the nucleus. To address this question we have selected Brn1p, a subunit of the yeast Condensin Complex, and asked if chromosome decompaction entails loss of Brn1p or interaction with a novel partner. The observation that the levels of Brn1p remained constant throughout the cell cycle led us to the search of interacting partners for Brn1 by immunoprecipitation experimetns. Among all the putative partners, we focused on Lte1p, a guanine nucleotide exchange factor that activates the mitotic exit network (MEN) during late anaphase. Activation of MEN kinase cascade leads to the release of the Cdc14 phosphatase from its inhibitor Net1p in the nucleolus. Cdc14p causes Clb2p degradaction and accumulation of Sic1p, promoting exit of mitosis and entry in G1. The lte1 deletion mutant showed a defect in decompaction of the ribosomal DNA and in nuclear organization. Analysis of other mutants downstream Lte1p suggested a role for MEN pathway in promoting chromosome decompaction, whereas, the nucleolar Net1p would play a role in maintenance of chromosome decompaction during interphase.

164

Poster 101

Probing the kinome with kinase inhibitors: chemical genomics based screening, selectivity profiling, and mode-of-action studies D. Eberhard, M. Bantscheff, Y. Abraham, S. Bastuck, G. Bergamini, V. Reader*, T. Werner, U. Kruse, G. Neubauer, N. Ramsden*, B. Küster, G. Drewes Cellzome AG, Meyerhofstrasse 1, D-69117 Heidelberg. [email protected] * Cellzome (UK) Ltd., Chesterford Research Park, Cambridge, CB10 1XL United Kingdom To predict efficacy and side effects of kinase inhibitors, it is advantageous to determine the target profile directly in the target cell or tissue, as opposed to the more conventional assays with recombinant enzymes. We have developed a range of Chemical Genomics-based methods that allow us to study the interaction of compounds with their targets directly in tissue culture or even tissue samples from patients. The drug or lead compound is applied over a range of concentrations to living cells or to cell lysates, and the lysate is subsequently captured on a mixed affinity matrix ('Kinobeads') which specifically binds hundreds of protein kinases and kinase-associated proteins. The target affinity profiles of the compound are then determined by differential analysis of the captured proteins from treated and untreated samples, by quantitative mass spectrometry using stable isotopes. The application of this technology to library screening, selectivity profiling, and mode-of-action studies on compounds will be presented.

165

Poster 102

Characterisation of activation of the Akt/mTOR pathway in various human malignancies reveals a wide heterogeneity between individual tumours Nelly Marmy Conus, Richard Young, Wayne Phillips, Melanie Trivett, Rick Pearson, Ian Campbell and Grant McArthur Peter MacCallum Cancer Centre, Translational Research Laboratory, St Andrews Place, East Melbourne 3002. Tel: 61 3 9656 1274. Email: [email protected] The PI3K/Akt/mTOR pathway plays a critical role in regulating multiple cellular functions including cell growth, proliferation, survival, mobility and angiogenesis. Activation of this pathway through a variety of mechanisms including mutation or overexpression of PI3K, or via inactivation of the negative regulator PTEN is a common occurrence in a high proportion of tumours. Specific inhibitors of mTOR are promising antitumour agents both in in vitro and in vivo models. We hypothesise that tumours with activation of the PI3K pathway will display enhanced sensitivity to mTOR inhibitors. Our aim is to develop assays to measure the activation of this pathway to assist in targeting those patients who will benefit from mTOR directed therapy.

Using a panel of 134 human tumours of diverse histologies, we have developed a range of assays to assess activation of the PI3K pathway. We have used quantitative Western blotting (WB) and immunohistochemistry (IHC) to examine tumours for proteins whose phosphorylation may reflect activity of the PI3K pathway including Phos-S6K, Phos-Akt, Phos-ribosomal protein S6 and Phos-ERK. We have also screened exons 9 and 20 of PIK3CA and exon 5 of PTEN for somatic mutations.

Our results indicate that Phos-S6 is the most robust assay across a range of tumour samples with approximately 70% expressing relatively high levels as detected by WB and by IHC. Phos-S6 levels vary across individual tumours of each type and also across tumours of different types. Moreover, Phos-S6 shows differential staining in epithelial cells, stroma, endothelial cells and lymphocytes. Phos-Akt levels appear to be surprisingly quite low in general as measured by WB and there was no correlation between the levels of Phos-S6 and Phos-Akt. PIK3CA mutation has been found in 11/134 tumours: 4/20 breast, 4/20 colorectal and 3/20 ovarian tumours. Although the numbers are small, surprisingly we found no correlation between PIK3CA mutation and the levels of Phos-S6 and Phos-Akt. No PTEN mutations were found in this cohort.

The results reveal the complexity and heterogeneity of the mTOR pathway in human tumours. However these validated biomarker assays could be used in a randomized mTOR inhibitor clinical trial to potentially identify a signature of response to treatment.

166

Poster 103

Structure of the catalytic domain of human Protein Kinase C-beta II with a bisindolylmaleimide inhibitor Stephan Grant Biochemical Pharmacology, Pfizer La Jolla Laboratories The conventional protein kinase C isoform, PKC-beta II (PKCbII), is a lipid-dependent Ser/Thr kinase. PKCbII and other conventional PKC-isoforms are activated through a signaling cascade downstream of growth-factor receptor tyrosine kinases. Activation of PKCbII has been implicated in oncologic pathways such as cell cycle, differentiation, and angiogenesis. Furthermore, PKCbII is activated during the hyperglycemic state and has been associated with diabetic microvascular abnormalities. Here, we describe the crystal structure of the kinase or catalytic domain of PKCbII in complex with an inhibitor at 2.6 Å resolution. The kinase domain of PKCbII was cleaved and purified from full-length PKCbII expressed in baculovirus-infected insect cells. The kinase domain displayed kinetic properties similar to full-length enzyme but did not require lipids for full activity. The overall catalytic domain structure follows the classical bilobal fold for protein kinases and is in its fully activated conformation with three well-defined phosphorylated residues: Thr-500, Thr-641, and Ser-660. Different from the crystal structures of nonconventional PKC-isoforms, the turn motif of the PKCbII catalytic domain is almost fully ordered and features a novel a-helix near the ATP binding site. An ATP-competitive inhibitor, 2-methyl-1H-indol-3-yl-BIM-1 was crystallized with PKCbII catalytic domain as an asymmetric pair of two identical molecules. The bound inhibitor adopts a nonplanar conformation in the ATP binding site with the kinase domain taking on an intermediate, open conformation. This PKCbII-inhibitor complex represents the first structural description of any conventional PKC kinase domain. Given the pathogenic role of PKCbII in the development of cancer and diabetic complications, this co-crystal structure may serve as a template for the rational design of inhibitors as potential therapeutic agents.

167

Poster 104

MAPKKK6 is a negative regulator of apoptosis Elisabeth Ortner and Karin Moelling Institute of Medical Virology, University of Zurich, Switzerland MAP kinase kinase kinase 6 (MAPKKK6) belongs to the family of MAPKKK, which comprises 20 members including the Raf kinase. It was initially identified as an interaction partner of another MAPKKK, the apoptosis signal-regulating kinase 1 (ASK1). These two kinases share a similar structure with a central kinase domain and a C-terminal coiled coil sequence and have 45% amino acid (aa) identity. MAPKKK6 is shorter than ASK1 lacking 32aa near the N-terminus and 54aa near the C-terminus. Both kinases are ubiquitously expressed regarding tissue distribution and show a similar intracellular localization in the cytoplasm, nucleus and mitochondria. Due to the homology with ASK1, MAPKKK6 was suggested also to be an apoptosis inducing kinase. This is true for the overexpression of MAPKKK6, which leads to apoptosis similar to overexpressed ASK1. Matched levels of overexpression of both kinases abolish induction of apoptosis after starvation. However, we show that MAPKKK6 differs from ASK1 by exhibiting an anti-apoptotic function. Endogenously expressed and overexpressed MAPKKK6 can form homo-oligomers similar to ASK1. MAPKKK6 and ASK1 also exist as hetero-oligomers when expressed at their endogenous levels. Furthermore, knockdown of MAPKKK6 by two different siRNAs to about a third of protein expression also induces apoptosis already in non-stressed cells, which is increased by stress induction by starvation, TNFα treatment or oxidative stress. This is in contrast to the behaviour of ASK1, since ASK1 knockdown results in inhibition of apoptosis activation upon stress induction. The relative levels of these two kinases decide on the biological outcome of the cell. We propose a model in which ASK1 is negatively regulated by hetero-oligomerization with MAPKKK6. ASK1 is negatively regulated by AKT phosphorylation at Ser83 near the N-terminus. MAPKKK6 exhibits two putative phosphorylation sites near the N- and C-termini. Whether one of these sites is phosphorylated by AKT is currently under investigation.

168

Poster 105

Approach to molecular basis of Insulin/IGF ageing signalling on Drosophila melanogaster Tomoatsu Ikeya and Linda Partridge University College London, UK Reduction of Insulin/IGF signalling (IIS) kinase activities results in extension of lifespan. For examples, lifespan of Drosophila melanogaster was extended by heteroallelic mutation of Insulin receptor (InR) tyrosine kinase regulating Insulin Receptor Substrate (IRS/chico). chico mutant flies also showed significant extension of the lifespan, which indicates that InR activity is required for the ageing process to activate the down-stream targets via chico. Indeed adipo-tissue specific de-phosphorylated form of the FOXO protein, a target of Akt/PKB kinase, extended the lifespan. However, puzzlingly, mutants in the gene encoding the Akt/PKB kinase did not extend lifespan despite their effect on growth and size. As IIS has a very complicated signal network and the ageing IIS may be slightly different from the growth IIS, we are now keen to figure out the molecular basis of the control of fly lifespan by IIS. To achieve this goal, we have several approaches; 1) Overexpression of chico- RNAi, 2) Overexpression of InR dominant-negative form and PTEN which is an antagonist of PI3 kinase, 3) molecular analysis of long-lived chico mutant. We shall present recent results.

169

Poster 106

A functional interaction between Sprouty proteins and Caveolin-1 Miguel A. Cabrita, Fabienne Jäggi, Sandra P. Widjaja and Gerhard Christofori Institute of Biochemistry and Genetics, Department of Clinical-Biological Sciences, University of Basel, Mattenstrasse 28, 4058, Basel, Switzerland. Growth factor-mediated signal transduction cascades can be regulated positively and negatively in a spatio-temporal manner. Such modulators include the Sprouty proteins and Caveolin-1. The four mammalian Sprouty family members are palmitoylated phosphoproteins that can attenuate or potentiate numerous growth factor-induced signaling pathways. Previously, we have shown that Sprouty-1 and Sprouty-2 associate with Caveolin-1, a cholesterol-binding protein that acts as a scaffolding protein in caveolae where it is the primary structural component. Caveolin-1, like Sprouty, inhibits growth factor-induced mitogen-activated protein kinase activation. We now show that all four mammalian Sprouty family members bind Caveolin-1. The C-terminus of Caveolin-1 is the major Sprouty-interaction site, while Sprouty binds Caveolin-1 via its conserved C-terminal domain. A single amino acid point mutation in this domain ablates Caveolin-1 interaction. Furthermore, the various Sprouty isoforms differ dramatically in their cooperation with Caveolin-1-mediated inhibition of mitogen-activated protein kinase activation in human breast cancer cells and such cooperation is also highly dependent on cell density and on the growth factor being examined. In summary, the data suggest that the Sprouty/Caveolin-1 interaction modulates signaling in a cell density-, mitogen- and Sprouty isoform-specific manner.

170

Poster 107

Phosphoproteomic indication of a MAPK kinase-mediated phosphorylation of an antioxidant enzyme in tomato Tim Xing, Christof Rampitsch, Natalia Bykova Department of Biology, Carleton University, Ottawa, Canada (TX), Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Canada (CR, NB) (Email: [email protected]) In plants, mitogen-activated protein kinase (MAPK) pathways play multiple roles in signal transduction-mediated events, such as response to pathogens, wounding, other abiotic stresses, or plant hormones. Tomato fruit is considered a functional food for its effect to combat cancers. It is a reservoir of deverse antioxidant molecules, such as ascorbate (vitamin C), α-tocopherol (vitamin E), and flavonoids. These antioxidants can help protect against the damage that gets cancer started, and recent evidence has suggest that tomatoes may help combat cancer in additional ways, at later stages of the process. Antioxidant enzymes such as glutathione peroxidase also protects the body from free radicals and possibly cancerous agents. We found that glutathione peroxidase was phosphorylated by tMEK2, a tomato MAPKK.

