DOI: 10.1002/cbic.200900594

FRET-Based Activity Biosensors to ProbeCompartmentalized SignalingXinxin Gao[b] and Jin Zhang*[a, b]

Introduction

The ability of a cell to properly respond to environmentalchanges is important for cell growth and survival. Appropriatecellular responses are mediated through exquisitely organizedregulatory networks, which consist of highly dynamic signalingmolecules. For the purpose of achieving signaling specificityand efficiency, it is crucial that activities of these signaling mol-ecules are spatially compartmentalized within a cell. For exam-ple, the intracellular second messenger, cyclic AMP (cAMP),[1] isknown for its ability to modulate a wide variety of fundamen-tal cellular processes, including metabolic, electrical, cytoskele-tal, and transcriptional responses.[2, 3] To account for specificregulation of these diverse processes, subcellular compartmen-tation of cAMP signaling was suggested more than 20 yearsago.[4] As shown in cardiomyocytes, the binding of two extra-cellular ligands, prostaglandin E1 (PGE1) and isoproterenol, todifferent G protein-coupled receptors (GPCR) results in similarlevels of cellular cAMP accumulation but distinct physiologicaloutcomes. Isoproterenol stimulation enhances contractile activ-ity through activation of particulate or membrane-boundcAMP-dependent protein kinase (PKA), whereas PGE1 stimula-tion causes no changes in contractile activity, correlated withcAMP elevation and PKA activation in the soluble fraction ofheart homogenates.[4, 5] Recent studies[6] have provided newevidence for compartmentalized cAMP signaling at differentlevels of the signaling cascade. At the level of cAMP, compart-mentalized phosphodiesterases (PDE) restrict cAMP accumula-tion within domains in correspondence with the transversetubule region to limit the activation to a specific population ofPKA.[7] At the PKA level, anchored by A-kinase anchoring pro-teins (AKAP),[8] different pools of PKA lie in proximity to distinctsubstrates to modulate their phosphorylation. Therefore, it ap-pears that cAMP accurately mediates various cellular processesthrough a signaling network in which the action of each sig-naling molecule is tightly controlled in defined subcellularcompartments.

cAMP compartmentation is not an exception to kinase- andsecond messenger-mediated signal transduction. It hasbecome increasingly clear that spatial compartmentalization isa general theme in signal transduction and plays pivotal rolesin ensuring specific signal processing by various signalingpathways, such as CaII,[9] phosphoinositide,[10] mitogen-activat-ed protein kinase (MAPK),[11] and Rho GTPase pathways.[12]

Given the dynamic nature of signaling molecules as well as thecritical involvement of cellular parameters and constraints informing various signaling compartments, a better understand-

ing of compartmentalized signal transduction requires meth-ods for tracking activity dynamics of signaling molecules withhigh spatiotemporal resolution in the native cellular environ-ment. To meet this challenge, a series of genetically encodedfluorescence resonance energy transfer (FRET)-based activitybiosensors have been developed for tracking dynamics of vari-ous signaling molecules, such as GTPases,[13] protein kinases,[14]

second messengers,[14] and membrane receptors.[15] OtherFRET-based approaches, including visualization of enzyme–sub-strate interactions by utilizing fluorescent protein-taggedenzyme and fluorophore-labeled substrate[16] have also beenapplied to study signaling compartmentation. In this shortreview, we discuss applications of genetically encoded fluores-cent biosensors with a focus on understanding compartmen-talized signaling of kinase and second-messenger dynamics.For general reviews regarding the development and applica-tion of these biosensors, please refer to recent review arti-cles.[15, 17–21]

Subcellular Targeting of Biosensors TrackingKinase and Second-Messenger ActivityDynamics

Kinases and second messengers mediate diverse cellular func-tions through subcellular compartmentation. As such, geneti-cally encodable FRET-based biosensors provide powerful toolsfor understanding the molecular mechanisms underlying sig-naling specificity and diversity of these important molecules. Aseries of biosensors have been developed by utilizing “molecu-lar switches”, a protein domain or chimera that senses the sig-naling activity of interest and changes conformation, sand-wiched between a FRET pair.[22–26] The conformational changeof the molecular switch, induced by kinase phosphorylation orsecond-messenger binding, can result in a change in FRET. Onekey feature of these genetically encodable biosensors is their