171

Poster 108

Chimeric receptor models of CD5-signalling in chronic lymphocytic leukemia (CLL) Michaela Patz, Aditya Veldurthy, Michael Hallek and Günter Krause Clinic I for Internal Medicine, University Hospital Cologne, Germany The undoubted importance of the microenvironment for the survival of CLL cells is due to signal transduction emanating from cell surface receptors. Since the B-cell receptor (BCR) and many of its co-receptors as well as cytokine receptors are devoid of intrinsic kinase activity, non-receptor kinases, e.g. Src family kinases (SFK), play crucial roles for the intracellular transduction of survival signals. Contrary to normal B-cells in bone marrow and blood, CLL cells express high levels of the trans-membrane receptor CD5, which is well known as a T-cell antigen and co-receptor of the T-cell receptor, and for which currently no specific ligand is available. The BCR and CD5 are known to be associated with and functionally linked to the SFK Lyn and Lck, respectively, both of which are aberrantly expressed in CLL cells. Studies of primary CLL cells treated with anti-CD5 antibodies strongly suggested that CD5 signalling is involved in the regulation of the survival of CLL cells, but reported contrasting effects of CD5 stimulation, namely prolonged survival versus apoptosis induction. To resolve such discrepancies we aim at establishing cell culture models expressing specifically stimulatable chimeric receptors containing the intracellular part of CD5. For this purpose a chimeric EGFR-CD5 receptor was constructed and transfected into two cell lines without intrinsic CD5 expression, namely into the human CLL cell line, Mec1, and into 2G5 cells, a derivative of the human T-cell lymphoma cell line Jurkat. In both cell lines, clones exhibiting stable expression of EGFR at the cell surface were obtained. Chemical crosslinking studies of Mec1-transfectants revealed that EGF-treatment induced dimerization of native EGFR and the chimeric EGFR-CD5 construct. While EGF-treatment of Mec1 or Jurkat 2G5 cells expressing native EGFR led to strong autophosphorylation of the receptor, changes in the tyrosine phosphorylation patterns in EGF-treated transfectants expressing chimeric EGFR-CD5 were smaller and more complex. Using immunocomplex kinase assays the SFK activities in Mec1 and 2G5 cells expressing EGFR-CD5 or not were determined with and without EGF stimulation. For studying the impact of CD5 signalling on the regulation of survival it will be interesting to monitor changes in proliferation, percentage of apoptotic cells and cytokine production upon induction of chimeric EGFR-CD5.

172

Poster 109

TGFβ1-mediated activation of protein kinase B/Akt is important for epithelial-mesenchymal transition in diabetic nephropathy Kattla J.J., Godson C, Brazil DP University College Dublin, UCD Conway Institute, University College Dublin Belfield, Dublin 4, Ireland The cellular signaling events driving the pathogenesis of diabetic nephropathy are complex and not fully elucidated. Transforming growth factor-β1 (TGF-β1) is a primary mediator of cellular damage during DN, and its upregulation is implicated in epithelial-mesenchymal transition (EMT) associated with DN-associated renal fibrosis. Protein kinase B (PKB/Akt) is a multi-functional regulator of multiple key cellular processes, and some of its downstream targets have been implicated in the onset and progression of EMT in renal fibrosis. Importantly, the regulation of Snail, a transcription factor that represses the expression of e-cadherin during renal fibrosis has been shown to be regulated by GSK3β, a downstream target of PKB/Akt. To investigate the relevance of PKB/Akt activation downstream of TGFbeta1 in tubulointerstitial fibrosis, kidney epithelial cells (NRK-52E and HK2 cells) were exposed to TGFβ1 for various periods. Phosphorylation of PKB/Akt was evident in cells exposed to TGFbeta1 for 60 min. Activation of downstream targets of PKB/Akt such as GSK3β and p27Kip1 was also detected. PI3K inhibitors such as LY294002 and wortmannin reduced this TGFbeta1-mediated activation. TGFbeta1 treatment of epithelial cells induces epithelial-mesenchymal transition as judged by decreased levels of epithelial markers e-cadherin and ZO-1, and increased levels of mesenchymal markers such as vimentin and alpha smooth muscle actin. Inhibition of PKB/Akt using LY294002 significantly reduced the ability of TGFβ1 to induce EMT in these cells, suggesting that PKB/Akt activity is involved in this response. To examine the role of PKB/Akt activation in DN in vivo, we analyzed PKB/Akt activation in kidney sections from both control Wistar and type 2 diabetic Goto-Kakizaki rats (GK). GK rats displayed mild to moderate diabetic nephropathy at 9-10 months of age. Elevated levels of phosphorylated PKB/Akt and its downstream target pGSK3β were observed in the kidneys of diabetic, but not control Wistar rats, with pronounced staining in the kidney tubules. These data suggest that TGFβ1 mediated PKB/Akt signaling plays an important role in the fibrotic response triggered by the extracellular milieu in diabetic nephropathy.

173

Poster 110

ECO-4601, a novel antitumor compound targeting the Ras-MAPK pathway induces apoptosis H. Gourdeau, V. Zvereff, M-A. Wioland, D. Lobreau, and P. Falardeau Ecopia BioSciences Inc., Ville Saint-Laurent, Canada H4S 2A1

ECO-4601 (MW 462), a farnesylated dibenzodiazepinone, is a promising new chemical entity discovered through Ecopia’s proprietary Decipher® drug discovery platform. The compound was shown to have a broad cytotoxic activity (low μM) when tested in the NCI 60 cell line panel. Antitumor evaluation in human glioma, breast and prostate xenografts indicated that ECO-4601 has potent in vivo antitumor activity. As the compound was identified through in vitro cytotoxic assays, its molecular target(s) were unknown at the time of discovery. Based on the structural characteristics of ECO-4601, we first investigated its binding affinity to the central (CBR; GABAA) and peripheral (PBR) benzodiazepine receptors. The CBR is restricted to the CNS and mediates the anxiolytic and anticonvulsant properties of benzodiazepines. On the other hand, while the PBR was originally discovered as an alternative-binding site for the benzodiazepine diazepam (Valium®), it is a critical component of the mitochondrial permeability transition pore (MPTP). This multiprotein complex is located at the contact site between inner and outer mitochondrial membranes and is involved in the initiation and regulation of apoptosis. ECO-4601 was tested on PBR in a radioligand-binding assay using 3H PK11195 as the specific ligand. Binding studies indicated that ECO-4601 had a selective affinity for the PBR (IC50 = 0.291 μM) with no specific binding to the CBR (IC50 > 10 μM). We further show that ECO-4601 exposure induces ROS, caspases 3 and 7 and induces DNA ladder. Although potent and selective PBR ligands have been developed for diagnostic use in cancer, none have been successful in demonstrating therapeutic benefit in oncology as indicated by lack of anticancer activity in animal models when tested as single agents. The potent antitumor activity of ECO-4601 as a single agent thus suggests that other targets are involved in its mechanism of action. Related to its farnesylated moiety, the effect of ECO-4601 was assessed on the Ras signalling pathway. We verified if ECO-4601 interfered with Ras processing by monitoring FTase and GGTase I activities. Downstream Ras signalling events, such as Raf-1 and ERK1/2 phosphorylation, were also evaluated by immunoblots in prostate (PC-3), glioma (U-87 MG), and breast (MCF7) cell lines. No mobility shift of either HDJ2 or Rap1A (surrogate markers of FTase and GGTase I, respectively) were observed in cells exposed to ECO-4601 for up to 48h. In contrast, a strong inhibition of EGF-induced phosphorylation of c-Raf-1 and ERK1/2 in the four cell lines tested was shown. This effect was time dependent with complete inhibition of protein phosphorylation within 6h. The dual activity of ECO-4601, where binding to the PBR, resulting in apoptosis, and inhibition of the Ras-mitogen-activated protein kinase pathway, involved in cell proliferation and migration, make ECO-4601 a unique and new targeted-therapeutic anticancer drug candidate. ECO-4601 is presently being tested in a Phase I clinical trial against solid tumors.

174

Poster 111

Conditional knock down of c-Myc in the mouse mammary gland Tina Stoelzle1, Andreas Trumpp2 and Nancy E. Hynes1

1 Friedrich Miescher Institute for BioMedical Research, Basel, Switzerland 2 Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland The c-Myc protein, a basic helix-loop-helix/leucine-zipper transcription factor, regulates many biological processes, like cell cycle progression, transcription, protein biosynthesis and metabolism. We are aiming to investigate the role of c-Myc during mammary gland development, using a conditional knock out approach. To address this question, mice homozygous for the conditional floxed allele (c-mycfl/fl) were crossed with mice heterozygous for the WAPiCre transgene to generate c-mycfl/fl;WAPiCre+/- (mutant) and c-mycfl/fl;WAPiCre-/- (wildtype) littermates. The Cre recombinase, under the control of the WAP (whey acidic protein) promoter, is expressed exclusively in the luminal alveolar cells starting from mid-pregnancy throughout lactation. Following Cre mediated recombination, we can see downregulation of c-Myc mRNA levels in mutant glands, starting from day 14.5 of pregnancy and staying low during the entire lactation period and involution. In a first pregnancy, we observed reduced bodyweight in pups nursed by mutant mothers, suggesting a problem with lactation. This lactation defect was not due to changes in milk protein composition and we are currently investigating this in more detail. Histological examination of mutant and wildtype glands showed almost no morphological differences at the onset of lactation (day 0.5). By day 3.5, some mutant glands displayed a slight reduction of alveolar density compared to wildtype glands, which became more pronounced at day 10.5 of lactation, suggesting a defect in proliferation. A microarray analysis of glands from 4 wildtype and 4 mutant animals at lactation day 3.5 revealed a 4 fold upregulation of the cyclin dependent kinase inhibitor p21, which is a known target of c-Myc mediated repression of expression. To check which effects of c-Myc are mediated via p21 in our system, we are currently crossing the mutant mice into a p21-/- background. When c-Myc deficient females were allowed to undergo a second pregnancy, the phenotype became more severe. In mid-pregnancy (day 10.5 and 14.5) the wildtype glands showed expanding alveoli with lipid droplets, whereas in the mutant glands the differentiation and milk production seemed to be delayed in total. By the time of gestation, the mutant glands contained milk and mutant mothers were able to nurse pups, however with a stronger reduction in bodyweight. When examining the histology of the glands at day 4.5 of second lactation, the mutant glands contained much fewer alveoli than the wildtype glands. These effects suggest a role for c-Myc in proliferation and/or maintenance of mammary progenitor cells, a question we are currently investigating using transplantation technique into cleared fat pad.

175

Poster 112

Lymphoid microenvironment inhibits apoptosis in B-CLL cells through activation of PI3K/Akt pathway Medhat Shehata, Dita Demirtas, Susanne Schnabl, Martin Hilgarth, Rainer Hubmann, Josef D. Schwarzmeier, Ulrich Jaeger. Medical University of Vienna, Clinic of Internal Medicine I, Department of Hematology/Hemostaseology, L. Boltzmann Institute for Cytokine Res., K. Landsteiner Institute for Cytokine and Leukemia Res. A-1090, Vienna, Austria. The accumulation of the malignant B cells in chronic lymphocytic leukemia (B-CLL) appears to be due to inhibition of apoptosis and long survival of the leukemic cells. This could be due to activation of anti-apoptotic cascades through interaction with the lymphoid microenvironment. The aim of this study was to elucidate the role of tumor microenvironment in activation of the potent anti-apoptotic PI3-K/Akt signal transduction pathway and prolongation of survival of B-CLL cells. Stromal fibroblasts of bone marrow (BMFs) were used as an in vitro model for lymphoid microenvironment and to test their ability to inhibit spontaneous apoptosis of B-CLL. Co-cultivation of B-CLL cells with human BMFs significantly inhibited apoptosis of B-CLL cells. Trans-well culture experiments indicated that the supportive effect is provided by cell-cell contact and soluble mediators produced by viable fibroblasts. Experiments using TGF-beta1 or neutralizing anti-TGF-beta1 antibodies showed that this cytokine inhibits apoptosis of B-CLL cells in presence of BMF. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K (p110 subunit) and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. Induction of apoptosis was associated with a significant decrease in the intracellular PIP3, PI3-K, PDK1 and Akt1, NF-kappa B, IKK, and de-phosphorylation/activation of tumour suppressor protein PTEN. Studies using phosphospecific anti-PTEN antibody demonstrated that PBMC of CLL patients (n=40) highly express a phosphorylated (inactive) form of PTEN. The results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment maintains the activation of this pathway. The data also suggest that targeting this pathway represents a new therapeutic approach in B-CLL.