[a] Prof. J. ZhangThe Solomon H. Snyder Department of Neuroscience,Department of OncologyThe Johns Hopkins University School of MedicineBaltimore, MD 21205 (USA)Fax: (+ 1) 410-955-3023E-mail : jzhang32@bs.jhmi.edu

[b] Dr. X. Gao, Prof. J. ZhangDepartment of Pharmacology and Molecular SciencesThe Johns Hopkins University School of MedicineBaltimore, MD 21205 (USA)

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targetability. Genetically targeting the biosensors to specificsubcellular locations through various targeting motifs enablesus to capture signaling dynamics and detect the subtle differ-ences in kinetics or activity patterns in these signaling com-partments (Figure 1). Compartmentalized signaling has beenstudied at several different levels. Biosensors targeted to cellu-lar regions such as the plasma membrane, cytosol and nucleushave been used to reveal signaling dynamics in these regions(Figure 1, level I).[27–30] Signaling activities have also been visual-ized at finer levels of cellular regions, such as plasma mem-brane microdomains (Figure 1, level II), with the help of fluo-rescent biosensors specifically targeted to these microdo-mains.[31–33] Furthermore, at the cellular nanomachinery level(Figure 1, level III), dynamics within signaling complexes havebeen studied by using fluorescent biosensors with motifs thatbind to proteins of interest.[34, 35]

Signaling Dynamics in Cellular Regions

Spatial organization of signaling molecules in different subcel-lular compartments is critical for achieving efficient and specif-ic signal transduction responsible for desired cellular functions.For example, in migrating cells, signaling molecules are recruit-ed to various regions of the cells to regulate critical steps ofcell migration. It is well known that phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3), the product of phosphoinosi-tide 3-kinase (PI3K),[36] accumulates locally at the leading edgeof a migrating cell.[37] The elevated PI(3,4,5)P3 level in this cellu-lar region is necessary for new actin polymerization and pseu-dopodia extension at the front.[38, 39] A recent study has shownthat the activity of PKA is also locally present in migratingcells.[40] To visualize PKA activity in migrating cells, a plasmamembrane-anchored biosensor for monitoring PKA activity,

pmAKAR (plasma membrane A-kinase activity report-er), was utilized. The membrane targeting restrictedthe diffusion of AKAR, thereby enhancing its abilityto detect localized PKA activity. Imaging by usingpmAKAR revealed high PKA activity at the leadingedge of migrating cells, concurrent with the forma-tion of membrane protrusions (Figure 1). Blocking in-tegrins with function-blocking antibodies abolishedenrichment of PKA activity at the leading edge aswell as cell migration; this suggests that integrinsmediate PKA activation at the leading edge of mi-grating cells.[30] Interestingly, PI3K inhibition hinderedcell migration, but did not block polarized PKA acti-vation; thus, this integrin-mediated PKA activation atthe leading edge is independent of PI3K. Inhibitionof PKA, on the other hand, reduced accumulation ofa sensor for PI(3,4,5)P3, comprised of a GFP-labeledpleckstrin homology (PH) domain of Akt that bindsto 3’-phosphoinositides, to the leading edge of mi-grating cells ; this suggests that PKA might controlPI(3,4,5)P3 level at the cell front.[30] Along the samelines, a recent study has revealed that PKA promotesmembrane ruffling by modulating PI(3,4,5)P3 dynam-ics at the leading edge during PDGF induced chemo-taxis.[41] These findings point the way to future stud-ies investigating the functional roles as well as themolecular mechanisms of the dynamic interactionsamidst various signaling pathways during cell migra-tion.

Kinase Activity Dynamics within thePlasma Membrane Microdomains

The plasma membrane mediates physiological pro-cesses through a mosaic of functional microdomains.The spatial organization of signaling molecules inthese discrete microdomains is key to achieving spe-cific interactions between individual components.[42]

Among these microdomains, the cholesterol-rich, de-tergent-resistant microdomains, lipid rafts, have re-ceived much attention in recent years, due to their

Figure 1. Compartmentalized signaling activity at different levels. At the cellular regionlevel, imaging by using pmAKAR revealed high PKA activity at the leading edge of mi-grating cells. At finer levels of cellular regions, such as plasma membrane microdomains,kinase activity dynamics has been visualized with the help of biosensors targeted tothese microdomains. At the cellular nanomachinery level, dynamics within signaling com-plexes, such as cAMP/PKA signaling in PKA isoform specific compartments, has also beenstudied using fluorescent biosensors. PKA I and PKA II are anchored to different subcellu-lar compartments via AKAPs. In these isoform specific compartments, distinct cAMPpools are generated through activation of various GPCRs and restricted by different sub-sets of PDEs. Accumulation of cAMP in PKA II compartment leads to activation of this iso-form, resulting in phosphorylation of a specific subset of PKA substrates (such as phos-pholamban and troponin), which remain unaffected upon PKA I activation.

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roles as platforms for many signaling events.[42, 43] However, themolecular mechanism underlying lipid raft-specific regulationis still not well understood, partly due to the dynamic natureof lipid rafts and their small size, which is below the classicaldiffraction limit. Powerful fluorescence microscopy techniquessuch as FRET, which have high spatial resolution, providedunique answers to this challenge. FRET can be used to detectmolecular interactions at the nanoscale level because FREToccurs only when two molecules (the donor and acceptor) arein proximity (that is, < 10 nm). Fluorescence correlation spec-troscopy (FCS) quantifies fluorescence intensity fluctuation inthe focal volume of a confocal or multiphoton microscope andhas also been applied to study nanoscale membrane organiza-tions in living cells, with some success.[44, 45] More recently, fluo-rescent biosensors equipped with membrane microdomain-tar-geting motifs have emerged as novel tools to study raft-associ-ated signaling dynamics with high spatial resolution.[32, 33] Herewe illustrate the application of such biosensors with two exam-ples.

Differential Akt Regulation within the Plasma MembraneMicrodomains

Identified as an oncogene in 1991, the serine/threonine kinaseAkt[46, 47] appears to be a central player in a variety of cellularresponses, including cell survival, growth, motility, and metab-olism. The activation of Akt involves its recruitment to theplasma membrane through 3’-phosphoinositides and phos-phorylation in the activation loop and the hydrophobicmotif.[48] It has been shown that a constitutively active form ofAkt, myristoylated Akt, is associated with the plasma mem-brane and highly enriched in lipid rafts.[49, 50] Several compo-nents upstream of Akt signaling have also been reported aspartitioned into the detergent-resistant membrane compart-ments.[51] These observations suggest a possible involvementof lipid rafts in Akt activation.

To study the roles of membrane microdomains in Akt signal-ing, we generated fluorescent Akt activity reporters (AktAR) tospecifically monitor Akt signaling dynamics in two differentplasma membrane microdomains.[32] Activities of raft- and non-raft-associated Akt activity were monitored with Lyn-AktAR (byusing a targeting motif derived from Lyn kinase) and AktAR-KRas (by using a targeting motif derived from K-Ras), respec-tively.[33] We demonstrated Akt activity is turned on more rapid-ly in lipid rafts upon growth factor stimulation. Importantly,differential activation patterns of membrane Akt induced byPDGF or IGF-1 were revealed, with Akt signaling in the nonraftregions dependent upon that in the raft regions with IGF-1stimulation but not PDGF stimulation. Thus, we proposed thatrafts are the exclusive sites for IGF-1-stimulated Akt signaling,while PDGF activates two relatively independent pools of Akt(Figure 2).[32]

This study revealed that lipid rafts play a critical role in regu-lating Akt activities in such a way that the overall Akt activityis raft-dependent. Consistent with these findings, it has beenshown that in small cell lung cancer (SCLC) cells, raft disruptioninhibited activation of Akt.[52] A recent investigation with FCS

also showed membrane microdomain impediment by inhibi-tion of sphingolipid and cholesterol synthesis reduced Aktrecruitment to the membrane in Jurkat cells.[45] Interestingly,studies in several cell lines demonstrated endogenous mem-brane Akt is enriched in the nonraft regions.[50, 52] Therefore, thehigh activity of raft Akt might be due to efficient interactionsamong components within the pathway including the recep-tors, PI3K, phosphoinositide-dependent kinase-1 (PDK1) andAkt rather than the abundance of total Akt in lipid rafts. Futurestudies regarding the roles of lipid rafts in regulating these sig-naling molecules will help us understand the molecular basisof membrane microdomain-mediated Akt activation.