176

Poster 113

A cellular test sytem for assaying inhibitors of the kinase activity of B-Raf VE600 Jan E. Ehlert, Bettina Mutschler, Melanie Müller, and Michael H.G. Kubbutat ProQinase GmbH, Breisacher Str 117, 79106 Freiburg, Germany B-Raf VE600 is the most common activating mutation of B-Raf and plays an important role in oncogenic transformation of melanoma cells. In order to characterize B-Raf VE600 kinase inhibitors we went to develop intermediate throughput test sytems that could be used to test compounds in a cellular context. We report on establishing a cellular assay that allows to assess B-Raf activity by measuring phosphorylation of B-Raf downstream target MEK-1. This assay is based on Rat1 cells in which B-Raf VE600 activity can be regulated by 4-OH-Tamoxifen. Moreover, we report about an approach of generating an inducible B-Raf VE600-in vivo tumor model using the murine melanocyte cell line Melan-A.

177

Poster 114

Increases in c-Src expression level and activity do not promote growth of human colorectal carcinoma cells in vitro and in vivo Arkadiusz Welman, Christopher Cawthorne, Lourdes Ponce-Perez, Jane Barraclough, Sarah Danson, Stephen Murray, Jeff Cummings, Terry D. Allen and Caroline Dive Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom The levels and activity of c-Src in colorectal cancer cells increase steadily during the course of colorectal carcinogenesis and are most highly elevated in advanced metastatic disease. However, the effects of increases in c-Src activity on proliferation of colorectal cancer cells during early and late stages of tumorigenesis remain elusive. To study the consequences of increases in c-Src levels and activity on the growth of colorectal cancer cells in the later stages of colorectal carcinogenesis we developed human colorectal cancer cell lines in which c-Src levels and activity could be inducibly increased by tightly controlled expression of wt c-Src or the constitutively active mutant of c-Src, c-SrcY527F. Src induction activated multiple signaling pathways (often associated with a proliferative response) but did not promote cell proliferation in vitro nor tumor growth in a xenograft model in vivo. These results indicate that in more advanced stages of colorectal carcinogenesis, increases in c-Src levels and activity are likely to have functions other rather than directly promoting tumor growth.

178

Poster 115

Bcl10 controls TCR- and FcγR-induced actin polymerization

Daniel Rueda1, Olivier Gaide1,8, Liza Ho2, Elodie Lewkowicz6, Florence Niedergang6, Fabien Rebeaud1, Stephan Hailfinger1, Montserrat Guzzardi1, Béatrice Conne2, Marcus Thelen3, Jerome Delon6, Uta Ferch4, Tak W. Mak5, Jürgen Ruland4, Jürg Tschopp1, Jürg Schwaller2,7 and Margot Thome1

1 Department of Biochemistry, University of Lausanne, BIL Biomedical

Research Center, Epalinges, Switzerland 2 Department of Clinical Pathology, CMU, Geneva, Switzerland 3 Institute for Research in Biomedicine, Bellinzona, Switzerland 4 Third Medical Department, Technical University of Munich, Klinikum rechts

der Isar, Munich, Germany 5 The Campbell Family Institute for Breast Cancer Research and Ontario

Cancer Institute, University of Toronto, Toronto, Canada. 6 Institut Cochin, INSERM U 567, CNRS UMR 8104, Université René

Descartes ParisV, Department of Cell Biology, Paris, France 7 Present address: Department of Research, University Hospital, Basel,

Switzerland 8 Present address: University Medical Center, Geneva, Switzerland The protein B-cell lymphoma / leukaemia-10 (Bcl10) is an adaptor protein that plays a crucial role in responses triggered by immune recognition receptors as the TCR, BCR or FcR, by connecting them to the activation of the NF-kB pathway and cytokine secretion. Bcl10 is a phosphoprotein, but the physiological relevance of this post-translational modification remains partially understood. Here we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify one particular serine residue as a key target of Bcl10 phosphorylation. We also show that a phosphorylation-deficient Ser /Ala mutant specifically inhibits TCR-induced actin polymerization yet does not affect NF-kB activation. Moreover, repression of Bcl10- but not of Carma1- expression induces a clear defect in TCR-induced F-actin formation, cell spreading and conjugate formation. Remarkably, Bcl10 repression also impairs FcγR-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages.

179

Poster 116

New transition state-based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells Fang Wu, Heinz Gehring Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich Switzerland Pyridoxal 5’-phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential cytostatic or cancer chemopreventive target. Here we show the design and synthesis of a coenzyme-substrate analog, a kind of transition state-based novel precursor inhibitor of ODC which is taken up by cells, blocks ODC activity in cells and inhibits proliferation of a wide range of tumor cell lines. The designed pyridoxyl-ornithine analog termed POB showed favorable interaction in the active site of human ODC by molecular modeling methods. POB was synthesized, purified and tested for its cell growth inhibition. POB at 100 μM concentration inhibited the activity of newly induced ODC activity in glioma LN229 and COS7 cells more than 90%. In correlation with the inhibition of ODC activity, a time dependent inhibition of cell growth was measured in myeloma, glioma LN18 and LN229, glioblastoma multiforme, Jurkat, COS7 and SW2 small-cell lung cancer cells but not in human aortic smooth muscle cells if DNA synthesis and cell number were measured. The IC50 of LN229 growth was obtained at 50 μM. In conclusion, POB is an effective inhibitor for ODC in cells and much more efficient in the proliferation-inhibiting potential of several types of tumor cells than α-DL-difluoromethylornithine, a well known irreversible ODC inhibitor. The present strategy might serve to develop inhibitors of this type for other PLP-dependent enzymes of pharmacological interest.

180

Poster 117

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice

Arash M Tehrani1, Soon-Kyung Hwang1, Tae-Hee Kim2, Chong-Su Cho2, Jin Hua1, Woon-Sung Nah1, Jung-Taek Kwon1, Jun-Sung Kim1, Seung-Hee Chang1, Kyung-Nam Yu1, Sung-Jin Park1, Dilli R Bhandari1, Kee-Ho Lee3, Gil-Hwan An4, George R. Beck Jr.5, Myung-Haing Cho1

1Laboratory of Toxicology, College of Veterinary Medicine and Nano Systems Institute-National Core Research Center, 2Laboratory of Biomedical Polymer and Tissue Engineering, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea, 3Laboratory of Molecular Oncology, Korea Institute of Radiological & Medical Sciences, Seoul 139-240, Korea, Department of Food Science & Technology, Chungnam National University,

Taejeon 305-764, Korea,4

5Emory University School of Medicine, Division of Endocrinology, Metabolism and Lipids, Atlanta GA 30322, USA.

Lung cancer has emerged as a leading cause of cancer death in the world, however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has lead to re-emergence of aerosol gene delivery as a nonivasive method for lung cancer treatment. In this study, nanosized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wilt type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nanosized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals while aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation while a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

181

Poster 118

A novel role of AMP-activated protein kinase in ginsenoside-Rh2-induced apoptosis in cancer cells Min-Jung Kim and Joohun Ha Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine, Seoul 130-701, Korea

Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases including cancer. Ginsenoside-Rh2, a ginseng saponin that is isolated from the root of Panax ginseng C. A. Meyer, has exhibited a potential as an anti-cancer reagent, but the underlying mechanisms remain practically almost elusive. In the present study, we have shown that some cancer cells have differential sensitivity to ginsenoside-Rh2-induced apoptosis, raising questions regarding the specific mechanisms responsible for the discrepant sensitivity to ginsenoside-Rh2. Here we demonstrate that a degree of responsiveness of AMP-activated protein kinase (AMPK) to ginsenoside-Rh2 is a novel factor responsible for the discrepant sensitivity to apoptosis. AMPK regulates energy balance by monitoring cellular energy status. Cancer cells with acute responsiveness of AMPK display a relative resistance to ginsenoside-Rh2, but co-treatment with AMPK inhibitor resulted in a marked increase of ginsenoside-Rh2-induced apoptosis. We also studied relevant signaling pathways, revealing a possibility that AMPK is closely associated with p38 MAPK signaling pathway. In conclusion, we suggest that further studies on AMPK may contribute to reveal an anti-cancer nature of ginsenoside-Rh2.

182

Poster 119

AMP-activated protein kinase suppresses apoptosis via feedback inhibition of c-Jun N-terminal kinase in glucose-deprived cancer cells Hee Yun and Joohun Ha Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine, Seoul 130-701, Korea As solid tumors outgrow the existing vasculature, they are continuously exposed to microenvironments where the supply of nutrients is limited, and adaptation to such environment as glucose deprivation is critical for survival of tumor cells. Nevertheless, the underlying mechanisms by which tumor cells either undergo apoptosis or survive under the glucose-deprived condition are poorly understood. In the present study, we examined the signaling pathways leading to apoptosis after glucose deprivation in cancer cells, focusing on the role of AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK). Here, we show that glucose deprivation activates JNK with biphasic kinetics; early JNK activation promotes cell survival whereas late JNK activation induces apoptosis. Inhibition of JNK at early stage under glucose-deprived condition decreased activity of AMPK, which plays a critical role for cell survival under ATP-depleted conditions, resulting in enhanced apoptosis in DU145 cells. Furthermore, we demonstrate that AMPK activation leads to cell survival via feedback inhibition of apoptosis-inducing property of JNK. Taken together, our data suggest that early JNK-mediated activation of AMPK and AMPK-mediated inhibition of late JNK activity plays a critical role in the tumor cell survival. Thus, our results demonstrate a molecular mechanism underlying the different biological function of JNK through AMPK activation signaling.

183

Poster 120

Fas (APO-1/CD95) activates AMP-activated protein kinase in DU145 human carcinoma: a novel role of AMPK in Fas-induced apoptosis Seung-Nam Jung, Sung-Soo Kim, and Joohun Ha Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine, Seoul 130-701, Korea The death receptor Fas (APO-1/CD95) induces apoptosis upon cross-linking by its ligand (FasL) in many tissues, but a number of cancer cells exhibit resistance to such an apoptosis-inducing agent. Indeed, resistance to apoptosis is believed to be one of the hallmarks of cancer, and therefore, it is clinically crucial to understand the underlying mechanisms how cancer cells acquire such a resistance. In the present study, we demonstrate that Fas signaling leads to a rapid activation of AMP-activated protein kinase (AMPK), which plays a major role in adaptive responses to ATP-depleting conditions, in DU145 human prostate cancer cells; prostate cancer is resistant to Fas-mediated apoptosis induced by anti-Fas antibodies despite high levels of Fas surface expression and no mutation in the Fas gene. We further demonstrate that inhibition of AMPK sensitizes DU145 cells to Fas-induced apoptosis via caspase-dependent manner. We also investigated a role of a transcription factor NF-κB and a cross-talk with AMPK in Fas signaling pathway. In conclusion, our data provide novel evidence that AMPK contributes to increased resistance to Fas-mediated apoptosis.

184

Poster 121

Anti-diabetes and anti-obesity effect of cryptotanshinone via activation of AMP-activated protein kinase Eun Ju Kim, Sung-Soo Kim, Joohun Ha Department of Biochemistry and Molecular Biology, Medical Research Center for Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine, Seoul 130-701, Korea Metabolic disorders, including type 2 diabetes and obesity, represent major health risks in industrialized countries. Recently, AMP-activated protein kinase (AMPK) has become the focus of a great deal of attention as a novel therapeutic target for the treatment of metabolic syndromes, as AMPK has been demonstrated to mediate, at least in part, the effects of a number of physiological and pharmacological factors that exert beneficial effects on these disorders. Thus, the identification of a compound that activates the AMPK pathway would contribute significantly to the treatment and management of such syndromes. In service of this goal, we have screened a variety of naturally occurring compounds, and have identified one compound, cryptotanshinone, as a novel AMPK pathway activator. Cryptotanshinone was originally isolated from the dried roots of Salvia miltiorrhiza, an herb which is used extensively in Oriental medicine, and which is known to exert beneficial effects on the circulatory system. For the first time, in the present study, we have described the potent anti-diabetic and anti-obesity effects of cryptotanshinone, both in vitro and in vivo. Our findings suggest that the activation of the AMPK pathway might contribute to the development of novel therapeutic approaches for the treatment of metabolic disorders such as type 2 diabetes and obesity.

185

Poster 122

Interleukin-6-induced activities of the Src family kinases Hck, Lyn and Fyn in myeloma cells are blocked by an acidic petide

A. Hausherr (a,b), R. Tavares (a), M. Hallek (b) and G.F. Krause (b) (a) Clinical Cooperation Group Gene Therapy, GSF-National Research Institute for Environment and Health (Munich, Germany), (b) Clinic for Internal Medicine I, University of Cologne (Cologne, Germany) Background: Src Family Kinases (SFKs) play pivotal roles in normal B-cell development and in B-cell neoplasias. Our aim is to elucidate the roles of SFKs in signaling cascades resulting in cell proliferation or anti-apoptosis in multiple myeloma (MM). The pleiotropic cytokine interleukin-6 (IL6) is one of the major growth factors for MM cells. We have previously shown that IL6 induces the activation of the SFKs Hck, Lyn and Fyn and that Hck is associated with the IL6R beta chain (gp130) via an acid domain (AD) in gp130. Aim: We observed that an 18mer peptide (18AD), which is derived from the AD, inhibited the IL-6-dependent growth of myeloma cells. In order to develop a lead structure for small molecule inhibitors we wish to elucidate the cellular and molecular effects of peptide 18AD. Results: On the cellular level, 50-100μM of a membrane-permeable myristoylated peptide 18AD inhibited factor-dependent proliferation in 7TD-1 (mouse) and INA-6 (human) cells by ~75%. Treatment with 100 µM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. A control peptide (18sc) with an identical amino acid composition but an arbitrarily scrambled sequence had no effect on proliferation and apoptosis. A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck. While the peptide 18AD concentration inhibiting 50% was ~30 µM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM.1 myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. In 7TD1 cells the expression of a kinase-inactive Hck mutant led to a reduced proliferative response to IL6. On the molecular level, immunoprecipitation experiments in 7TD1 cells showed that peptide 18AD decreased the complex formation between Hck and gp130 in a concentration dependent way. Immunocomplex kinase assays in INA-6 cells showed that the IL6 induced activation of Hck, Lyn and Fyn was blocked by peptide 18AD, but not by peptide 18sc. Conclusion: We characterized peptide 18AD, which inhibits the association of Hck and gp130 as well as IL6-induced SFK activity. Thus, the observed anti-proliferative and pro-apoptotic effects of peptide 18AD may be due to inhibition of SFK-mediated pathways.

186

Poster 123

Protein Kinase B, a Survival Kinase In Chronic Lymphocytic Leukaemia of B Cells Sebastian Hofbauer, Inge Tinhofer, Richard Greil Laboratory of Immunological and Molecular Cancer Research (LIMCR), 3rd Medical, Dept., University Hospital Salzburg, Austria In B-chronic lymphocytic leukemia (B-CLL), a relevant number of tumor cells home to and grow within the bone marrow. There, the support for tumor growth and survival is provided by stromal cells. These cells are capable of secreting high amounts of cytokines, chemokines, and/or tumor growth factors. In the signaling cascades activated by these factors, protein kinase B/Akt (PKB/Akt) is thought to be a central player. We therefore aimed to characterize the role of PKB in modulating the downstream cellular processes that decide on survival or death of these neoplastic B-cells within their microenvironment. Analyses of the expression levels of PKB/Akt isoforms (Akt-1, -2, and -3) in B-CLL demonstrated that significant mRNA expression of all isoforms could be detected in primary tumor cells. Blocking PKB activity either directly by using the specific PKB inhibitor SH-5, or indirectly by targeting the upstream activator phosphoinositol-3 kinase (PI3K), efficiently triggered apoptosis in B-CLL cells. While B-CLL cells in vitro are prone to spontaneous apoptosis, we could observe strong protection of these tumor cells from death when co-cultured with stromal cells. This protection was mainly mediated by soluble factors and allowed the long-term culture of B-CLL cells for up to 14 days. The enhanced survival of the tumor cells in co-culture with the stromal cell line HS-5 was associated with increased PKB activation as detected by flow cytometric analysis. This effect could be repressed by pre-incubation of CLL cells with the irreversible PI3K-inhibitor Wortmannin. We also investigated the possible role of PKB in the survival of B-CLL cells under cytotoxic stress. Under low serum conditions that was accompanied by increasing levels of reactive oxygen species (ROS), blocking PKB induced apoptosis in a very effective manner. Treatment of B-CLL cells with H2O2 resulted in activation of PKB as detected by Western blot analysis using a pS(473)-PKB antibody. Congruently, co-administration of SH-5 with the complex I inhibitor rotenone resulted in a synergistic increase in the levels of apoptosis in these cells. PKB can therefore be seen as a rescue signal in ROS-stressed B-CLL cells. In summary, we have demonstrated that targeting PKB activity may repress tumor survival mediated by the stromal microenvironment and also increase cytotoxic capabilities of ROS-generating drugs used in the treatment of B-CLL. The next phase of the project will focus on the translation of these results into a feasible therapeutic strategy for B-CLL.

187

Poster 124

PI3Kγ Adaptors Relay PI3Kγ Activity To Specific Cell Responses Bohnacker T*, Marone R*, Calvez R*, Clément E*, Hirsch E°, Wymann MP* *Institute of Biochemistry and Genetics, DKBW, Centre of Biomedicine University of Basel, Switzerland, ° Universita di Torino, Turin, Italy Phosphoinositide 3-kinase γ (PI3Kγ) is activated via G protein-coupled receptors (GPCR) by Gβγ subunits of trimeric G proteins. It has been shown that PI3Kγ activity is increased in presence of its adaptors, p101 and the recently described p84/p87PIKAP, in vitro, but their role in vivo was disputed. In mast cells PI3Kγ relays autocrine/paracrine signaling through GPCR, which synergize with IgE/antigen to trigger maximal mast cell degranulation. We found that mast cells express high levels of p84/p87PIKAP and lack p101. Moreover, loss of PI3Kγ goes along with reduced p84/p87PIKAP levels. Complementation of both, PI3Kγ and p84/p87PIKAP in PI3Kγ null bone marrow derived mast cells (BMMC), but not PI3Kγ alone, completely restored adenosine enforced mast cell degranulation. The PI3Kγ.p101 complex on the other hand was not able to support degranulation. Adenosine triggered PI3Kγ activation as measured by phosphorylation of Akt/PKB and by relocalization of a PtdIns(3,4,5)P3-specific probe (PHBtk-GFP). While PI3Kγ.p84/p87PIKAP-derived PtdIns(3,4,5)P3 was localized to the plasma membrane, PI3Kγ.p101 complexes deposited their product in sub-membrane speckles. To conclude, PI3Kγ acts in an adapter-specific context, and thus adapters deviate PI3Kγ signalling to specific cell responses, possibly by spatial differences of PIP3 production. These findings have an importance in future strategies to target PI3Kγ in tissue-specific manner or with functional specificity.

188

Poster 125

PhosphoSite® update 2007: global analyses of the phosphoproteome Peter Hornbeck, John Rush, Jon Kornhauser, Julie Nardone, Ela Skrzypek, And Bin Zhang Cell Signaling Technology PhosphoSite® (www.phosphosite.org) is an expert-curated bioinformatics resource containing over 16,000 public non-redundant phosphorylation sites on over 5,000 proteins. Over 10,000 of these sites have been curated from the literature. Approximately 6,000 novel phosphorylation sites discovered at Cell Signaling Technology (CST) using PhosphoScan® (Nat. Biotechnol. 2005 23:94) have been recently made public through PhosphoSite. Over 80% of these new sites are tyrosine phosphorylated and have been discovered in specific tumor models. Bioinformatics analyses of the phosphoproteome as represented by PhosphoSite will be presented, including analyses of kinase signature motifs, positional analyses of phosphorylation sites, comparison of the proteomic distribution of tyrosine vs. serine/threonine, and the generation of regulatory phosphorylation cascades from the information aggregated in PhosphoSite.

189

http://www.phosphosite.org

Poster 126

Human DNA polymerase λ is phosphorylated by the cyclin-dependent kinase Cdk2/cyclin A complex Ursula Wimmer, Isabelle Frouin, Elena Ferrari, and Ulrich Hübscher Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Polymerase λ (pol λ) belongs to the X family of DNA polymerases and possesses four different enzymatic activities, being DNA polymerase, deoxyribose phosphate lyase, terminal transferase, and polynucleotide synthetase. Based on its biochemical properties, pol λ has been implicated in base excision repair as well as non-homologous end joining and abasic site translesion synthesis (reviewed in Ramadan, K., Shevelev, I. and Hübscher, U.: Nature Reviews Mol. Cell. Biol., 5, 1038-1043, 2004). However, its in vivo role(s) remain to be elucidated. Using affinity chromatography, we identified cyclin-dependent kinase Cdk2 as a novel interaction partner of human pol λ. Phosphorylation of human pol � by Cdk2/Cyclin A appears to be regulated by its association with proliferating cell nuclear antigen (Frouin, I., Toueille, M., Ferrari, E., Shevelev, I. and Hübscher, U. Nucl. Acids. Res, 33, 5354-5361, 2005). Analysis of single, double, and triple mutants of pol λ for the four conserved putative Cdk phosphorylation sites Ser-167, Ser-177, Ser-230, and Thr-553 revealed only a partial reduction of phosphorylation by Cdk2/Cyclin A in vitro. However, mutation of all four residues reduced phosphorylation significantly, indicating that all four sites are phosphorylated in vitro. Mass spectroscopy analysis of in vitro phosphorylated pol λ showed two phosphorylated residues for the peptide including Ser-167 and Ser-177. Unfortunately, peptides with Ser-230 or Thr-553 could neither be detected in unphosphorylated nor in phosphorylated state. Pol λ phosphorylation was also found in vivo in human cells, and the phosphorylation status was modulated during the cell cycle. Comparison of electrophoretic mobility of pol λ and its phosphorylation mutants in 293T cells revealed a similar increase in mobility for pol λ wild-type upon dephosphorylation, the pol λ quadruple mutant with mutations in Ser-167, Ser-177, Ser-230, Thr-553, and, surprisingly, the Ser-177 single mutant. In contrast, the Ser-167, Ser-230, and Thr-553 single mutants showed no change in mobility compared to phosphorylated pol λ wild-type. Thus, Ser-177 appears to be the crucial site for phosphorylation of pol λ in vivo. To further elucidate the function of pol λ phosphorylation by Cdk2/Cyclin A, a detailed characterization of pol λ wild-type and phosphorylation mutants in vitro and in vivo is under way. This work is supported by the Swiss National Science Foundation.

190

Poster 127

Chemical genetics: functional analysis of the analog sensitive allele of S.pombe p34cdc2 (cdc2-as) and the role of cdc2 in regulating cytokinesis Sandra Dischinger*, Andrea Krapp*, Lifeng Xie§, James R. Paulson§ and Viesturs Simanis* *Cell cycle control laboratory, ISREC, 1066 Epalinges, Switzerland and Faculty of Life Sciences, Ecole Polytechnique Fedérale de Lausanne, Switzerland; §Department of Chemistry, University of Wisconsin-Oshkosh, 800 Algoma Boulevard, Oshkosh, WI 54901 USA We have constructed an analog sensitive allele of the fission yeast cdc2 kinase (cdc2as), which is a key master of the S.pombe cell cycle, to study the role of cdc2 in regulating the Septation Initiation Network (SIN)1. This network functions to regulate initiation of cytokinesis at the end of mitosis after chromosome segregation has been completed and presumably the key promoter of mitosis- the mitotic cyclin dependent kinase (CDK) cdc2p-cdc13p complex –is inactivated. The strategy is to sensitize a protein kinase of interest to a cell-permeable inhibitor molecule that does not inhibit wild-type kinases2. A genetically engineered mutation in the ATP-binding site of the kinase of interest enlarges the ATP binding pocket, so that a chemically synthesised ATP analog can only bind to the ATP-binding site of the engineered protein kinase, but not wild type kinases3. This approach has been chosen to permit reversible, rapid, temperature independent inactivation of cdc2. Our initial analysis indicated that cdc2as is functional and analog sensitive. We have now the ability to inactivate cdc2p in mitotically arrested cells by the addition of the analogue. Currently we are testing the behaviour of the SIN proteins in these circumstances to determine which aspects of SIN function depend upon normal anaphase progression and which are directly regulated by cdc2p. Since delayed inactivation of cdc2 kinase activity in meiosis is known to delay spore formation4 and that the spore formation depends on the SIN5 we are also interested in the function of cdc2 in meiosis. This may be a similar mechanism to the situation in the mitotic cycle, where cdc2p activation may prevent septum formation. 1. Krapp A., Gulli MP. and Simanis V. (2004). SIN and the Art of Splitting the Fission Yeast Cell.