Plasma Membrane Microdomain-Regulated Src KinaseActivity Dynamics

Src kinase regulates cell adhesion, mitosis, and motility byphosphorylating many downstream protein substrates.[53, 54] Ithas been shown that the activation of Src kinase requires itstranslocation to the plasma membrane through actin rear-rangements.[55] However, the roles of plasma membrane micro-domains in mediating Src kinase activation remain unknown.To investigate Src kinase activity dynamics in membrane micro-domains, a Src activity biosensor was targeted to lipid raftsand nonraft regions through Lyn- and KRas-targeting motifs,respectively.[33]A faster and stronger response was observedwith the nonraft Src biosensor than with the raft Src biosensorin response to pervanadate (PVD) stimulation; this suggeststhat Src is activated more potently in the nonraft regions. Fur-

Figure 2. Akt and Src activity dynamics within plasma membrane microdo-mains. Lipid rafts are critical in regulating both PDGF-and IGF-1-stimulatedAkt activation. PDGF stimulates raft and nonraft Akt activation independent-ly, whereas IGF-1-stimulated Akt signaling in the nonraft regions is highlydependent on that in the raft regions. On the other hand, nonraft Src kinaseis activated more quickly upon stimulation of pervanadate, and raft Srckinase is translocated to lipid rafts through actin filaments upon stimulationand becomes activated. Future studies of the interplay between Akt and Srcsignaling pathways in plasma membrane compartments will provide insightsas to how these microdomains coordinate various signaling pathways toachieve specific functions.

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thermore, PVD-induced response of the raft Src biosensor wassignificantly inhibited by actin microfilament disruption, butnot by inhibition of microtubule polymerization. By contrast,inhibition of microtubule polymerization, but not actin fila-ment disruption, caused a significant delay in the response ofthe nonraft Src biosensor upon PVD treatment. A model waspresented to suggest that one pool of Src kinase is prestoredoutside of lipid rafts on the plasma membrane and can be rap-idly activated upon stimulation, and another population of Srckinase is translocated to lipid rafts through actin filamentsupon stimulation and become activated (Figure 2).[33] Given thedistinct roles of lipid rafts in regulating Akt and Src, it is inter-esting to note that Src kinase has been shown to directly asso-ciate with and activate Akt.[56] Previous studies have alsoshown that raft-associated Src is critical for maintaining Akt ac-tivity.[52] However, the spatial regulation of the interaction be-tween the two kinases remains largely unknown. Future stud-ies of the interplay between these two signaling pathways inplasma membrane compartments will likely provide insights asto how these membrane microdomains coordinate various sig-naling pathways to achieve specific functions (Figure 2).

Signaling Dynamics at the Cellular Nano-machinery Level

Signal transduction is mediated by assembly, rearrangement ordisassembly of nanomachine-like signaling complexes in spe-cific cellular compartments.[57] These dynamic nanomachinesensure specific and efficient signal processing and properphysiological functions. A prototypical example is the compart-mentation of PKA signaling by AKAP-assembled signaling com-plexes in various subcellular compartments.[8, 58] Binding to theR subunits of PKA, AKAPs target PKA to various subcellularregions, where specific signaling molecules, such as receptors,other kinases, phosphatases, or distinct PKA substrates, interactwith PKA. For instance, it has been shown that anchoring ofPKA by AKAP is required for the regulation of synaptic func-tion.[59]

With the help of fluorescent biosensors, signaling dynamicsin these signaling complexes have been investigated. For ex-ample, a biosensor incorporating the docking sites for the PKAholoenzyme and PDEs derived from AKAPs allowed local cAMPsignaling events to be unraveled.[34] The transient responsefrom this biosensor, which was tethered to both PKA and PDE,became more sustained in the presence of constitutively activeMAP-kinase kinase (MEK); this indicates that the presence ofMEK inhibited cAMP degradation by locally tethered PDEs. Fur-ther biochemical analysis conveyed that PKA, PDE, extracellularsignal-regulated kinase (ERK) and other cAMP-dependent regu-lators of ERK all resided in an AKAP regulated signaling com-plex.[34] ERK activation results in PDE inhibition, which enhan-ces cAMP generation and PKA activation in local compart-ments. On the other hand, sustained cAMP accumulation canlead to inhibition of ERK signaling, providing local feedback in-teractions. This study demonstrated that the dynamic interac-tion between cAMP and ERK signaling mediated through AKAPfine-tunes signaling dynamics in microcompartments.