Curr Biol 14: R722-30. 2. Bishop AC., Shah K., Liu Y., Witucki L., Kung C., Shokat KM. (1998). Desing of allele-specific

inhibitors to probe protein kinase signalling. Curr. Biol. 8: 257-266. 3. Bishop AC., Ubersax JA., Petsch, DT., Matheos, DP., Gray, NS., Blethrow, J., Shimizu, E.,

Tsien, JZ., Schultz, PG., Rose, MD. (2000). A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407: 395-401.

4. Blanco MA, Pelloquin L., Moreno S. (2001). Fission yeast mfr1 activates APC and coordinates meiotic nuclear division with sporulation. J Cell Sci. 114 (Pt 11): 2135-43.

5. Krapp A, Collin P, Cokoja A, Dischinger S, Cano E, Simanis V. (2006). The Schizosaccharomyces pombe septation initiation network (SIN) is required for spore formation in meiosis. J Cell Sci. 119: 2882-91.

191

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16787941&query_hl=1&itool=pubmed_docsum

Poster 128

Generation of new powerful and selective inhibitors of protein kinase CK2 by in silico analysis and molecular derivatization of cinnamic and coumarinic scaffolds G. Cozza1, M.A. Pagano1, G. Poletto1, F. Meggio1, S. Moro2, A. Guiotto2, A. Chilin2, G. Zagotto2 and L.A. Pinna1

1Dipartimento di Chimica Biologica and 2Dipartimento di Scienze Farmaceutiche, Università di Padova, Padova, Italy Protein kinase CK2 is a ubiquitous Ser/Thr-specific protein kinase which has been found constitutively active in most organisms and whose overexpression correlates in mammals with abnormal cell proliferation (1). The physiological role of CK2 is still poorly understood although more than 300 physiological substrates of CK2 have been already identified; these are involved in signal transduction, gene expression and development, metabolism, DNA repair and apoptosis (2). It has been suggested that CK2 plays a role as a transducer of survival signals in the cell (3). Given these premises it is not surprising that the search of compounds usable in vivo to block the activity of CK2 recently received a strong impulse. Among the already identified ATP site-directed CK2 inhibitors some anthraquinones, xanthenones and polybrominated benzimidazole and benzotriazole derivatives proved quite selective and their mode of binding was scrutinized by both crystallographic and modeling approaches (4, 5). In particular, it was found that the imidazole ring of the tetrabromobenzimidazole is apparently bridging the brominated benzene moiety through water molecules or ions to polar side chains of the kinase which otherwise are too remote. We have now extended our search by using both in silico virtual screening of selected databases and molecular fragmentation of tetrabromo-benzimidazole molecule. This work led to the identification of two new highly effective CK2 inhibitors among cinnamic acid and coumarins derivatives. In the first case, by opening the imidazole ring and by generating a flexible tether, we found that tetrabromo-cinnamic acid (TBCA) is a 5-fold more efficient inhibitor than TBB without any comparable efficacy on DYRK1A as well as on about thirty other protein kinases (6). Besides the apolar interactions of the polybrominated benzene ring with the two bulky residues Val66 and Ile174 of the kinase, the negative charge of its carboxylic end appears to be in electrostatic contact with the charged Lys68 residue as suggested by the binding of trans but not of cis derivative. In the second case, ellagic acid (EA), a naturally occurring tannic acid derivative, represents today the most potent known CK2 inhibitor with a Ki value of 20 nM (7). From a structural point of view, molecular docking shows that while TBCA accommodates in CK2 nucleotide pocket similarly to TBB, EA penetrates deeper reaching the hinge region of the kinase usually implicated in the binding of the adenine moiety of ATP, through a stabilizing interaction between its hydroxyl group at 3-position and the backbone carbonyls of Glu114. 1. Litchfield, D.W. (2003) Biochem. J. 369, 1-15. 2. Meggio, F. and Pinna, L.A. (2003) FASEB J. 17, 349-368. 3. Ahmed, K. et al. (2002) Trends Cell Biol. 12, 226-230. 4. De Moliner, E. et al. (2003) J. Biol. Chem. 278, 1831-1836. 5. Battistutta, R. et al. (2005) Chem. Biol. 12, 1211-1219. 6. Pagano, M.A. et al. (2006) ChemBioChem., in press. 7. Cozza, G. et al. (2006) J. Med. Chem. 49, 2363-2366.

192

Poster 129

Cell-based approaches to protein kinase assays using GFP-fusion substrates Brian Pollok, Thomas Machleidt, Matthew Robers, Kevin Vedvik, Steve Riddle and Kurt Vogel, Invitrogen Discovery Sciences, Madison Wisconsin USA The difficulties inherent in recreating relevant biological models within the confines of a microtiter plate often limits early discovery assay efforts to simplified systems using purified assay components. Obviously, such models lose the ability to interrogate the target of interest within the complex cellular milieu where it functions. Despite this key shortcoming, biochemical assays are often preferred to their cell-based counterparts due to perceived differences in complexity, time, and cost. We have recently focused efforts on developing time-resolved FRET (TR-FRET) based assays to monitor phosphorylation and ubiquitination of specific target proteins within the native cellular environment. By expressing the protein of interest as a fusion with green fluorescent protein (GFP), modification-specific antibodies labeled with a TR-FRET donor partner (e.g., terbium) can be used to quantitatively monitor these modifications following cell lysis, thereby replacing lower throughput methods such as western blots or ELISAs. We will present data from this technique as applied to analysis of MAP kinase pathways, and of the TNF-α stimulated activation of the NF-κB pathway, in which both phosphorylation and ubiquitination of IκB can be monitored in an HTS-compatible format.

193

Poster 130

The effect of statins on K-Ras localization and cellular proliferation H.Gbelcová1, Z.Knejzlík1, J.Zelenka2, M.Leníček2, L.Vítek2, 3, T.Ruml1

1 Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague Czech Republic,

2 Institute of Clinical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic

3 4th Department of Internal Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic

Statins are specific inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). They are used as hypolipidemics. In recent years these compounds were observed to exhibit also cancerostatic activity. The inhibition of HMG-CoA reductase results in inhibition of Ras protein farnesylation, which regulates cell proliferation and differentiation. Initially, the effects of statins on a proliferation of human pancreatic cancer cell lines with (CAPAN-2 and MiaPaca-2) and without (BxPc-3) activating mutations in K-ras protooncogenes and on survival of nude mice xenotransplanted with CAPAN-2 cells were studied. Then the impact of statins on the localization of K-Ras proteins was investigated. K-ras cDNA was generated from human HeLa cell line using the RT-PCR and inserted into pEGFP-CI vector enabling the expression of corresponding gene product in N-terminal fusion with GFP. Fluorescent microscopy revealed that K-Ras accumulated at a cytoplasmic membrane in different manner in the control cells and its accumulation was affected by statins (except pravastatin). Statins induced a redistribution of K-Ras proteins to the cytoplasm and thus inhibited downstream signaling pathway most likely due to the inhibition of protein farnesylation by HMG-CoA reductase.

194

Poster 131

Growth inhibition of a gefitinib resistant NSCLC cell line with the anti-EGFR monoclonal antibody cetuximab Denis Irmer, Jana Plagge, Christiane Amendt & Andree Blaukat Oncology Research Darmstadt, Global R&D, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany In 2004 remarkable clinical responses in Non-Small Cell Lung Cancer (NSCLC) patients treated with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839/Iressa from Astra Zeneca) were reported to correlate with the presence of certain EGFR kinase domain mutations in tumor tissues. Various mutations at different sites in or close to the EGFR kinase domain have been described so far, small deletions in exon 19 encompassing the LREA motive (~48% of mutations) and point mutations, such as L858R (~38%) being the most abundant ones. Shortly after this finding an additional mutation, EGFR-T790M, was observed in NSCLC patients that reverts gefitinib sensitivity and causes resistance to treatment. Further, a human NSCLC cell line, H1975, which carries T790M in addition to a sensitizing L858R mutation has been shown to be resistant to gefitinib mediated growth inhibition . To study the response of gefitinib resistant H1975 cells towards cetuximab (C225/Erbitux from ImClone Systems/Bristol-Myers Squibb/Merck KGaA), an anti-EGFR directed monoclonal antibody, several in vitro and in vivo studies were conducted. First, the gefitinib resistance of H1975 cells was confirmed in growth and colony formation assays. Unlike gefitinib, cetuximab effectively inhibited H1975 cell growth in a soft agar matrix, but not in a conventional cell monolayer proliferation assay. In contrast, the wild type EGFR expressing NSCLC cell line H292 was effectively inhibited by both drugs. In vivo examinations using xenotransplanted mice that were treated with gefintib or cetuximab reproduced the in vitro observations, i.e. activity of cetuximab in both, H292 and H1975 tumors and resistance of the latter one to gefitinib. In summary, our data show that growth of a gefitinib resistant NSCLC cell line can be effectively inhibited by treatment with cetuximab. Further, colony formation assays seem to be superior in characterizing growth inhibitory effects of cetuximab and predicting its in vivo activity.

195

Poster 132

Identification of MAT2A as a new substrate of the protein tyrosine kinase Pyk2 by a modified KESTREL approach Friederike Krop, Vanessa Jacob, Roland Kellner, Dirk Vocke and Andree Blaukat Oncology Research NCE, GP R&D, Merck KGaA, Frankfurter Str. 250, 63293 Darmstadt, Germany Proline-rich tyrosine kinase 2 (Pyk2), a calcium-dependent non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions we performed a substrate screen using a modified version of KESTREL (kinase substrate tracking and elucidation), which is a combination of protein fractionation, an in vitro kinase assay and tryptic mass fingerprinting. Upon its stimulation Pyk2 undergoes autophosphorylation at tyrosine 402 providing a binding site for the SH2 domain of Src family kinases, leading to full mutual activation of both kinases. Due to this close cooperation of Pyk2 and Src it is difficult to differentiate between substrates of Pyk2 and Src. Therefore a Pyk2 mutant, Pyk2 Y402F, that cannot bind Src but exhibits almost full in vitro kinase activity was included in the substrate screen facilitating the identification of Src independent substrates of Pyk 2. A protein identified by the KESTREL method as direct substrate of Pyk2 was methionine adenosyltransferase II alpha (MAT2A), an essential enzyme that catalyzes the formation of S-adenosylmethionine, the principal biological methyl donor. Through cotransfection experiments in combination with immunoprecipitation and immunoblotting we were able to verify MAT2A as a direct, Src-independent substrate of Pyk2. Currently, we are mapping the Pyk2 phosphorylation site(s) and studying the impact of this phosphorylation on MAT2A function.

196

Poster 133

Generation of biologically active Reg-4 and PAP-1 Fc-fusion proteins that activate kinase cascades in cancer cells Dirk Vocke, Silvia Jezek, Iris Mannberger and Andree Blaukat Oncology Research NCE, GP R&D, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany Reg-4 and PAP-1, secreted proteins and members of the regeneration islet-derived gene family, are upregulated during carcinogenenis of the gastrointenstinal tract. Using cell culture models both proteins have been described as trophic factors stimulating cell growth and also as survival factors reducing sensitivity to apoptosis. Their mitogenic and antiapoptotic effects seem to be mediated by NFkB and Akt, but a specific Reg receptor upstream of the kinase cascades is not known. We have produced highly pure Fc-fusion proteins from supernatants of transfected NS0 cells to study cellular events induced by Reg-4 and PAP-1 and as probes for the identification of specific receptors. Although the Reg proteins may act in an autocrine manner there was no evidence for an increased NFkB phosphorylation in CX-1 colon and AGS gastric cancer cells that both express endogenous Reg-4. However, upon challenge with purified Reg-4 and PAP-1 Fc fusion proteins we have observed a dose-dependent activation of the ERK/MAP kinase cascade in AGS cells. Further, both proteins led to an increase in NFkB phosphorylation in HT-29 colon cancer cells. Currently, we are mapping the signaling pathways triggered by Reg-4 and PAP-1 and use affinity chromatography approaches to identify their receptors. Our initial studies suggest that both Fc fusions are biologically active and Reg-4 may elicit mitogenic effects in an autocrine manner, while it has antiapoptotic effects on cells adjacent to the Reg-4 expressing cells.

197

Poster 134

Analysing the role of a novel cell-cycle-dependent modification of the yeast Chk2 homologue Rad53p Thomas Schleker, Kenji Shimada, Ragna Sack and Susan M. Gasser Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH 4058 Basel, Switzerland Although the checkpoint response to DNA damage occurs at all mitotic cell cycle stages, this response can differ during cell cycle. In S phase cells both a DNA damage sensing signaling pathway and a DNA replication checkpoint exist, and in budding yeast, both pathways lead to the activation of Rad53p as the predominant effector of checkpoint signaling. A S-phase specific threshold for Rad53p activation was observed showing that S phase cells have a higher threshold for damage, such that more signal is needed to activate Rad53p (Shimada et al. Genes Dev. 2002). We asked whether Rad53p itself is modulated in a cell-cycle dependent manner and found that a subfraction of Rad53p is phosphorylated during mitosis in the absence of any checkpoint activation event. This modification led to a slightly lower mobility band in SDS-Page during M and G1, but not S phase. This phosphorylation depends on both, Cdc5p (Polo-like kinase) and Cdc28p (Cyclin-dependent kinase). Cdc5p was previously shown to be important for checkpoint adaptation through inactivation of Rad53p in response to DNA damage treatment. Using the inducible expression of partial Rad53-protein fragments, we mapped the phosphorylation event to the C-terminal part of Rad53p and identified specific phosphorylation sites by mass spectrometry. Our results suggest that the cell cycle dependent phosphorylation of Rad53p at Ser 774 leads to a modulation of Rad53p-activity during the cell cycle.

198

Poster 135

Association of common haplotypes of the p85α subunit of phosphoinositide 3-kinase gene (PIK3R1) with glycemic response to 26 weeks of treatment with an insulin sensitizing PPAR-γ/α agonist in type 2 diabetic patients L. Hansen1, C. T. Ekstrøm1, R. Tabanara Y Palacios1, M. Anant2, K. Wassermann2, R. R. Reinhardt2 Novo Nordisk A/S, Krogshoejvej 53A, 9ES.17, DK-2880 Bagsvaerd, 1 Denmark, 2 USA The regulatory subunit p85� of the enzyme phosphoinistide 3-kinase is a critical component in insulin action. In mice reduced expression levels of p85� increase insulin sensitivity, and in humans variation within the p85α gene (PIK3R1) is associated with insulin resistance and an increased susceptibility for developing type 2 diabetes (T2D). The aim of the present study was to investigate if the PIK3R1 associated with glycemic response to 26 weeks of oral antidiabetic therapy in patients with T2D. 290 T2D patients were randomized, parallel, double-blind to monotherapy with either 1 or 2 mg/day of the dual acting PPAR-γ/α agonist ragaglitazar (n=165) or 2 g/day metformin (n=63) or 20 mg/day glyburide (n=62). At 26 weeks, the mean HbA1c % and fasting plasma glucose (FPG) of the ragaglitazar treatment groups (1 and 2 mg/day) had dropped by approximately 1.1 HbA1c %, and 2 mmol/l, compared to 0.7 HbA1c % and 0.4 mmol/l for glyburide and 0.9 HbA1c % and 0.9 mmol/l for metformin. Haplotypes were generated by genotyping (MALDI-TOF) 7 single nucleotide polymorphisms covering the coding region and the 5’upstream region of the PIK3R1. General linear regression models were run with Box-Cox transformed variables (HbA1c, FPG) of treatment as the endpoints, and with haplotypes h1, h2, h3, and hx as the markers of interest, adjusting for age, sex, treatment group, BMI, ethnicity, diastolic BP, the Met326Ile snp, and the baseline level of the endpoint. Haplotyping classified the 290 T2D patients into 4 common haplotypes: h1, 1111112, 22%; h2, 1112112, 14%; h3, 1112111, 10%; hx, 54%. A general test showed that these haplotypes had effect on FPG (p=0.002) and HbA1c (p=0.031) in patients randomized to ragaglitazar only. In pair-wise comparisons haplotypes h1, h2, h3 dropped 0.55, 0.93 and 0.73 mmol/l less in FPG (p=0.013, 0.0007 and 0.018) whereas h2 and h3 also dropped 0.27 and 0.43 HbA1c % less than the hx reference haplotype (p=0.038 and 0.012). The present study indicates that variation in the PIK3R1 gene may regulate the glycaemic response to antidiabetic PPAR-γ/α agonist therapy in patients with T2D.

199

Poster 136

Quantitative Western blot: step by step analysis F. Heidebrecht1, A. Heidebrecht2 and A. Bader1

1 Cell Technology and Applied Stem Cell Biology , Biomedical-Biotechnological Center, University of Leipzig; 2 DLR Braunschweig An important step in development of most tissue engineered constructs is the controlled replication of the cells in vitro. Although the liver is well known for its high regenerating capacity, in vitro cultured primary hepatocytes divide poorly even when stimulated with growth factors. A mathematical model can deliver a very precise representation of the temporal and spatial dynamics of signalling pathways and thus help to better understand cellular responses. To validate mathematical models, quantitative data of protein activity are necessary. Although the most typical quantitative detection method is ELISA, quantitative WB may also have advantages: the separation of proteins by size allows the evaluation of only specifically bound protein. For a quantitative analysis of Western blots, a correlation between the measurable results and the amount of protein in the examined samples must be established. This correlation is determined by the different processing steps: SDS-PAGE, western blot, primary and secondary antibody incubation, chemiluminescence reaction, image acquirement and processing, quantification of the image (densitometry). Parameters that influence the correlation are: blot transfer conditions, concentration of antibodies and ECL substrate, exposure time, camera sensitivity, colour format and adjustments, band definition, background correction. Given all these possible sources of variability from one experiment to another, a calibration is necessary. All elements in the processing chain were regarded, some were found to have only linear influence on the results, for others a procedure could be found that delivers linear behaviour, leaving only the antibody reactions as non-linear elements. Thus, the type of the calibration curve was derived from a mathematical model of the reaction kinetics. The resulting calibration function was validated with experimental data. A statistical analysis was done to evaluate reproducibility and determine the level of accuracy. A comparison with ELISA results is ongoing work. Using the presented method it is possible to measure relative quantities of proteins. With a reference sample of known protein concentration absolute amounts can be determined, and also absolute phosphorylation levels can be measured. The work presented herein was supported by the Bundesministerium für Bildung und Forschung (BMBF), Projekt Systembiologie; grant number 0313081

200

Poster 137

Coupled mchanism of activation of AGC kinases by linker and hydrophobic motif phosphorylation sites Camilla Hauge, Torben L. Antal, Ulrik Doehn, Kathrine Thorup, Leila Idrissova, Ricardo M. Biondi and Morten Frödin BRIC, University of Copenhagen, Fruebjergvej 3, 2100 Copenhagen, Denmark A significant portion of growth factor/insulin action is executed by a functionally diverse, but structurally related group of protein kinases that belong to the AGC kinase family. This group includes PKB, (also known as AKT), S6K, RSK, MSK, and several members of the PKC family. Collectively, these kinases phosphorylate a large number of cellular proteins and thereby regulate cellular division, growth, survival, metabolism, motility and differentiation. Several of the kinases are implicated in human disease, as e.g. PKB, which has important roles in diabetes and cancer. The activation mechanism of these kinases is complex, and involves phosphorylation of 3 highly conserved S/T residues, all of which are necessary for maximal kinase activity. Of these, only the role of the activation loop site in the kinase domain and the hydrophobic motif site in a linker/tail region are known. We identify the mechanism whereby the third phosphorylation site, also located in the tail, activates these kinases. We term this phosphate the zipper (Z) phosphate, since the phosphate binds a phosphoSer/Thr-binding site in the kinase domain close to the hydrophobic pocket, serving to guide the HM to its binding site in a zipper-like manner and stabilize it there. The Z phosphate thereby synergistically enhances the allosteric activation of the kinase domain by the hydrophobic motif and controls proper phosphorylation of this motif. In S6K and MSK, the Z phosphate-binding site protects the hydrophobic motif from dephosphorylation. In PKB, specific point mutations in the Z phosphate-binding site lead to hyperphosphorylation of the HM and constitutive activation. Intriguingly, the active mutants of PKB showed increased growth promoting activity of NIH 3T3 cells, compared to wild type PKB, indicating that such mutations in the Z phosphate-binding site of PKB might exist in tumours and promote cancer. Sequence conservation suggests that this mechanism is a key feature in activation of up to 26 human growth factor-activated AGC kinases. The Z phosphate-binding site may provide a new target for drugs aimed at activating or inhibiting these kinases.

201

Poster 138

Systems biology modelling of signalling pathways in drug discovery S. Reinker, J. Voshol, K. Mayawala†, C.R. Cho†, J. van Oostrum Department of Systems Biology, Genome and Proteome Sciences, Novartis Institutes for Biomedical Research, Novartis AG, CH-4002 Basel, Switzerland †Cambridge, MA, USA We present a systems biology method using quantitative proteomics data obtained from reverse protein array (RPA) proteomics for pathway and crosstalk analysis1 using mathematical modeling. RPA enables the simultaneous, quantitative measurement of multiple protein read-outs of up to 32 samples of cell or tissue lysates. For systems biology studies, we sample a time course of multiple perturbations. In anticipation of dendritic cell studies, we have validated antibodies for proteins and phosphorylation in the JAK/STAT, mTOR, and NFkB pathways, and developed an initial model-based analysis. The studies will examine single and multiple stimulation protocols in order to identify crosstalk between pathways. We use simulated data to demonstrate the validation of the presented mathematical models of signaling mechanisms. We have also implemented a method for analyzing crosstalk1. These methods are used to select antibody panels for RPA, stratify the most informative stimulation protocols, predict outcomes of new experiments, and suggest possible disease biomarkers and targets for drug interference.

1 M. Natarajan, K.-M. Lin, R.C. Hsueh, P.C. Sternweis, R. Ranganathan, A global analysis of cross-talk in a mammalian cellular signalling network, Nature Cell Biology 8, 571 - 580 (2006).

202

Poster 139

A KESTREL screen for substrates of the TORC2 effector kinase Ypk2 Alexandre Huber and Robbie Loewith Department Of Molecular Biology, University Of Geneva, Switzerland The Ypk1/2 genes encode very similar kinases belonging to the AGC family. Ypk2 was recently shown to be directly phosphorylated, and thereby regulated, by the Target Of Rapamycin Complex 2 (TORC2) which plays a central role in spatial regulation of growth (Kamada et al. 2005). Ypks also receive signals from Pkh1/2, the mammalian phosphoinositide-dependent kinases homologs in yeast. Although Ypks are implicated in different processes including endocytosis, actin polarization during the cell cycle and translation regulation, the only described YPK substrates are the type I myosins which function in endocytosis (Grosshans et al. 2006). Type I myosin mutants that mimic phosphorylation by YPKs do not suppress the phenotypes observed upon loss of Ypk function, suggesting that other Ypk substrates exist. Using a biochemical screen we have identified a 22 kDa protein that was specifically phosphorylated by Ypk2. Mass spectrometric analysis identified this protein as Rps7A. This novel substrate of Ypk2 provides a putative mechanistic link between Ypks and their translational readout.

203

Poster 140

Targeting the EGF-receptor with EGF-derived peptides fused to a multimeric enzyme

Annette Gaida, Illip Burmester and Kristian M. Müller Institut für Biologie III, Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany; E-mail: [email protected] Human Epidermal Growth Factor Receptor (EGF-R) is overexpressed in many tumors providing a validated target for anticancer therapy. To investigate basic principles of tumor targeting for prodrug activation therapy, cysteine free Epidermal Growth Factor (EGF) derived peptides were fused to a multimeric enzyme. Based on the structures of EGF and EGF-R we defined three peptides representing two contact sites of EGF, in a rational approach. Fragment 1 binds to EGF-R domain I. Fragments 2 and 3 bind to EGF-R domain III and were thus linked by three glycines. Cysteines within these fragments were replaced by serines. On the genetic level fragment 1 was fused to the N-terminus and fragments 2/3 to the C-terminus of the trimeric bacterial enzyme Chloramphenicol-Acetyltransferase (CAT), which served as a model scaffold. The resulting construct was named CATfragmentEGF. Trimerization of both peptide fragments bears the potential of increased avidity to high densities of EGF-R presented by many tumor cells (e. g. breast, ovary, prostate). We showed binding of CATfragmentEGF to immobilized EGF-R ectodomain and to A431 cells, a human epidermoid tumor cell line overexpressing EGF-R, by enzyme-linked immunosorbent assay (ELISA). Additionally, binding specificity was demonstrated by competition ELISA with a high affinity anti-EGF-R antibody fragment (scFv-425). On the cellular level binding and specificity of CATfragmentEGF to A431 cells was revealed by confocal microscopy. Wound healing assays showed that CATfragmentEGF is not promoting EGF-R stimulation. Genetically coupling multimeric enzymes with small binding motifs to the EGF-R and taking advantage of increased avidity may provide a powerful approach for tumor targeting. In addition, the generated peptides could serve as lead compounds for EGF-R inhibitors.

204

Poster 141

Identification of an EGF-receptor binding peptide for tumor targeting, based on phage display and random fragmentation Illip Burmester, Annette Gaida, Kristian Müller Institut für Biologie III, Universität Freiburg, Freiburg, Germany We are scrutinising the potential of short peptides as targeting modules for putative application in prodrug activation therapy. To obtain such a peptide the gene of epidermal growth factor receptor (EGF-R) binding scFv 425 was amplified by PCR and randomly fragmented by controlled DNase I digest. Gene fragments of about 30 base pairs were isolated and cloned into the M13 phage genome, resulting in peptides presented as N-terminal fusions with gene III proteins. Phages were panned on a secreted variant of the EGF-R (sEGF-R) and A431 tumour cells expressing EGF-R. Enriched clones contained a consensus sequence mapping to the hypervariable loop 3 of the scFv 425 light chain. Monoclonal phage ELISA demonstrated binding of this consensus sequence to immobilized sEGF-R. Consequently, the identified peptide was genetically fused to the N- and C-termini of the trimeric model enzyme Chloramphenicol Acetyltransferase (CAT), allowing for a 6 fold presentation of the targeting peptide potentially evoking the avidity effect. This EGF-R targeting enzyme demonstrated binding to immobilized sEGF-R in ELISA experiments. Furthermore, we were able to competitively inhibit binding of the targeting module with a scFv 425 construct. In wound healing experiments with A431 cells our fusion protein exhibited lower mitogenic activity than EGF, resulting in a protracted wound closure compared to EGF treated cells. Thus, the generated peptide fused to the model enzyme CAT might serve as a proof of principal for tumour targeted prodrug activation therapy. EGF-R overexpression is frequently observed in tumours and correlates with poor prognosis. Hence, EGF-R has become a validated target for tumour therapy and targeting EGF-R with antibodies has emerged as a major therapeutic approach. Monoclonal antibodies have been approved for therapy. However, discrimination between healthy and tumour cells may be better achieved via avidity rather than high affinity, because many tumour markers are overexpressed at the tumour but are still present at other sites. Accordingly, we are aiming for smaller peptides as targeting modules. Small peptides are more economical in comparison to antibodies or single-chain antibodies and result in manageable size if used as fusion proteins. The generated peptide is a minimal lead compound for tumor targeting. Prodrug activation therapy with enzymes targeted to a tumour marker is a promising approach, because local activation of a prodrug has the potential to provide access to many toxic compounds without adverse systemic effects.

205

Poster 142

Novel 2-acylaminopyridin-4-yl imidazoles: potent and metabolically stable p38 MAP kinase inhibitors Ziegler K, Laufer S Institute of Pharmacy, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany p38 mitogen-activated protein (MAP) Kinase is the key enzyme for the synthesis and release of pro-inflammatory cytokines like IL-1 and TNF α. Inhibition of p38 MAP kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders. Tri- and tetrasubstituted pyridinyl imidazoles are potent inhibitors of the p38 MAP Kinase. Our aim is to develop novel 1,2,4,5-tetrasubstituted imidazole derivates with high anti-inflammatory activity as well as better metabolic stability by preparing a series of substituted 2-acylaminopyridin-4-yl imidazoles. As substitution at this pyridinyl moiety allows both increase and anti-inflammatory activity as well as selectivity. This position should be metabolically stable. Biological datas demonstrate that 2-acylaminopyridin-4-yl imidazoles lead to highly efficient inhibitors. The preparations of 2-acylaminopyridin-4-yl imidazoles are described. Further possible interactions between the different substituents of 2-acylaminopyridines and the backbone region of the enzyme are to be demonstrated by docking experiments. First experiments suggest increased metabolic stability of 2-acylaminopyridines against 2-alkylaminopyridines.

206

Poster 143

Design, synthesis and selectivity profile of a novel structural class of p38 MAP kinase Inhibitors Ahrens G., Karcher S., Nieß R., Hering J., Laufer S. Institute of Pharmacy, University of Tuebingen, Auf der Morgenstelle 8, Germany p38 MAP Kinase is a key enzyme for signal transduction and release of pro-inflammatory cytokines and is therefore a valid and promising drug target. Prototype inhibitors of p38 MAP Kinase like SB 203580 are well investigated lead compounds but they are often suffering from series drawbacks like Cytochrom P 450 interaction and poor selectivity. Even second generation allosteric inhibitors (BIRB 796) are far away from being selective. Starting from LEO type benzophenone leads our design strategy was based by fixing the torsion angle between the two phenyl rings by introduction of condensed ring systems. In order to keep the molecular geometry and the spatial configuration similar to those of the benzophenones, we chose as linker moieties ethano, etheno, methylenoxy and methylsulfanyl. In this study we describe N-substituted 2-aminodibenzosuberones and 3- and 8- substituted 6,11-dihydrodibenzo[b,e]oxepin-11-ones as a novel class of p38 MAP Kinase inhibitors. One major challenge in the development of novel kinase inhibitor candidates is the selectivity profile with respect to other kinases. The compounds were tested for their inhibitory activity against seventeen disease relevant kinases from different families of the human kinome. Exemplified by 2-amino-10,11-dihydrodibenzo[a,d]cyclohepten-5-one this compound is clearly more selective than current inhibitors in clinical trials. Apart from the p38 MAP Kinase only the closely related kinase JNK3 is inhibited.

207

Poster 144

Profile and molecular modeling of 3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione as a highly selective VEGF-R2/3 inhibitor Christian Peifer, Gerd Dannhardt and Stefan Laufer University of Tübingen, Department of Pharmacy, Auf der Morgenstelle 8/B, D-72076 Tübingen, Germany The new combretastatin A-4 analogue 3-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-1H-pyrrole-2,5-dione was discovered to be an inhibitor of tubulin polymerization with an IC50 of 7.6 µM and reduced angiogenesis in the in vivo chick embryo model. Interestingly, in a series of 2,3-diarylmaleimides closely related to this lead, no other compound was found to be active in the tubulin polymerization assay. However, by screening in the in vivo chick embryo assay 3-(Indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (10) was identified as a potent angiogenesis inhibitor indicating an alternative target. Indeed, molecular modeling studies suggest a reasonable binding mode of 10 at the ATP-binding site of the model kinase CDK2. Motivated by these results, analogues of 10 were screened for inhibitory activity in a panel of 12 selected protein kinases and a high affinity of 10 to VEGF-R2 was found showing an IC50 of 2.5 nM. SAR for this compound series with the isolated enzyme and equivalent antiangiogenic activity in the chick embryo assay have been investigated. We report on selectivity profiling of 10 in a panel of 20 protein kinases and molecular modeling indicating 10 to be highly active and selective for VEGF-R2/3. Sequence alignment analysis and detailed insights into the ATP binding pockets of targeted protein kinases from the panel result in a unique structural architecture of VEGF-R2 mainly caused by the hydrophobic pocket I, determining the molecular basis for activity and selectivity of 10.

208

Poster 145

From five to six membered rings: 3,4-diarylchinolinone as lead for novel p38MAP-kinase inhibitors Peifer C, Abadleh M, and Laufer S Institute for Pharmacy, University of Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany In this study we describe design, synthesis and biological evaluation of 5 as a new inhibitor of p38α MAPK with an IC50 of 1.8 μM. By keeping the common vicinal pyridine/4-F-phenyl pharmacophore such as in prototypical inhibitor SB203580 (imidazole) or 13 (isoxazole) but in 5 connected to the six membered quinolin-core, we were particularly interested in comparing biological activity, details of molecular geometry and different binding modes of these compounds. SB203580 and 13 were active both for p38α and the closely related JNK3 whereas 5 was selective for p38α with no JNK3 inhibition. By comparing the X-ray structures of the compounds, we found a significant larger distance between the pyridine and the 4-F-phenyl moiety in five-membered core structures relevant for ligand-protein interactions. Furthermore, molecular modeling studies support the results based on differences in the ATP pockets of p38α and JNK3. Since most five-membered-core based p38α inhibitors show also activity for JNK3, compound 5 is an interesting lead to develop selective p38α inhibitors.

N

F

NH

N

S

CH3

O

F

N

NH

O

IC50μM N

O

N

CH3

CH3

F

5 SB203580 13 p38α 1.8 0,03 0,45 JNK3 - 0,79 0,54

209

Poster 146

Compound B1 leads to recovery from alcohol-induced fatty liver in rats Sanghee Shin1, Keum-Jin Yang1, Longzhen Piao1, Eulsoon Shin3, Li Yuwen1, Miran No1, Kyung Ah Park2, Hee Sun Byun2, Minho Won2, Gangmin Hur2, Jeong Ho Seok2, Jin Man Kim4, Minho Song5 and Jongsun Park1 1Cell Signaling Laboratory, Cancer Research Institute, Dept. of 2Pharmacology, 3Clinical Pathology, 4Pathology and 5Internal Medicine, College of Medicine, Chungnam National University, Taejeon, 310-131, South Korea. Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is deranged. Alcohol has long been thought to cause fatty liver by way of altered NADH/NAD+ redox potential in the liver, which, in turn, inhibits fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. Rats fed ethanol-containing diets for 30days showed remarkable increase in hepatic lipids and lipid droplet accumulation in the hepatocytes, indicating the onset of alcoholic fatty liver. Administration of compound B1 for the 30days of ethanol treatment markedly decreased hepatic lipids to a level lower than control group. Also, compound B1 was able to induce fatty acid oxidizing capacity in the ethanol-fed rats. In contrast, serum-lipids levels were increased with compound B1 Administration. Primary cultured hepatocytes prepared from the fatty liver retained lipid droplets after 8days culture. However, intracellular lipid concentrations were decreased and lipid secretion was enhanced, when primary hepatocyte were cultured in the presence of compound B1 consistent with these events, compound B1 stimulated the rate of protein synthesis of apolipoprotein B100 (apoB100), leading to enhance the subsequent mobilization of lipids. These results indicate that compound B1 administration induced recovery from the fatty liver, at least in part, by enhancing apoB100 synthesis and the subsequent mobilization of lipids from hepatocytes. Therefore, the possibility that compound B1 can be the potential clinical therapeutic value for subjects with an alcohol-related fatty liver warrants further attention. This work was supported by the SRC/ERC Program (Grant R11-2002-100-02006-0) and Basic Research Program grant (R01-2005-000-10240-0, F01-2005-000-10011-0) of MOST/KOSEF

210

Poster 147

Kinase inhibitors as antifungals: targeting Septoria tritici Marc Morgan, Jane Endicott, Martin Noble, Jane Wibley, Andy Corran Laboratory of Molecular Biophysics, Rex Richards Building, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, UK Septoria tritici (Mycospharella graminicola) is the cause of leaf blotch of wheat and affects over fifty million hectares of wheat crops worldwide. Conventional control techniques such as fungicide use and breeding for major gene resistance have not been wholly effective at containing Septoria tritici infections, and therefore a fresh approach to tackle the Septoria tritici plant pathogen is required. Cell cycle kinases have provided much promise as drug targets for a number of diseases, and could prove valuable in the search for new antifungal agents targeting Septoria tritici. In fungi, the essential cell cycle kinase is Cdc28, which governs the signalling of most cell cycle events by association with different cyclin molecules. As yet, there is no structure publicly available for any fungal Cdc28 in protein structure databases. The poster presented here reports work done to model Cdc28 from Septoria tritici by targeted mutagenesis of human CDK2 residues in and around the CDK2 ATP-binding site (generating CDK2-Cdc28 hybrid constructs). With the goal of expanding our knowledge of the Septoria tritici kinome, candidate cyclin partners for Septoria tritici cell cycle kinases have been identified in Septoria tritici genome reads. The CDK2-Cdc28 hybrid constructs with their associated Septoria tritici cyclins will then undergo ATP-competitive inhibitor screening in the hope of finding a potent inhibitor that will block Cdc28 activity, to arrest the Septoria tritici cell cycle and convey fungicidal activity. Structural studies of inhibitors bound to the surrogate Septoria tritici Cdc28:cyclin complexes may also support structure-based design of new fungicides.

211

Poster 148

Mammalian sterile 20-like kinase Mst1 is a multi-functional signal transducing in prostate cancer Martin H. Hager1, Bekir Cinar1, Elena Oancea2, Dolores DiVizio1 and Michael R. Freeman*1

1Depts of Urol, Surg and Biol Chem and Molec Pharmacol, 2Dept of Cardiol, Children’s Hospital Boston, Harvard Med School, Boston, MA The serine/threonine kinase Mst1, a homolog of the yeast Ste 20 kinase and a member of the group II GC kinase subfamily, is a caspase target and an amplifier of apoptotic signals. Mst1 has not been previously studied in the context of prostate cancer. The 56 kDa mature Mst1 protein is primarily located in the cytoplasm and, during apoptosis, is cleaved by a caspase-dependent mechanism into a N-terminal catalytic fragment (Mst1-N) and a C-terminal regulatory fragment (Mst1-C). Release of the 36 kDa Mst1-C domain, which performs a kinase inhibitor function in the whole protein, allows translocation of Mst1-N from the cytoplasm to the nucleus. In the nucleus, kinase-active Mst1-N phosphorylates histone H2B on serine 14 and causes chromatin condensation, DNA fragmentation and cell death. Consequently, Mst1 represents a mediator of apoptotic signals from the cytoplasm into the nucleus. Immunostaining analyses of human prostate cancer tissues in our laboratory indicate that Mst1 is aberrantly expressed in human prostate cancer tissues and that levels of the Mst1 protein increase with progression from the benign state to cancer. Biochemical and imaging experiments also show that Mst1 localizes to cholesterol-rich, lipid raft membranes in prostate cancer cells, which serve as membrane platforms for signal transduction. Furthermore, we found that expression of Mst1-C alone results in translocation of this fragment to the plasma membrane where it interacts with signal transduction proteins. These findings suggest that Mst1 plays a role in cell signaling and that the caspase-cleaved Mst1-C species exerts a novel function beyond its role as an inhibitor of kinase activity in the full-length form of the protein. To further analyze the fate of the mature protein and the caspase-cleaved fragments, we constructed GFP-tagged forms of Mst1 and are examining the response of the protein to apoptotic inducers in prostate cancer cells and other cell backgrounds.

212

Poster 149

The increased metastatic potential of human pancreatic cancer cell lines is associated with the expression of constitutively active KRAS Stephan Gysin, Byron Hann and Martin McMahon UCSF Comprehensive Cancer Center, San Francisco, USA Pancreatic cancer cells are characterized by a large occurrence of genomic instability and a high propensity to metastasize. KRAS is predominatly mutated in pancreatic cancer leading to the constitutive activation of various RAS effector pathways. Our focus is on the role of KRAS in the metastatic process of pancreatic cancer as well as its contribution to cell cycle regulation. We have been making use of an orthotopic mouse model. The human pancreatic cancer cell line COLO357 served as a basis for this model. The sequential injection of this cell line into the spleen or pancreas of athymic nude mice resulted in the derivation of cell lines with a continuously increased metastatic potential. We could show by using array based comparative genomic hybridization and sequencing that this increase in the metastatic potential was accompanied by the acquisition of focal amplifications in the KRAS locus on chromosome 12 and of oncogenic KRAS mutations. Our hypothesis is that oncogenic KRAS in the cell lines with increased metastatic potential is at least partially responsible for that phenotype. We attempt to verify our hypothesis by expressing constitutively activated RAS forms in the cell lines with low metastatic potential or by knocking down oncogenic KRAS using short hairpin RNAs in the cell lines with increased metastatic potential and by injecting these cell lines orthotopically into nude mice. Furthermore, we analyze the role of the RAS regulated RAF-MEK-ERK MAP kinase pathway in our pancreatic cancer cell lines by using small molecule inhibitors targeting MEK. Our studies show that inhibition of MEK blocks the outgrowth of primary tumors in our orthotopic mouse model. Finally, we could identify a number of genes differentially expressed after MEK inhibition that could be downstream mediators of the MAP kinase pathway using Affymetrix gene chip analysis.

213

Poster 150

PASKIN: link between energy metabolism and protein translation in mammals? Juliane Tröger, Katrin Eckhardt, Emanuela Borter, Patrick Spielmann, Gieri Camenisch and Roland H. Wenger Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland The PAS domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in β-cells of the pancreas. We previously reported that PASKIN is highly expressed in the testis and that PASKIN-deficient mice are normally viable and fertile. A role for glucose-stimulated insulin expression could not be confirmed so far. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN by mammalian two-hybrid assays and by in vitro interaction assays. Kinase assays revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation. However, general protein synthesis was not affected by PASKIN overexpression. With the help of newly generated monoclonal antibodies, PASKIN was localized in the spermatogonia of human testis. A speckled nuclear pattern was also observed for endogenous PASKIN in HeLa cells in addition to the cytoplasmic localization. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins showed a remarkably similar localization in the midpiece of the sperm tail, suggesting a translation-independent function of PASKIN-dependent eEF1A1 phosphorylation in sperm cells such as F-actin organization. By peptide and protein array screening, additional phosphorylation targets of PASKIN were identified. Among others, we could confirm the phosphorylation of mammalian glycogen synthase. The identification of these novel PASKIN phosphorylation targets will help to elucidate the physiologic function of PASKIN.

214

Poster 151

Haematopoietic progenitor cells utilise conventional PKC to suppress PKB/Akt activity in response to c-Kit stimulation Charlotte E. Edling1, Malin Pedersen4, Leif Carlsson2, Lars Rönnstrand4, Ruth H. Palmer3 and Bengt Hallberg1* 1Department of Medical Biosciences/Pathology, and 2Umeå Center for Molecular Medicine, Build. 6M, Umeå University, S-901 87 Umeå, Sweden. 3Umeå Center for Molecular Pathogenesis, Umeå University, S-901 87 Umeå, Sweden. 4Experimental Clinical Chemistry, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden Receptor tyrosine kinase (RTK) c-Kit signalling is crucial for the proliferation, survival and differentiation of haematopoietic stem cells (HSCs). To further understand the mechanisms underlying these events we have explored how the downstream mediators interact. In the present study we investigate the function of conventional protein kinase Cs (cPKC) in c-Kit mediated signalling pathways in HSC-like cell lines. This analysis supports earlier findings, that steel factor (SF) activates cPKC, extracellular signal-regulated kinase (Erk) and protein kinase B (PKB). The present results were consistent with an important role of cPKC in the positive activation of Erk and for proliferation. Further, we observe that cPKC negatively regulates PKB activity upon SF stimulation, indicating that cPKC acts as a suppressor of c-Kit signalling. Finally, these observations were extended to show that cPKC mediates the phosphorylation of the endogenous c-Kit receptor on serine 746, resulting in decreased overall tyrosine phosphorylation of c-Kit upon SF stimulation. Furthermore, we here report that this specific feedback mechanism of cPKC mediated phosphorylation of the c-Kit receptor has consequences for both proliferation and survival of HSC like cell lines.

215

Poster 152

Quantitative analysis of receptor tyrosine-kinase signaling exploited by Listeria monocytogenes Tobias Reinl3; Joseph Wissing3; Roman Fischer3; Claudia Hundertmark3; Frank Klawonn2; Henrik Daub1; Jürgen Wehland3; Lothar Jänsch 3

1Max-Planck Institute of Biochemistry, Martinsried, Germany; 2UAS Braunschweig/Wolfenbüttel, Wolfenbüttel, Germany; 3 Helmholtz Centre for Infection Research, Braunschweig, Germany Listeria monocytogenes, a food borne mammalian pathogen, exploits the c-Met-receptor tyrosine kinase pathway to induce its uptake into non-phagocytic cells. The listerial virulence factor Internalin B (InlB), a cell wall-attached Leucin-rich-repeat-containing protein, is sufficient to activate c-Met after binding and to promote a massive actin filament rearrangement with the ultimate effect of internalization. In contrast, the endogenous c-Met ligand, the Hepatocyte Growth Factor HGF, induces proliferation and differentiation in a paracrine fashion. Maintenance and tight control of this pathway is crucial to avoid development of metastasis. Signaling cascades are generally regulated by the activity state of transmembrane receptors and downstream signal molecules like GTPases, phosphatases and kinases, which act in concert to translate the input signal into a well-defined and unique cell response. Noteworthy, phosphoproteomics already allows the analysis of the phosphorylation state of abundant cellular components. However, the systematic characterization of substrates does not reveal directly the activity state of the responsible kinases and phosphatases and additionally requires sophisticated strategies for the reverse engineering of signaling networks. To define the potency of InlB to exploit host cell signaling, we (i) lysed InlB treated cell lines, (ii) purified systematically human kinases by applying highly unspecific kinase inhibitors for affinity chromatography and (iii) characterized their phosphorylation sites based on immobilized metal affinity chromatography (IMAC) and LC-MS/MS. To achieve relative quantification we developed a new bioinformatic algorithm for the evaluation of iTRAQ™-labeled peptides that facilitates the detection of unambiguously regulated phosphorylation events. To our knowledge, this study for the first time presents systematic quantitative data about phosphorylation sites at a significant portion of the human kinome to decipher InlB-dependent receptor tyrosine kinase signaling. First results will be presented and discussed.

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Poster 153

Activation by drugs of the PKCζ-LKB-AMPK signaling cascade is necessary for transcriptional regulation of cytochromes P450 Sharon M. Blättler, Michel R. Kaufmann, Franck Rencurel and Urs A. Meyer Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel Inside the liver cells sophisticated mechanisms have evolved over millions of years to metabolize toxic substances, many of which are fat-soluble compounds making them difficult for the body to excrete. Cytochromes P450 (CYPs) are drug-metabolizing enzymes predominantly expressed in the liver that catalyze the first step in the metabolism of lipophilic substances into water-soluble and thus excretable compounds. Transcriptional activation of CYPs and other drug-metabolizing enzymes by drugs and xenobiotics in the liver is mediated by nuclear receptors such as the constitutive androstane receptor (CAR) in mammalian hepatocytes. This phenomenon, called drug induction, is fully reversible and dose-dependent. By increasing the capability for metabolic detoxification, induction of CYPs is an integral part of the defense mechanism against xenochemical insult. However, it also has severe negative clinical consequences, such as altered pharmacokinetics of drugs and carcinogens, drug-drug interactions, and changes in the metabolism of steroids, vitamin D, and other endogenous compounds. For these reasons, it is of great importance to understand the molecular mechanisms leading to drug induced gene expression of hepatic drug-metabolizing enzymes. In this study, the role of the AMP-activated protein kinase (AMPK) in the drug-mediated transcriptional regulation of CYPs was studied in hepatoma cells and in primary cultures of hepatocytes. We show that AMPK activation by the prototypical inducer phenobarbital (PB) occurs via enhanced reactive oxygen species (ROS) formation in mitochondria and that increased AMPK activity is necessary for drug induction. Furthermore, we demonstrate that PB affects the AMPK upstream kinase LKB1 and the protein kinase C zeta (PKCζ) activity. Importantly, these studies reveal that the signaling cascade involved in drug responses is conserved in evolution from birds to mammals. In conclusion, these findings provide new insights into the so far poorly characterized molecular mechanism, by which drugs lead to transcriptional regulation of CYP enzymes.

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