Another example of probing signaling dynamics at thenanomachinery level with FRET-based biosensors is the studyof cAMP signaling in PKA isoform specific compartments. Inthis study, a cAMP biosensor was targeted to PKA-RI- and RII-specific compartments by incorporating specific motifs thatbind to different AKAPs.[35] By using these two spatially local-ized biosensors, it was revealed that the PKA-isoform-specificsignaling compartments are defined by specific cAMP signals,which are generated by distinct GPCRs and confined by select-ed subsets of PDEs. The functional outcome of this compart-mentalized cAMP/PKA signaling is that b-adrenergic receptorstimulation causes PKA-RII activation and phosphorylation of aspecific subset of PKA substrates, which remain unaffectedupon PKA-RI activation (Figure 1).[35] This study demonstratedthat cAMP/PKA signaling is spatially regulated at multiplelevels of the signaling cascade, from the receptors, to thesecond messenger, and to effectors.

Outlook

Engendered as powerful tools to study signaling dynamics inlive cells, genetically encoded fluorescent biosensors havebeen applied to probe compartmentalized signaling. Activitydynamics of individual molecules in various compartmentshave been revealed by fluorescent biosensors targeted to cel-lular regions, signaling microdomains or protein complexes.The biological significance of pronounced difference in signal-ing activities at various cellular regions, such as the trailingversus the leading edge of migrating cells, is generally appreci-ated. However, more subtle changes in signaling dynamics,such as the dynamic interplay between signaling moleculeswithin protein complexes, also play critical roles in cellularfunctions. Investigation of such subtlety in compartmentalizedsignaling with fluorescent biosensors will continue to expandour knowledge about the molecular mechanisms underlyingfundamental cellular processes.

Furthermore, the application of genetically targeted biosen-sors can be complemented with biochemical perturbationtechniques, which allow for manipulation of local signaling ac-tivities. One example of such a technique is the chemical dime-rization method which takes advantage of rapamycin-inducedheterodimerization between FK506 binding protein (FKBP) andFKBP12 rapamycin binding (FRB) domain.[60] This geneticallyencodable system can be targeted to various subcellular com-partments, thus enabling acute activation/deactivation of spe-cific signaling molecules in these compartments.[61, 62] On theother hand, rapid signaling activation can be achieved by pho-tolytic release (“uncaging”) of active second messengers (suchas CaII or cAMP) from inert precursors.[63, 64] More recently, sev-eral genetically encoded photoactivatable signaling mole-cules,[65, 66] including photoactivatable Rac1,[65] have been devel-oped. Targeting these photoactivatable signaling molecules todifferent subcellular compartments should enable precise con-trol of signaling dynamics in these locations. These biochemi-cal techniques, when combined with genetically targeted bio-sensors, provide powerful tools for probing the dynamic inter-play between signaling molecules in various compartments.

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In conclusion, with their unique features of genetic encoda-bility and targetability, fluorescent-protein-based biosensorsallow real-time tracking of activity dynamics with high spatio-temporal resolution. Applications of subcellularly targeted bio-sensors have expanded our knowledge of compartmentalizedsignaling. Continuing development of these fluorescent bio-sensors and their applications in living systems should lead toa better understanding of how signaling information is pro-cessed by the cell regulatory networks in a context dependentmanner.

Acknowledgements

We thank Charlene Depry and Vedangi Sample for critical read-ing of the manuscript. Work in the lab was supported by Nation-al Institutes of Health grants (R01 DK-073368 and R21 CA-122673), the Young Clinical Scientist Award Program of the FlightAttendant Medical Research Institute, a Scientist DevelopmentAward from the American Heart Association, and 3M (to J.Z.).

Keywords: biosensors · FRET · kinases · second messenger ·signaling compartmentation

[1] E. W. Sutherland, T. W. Rall, J. Biol. Chem. 1958, 232, 1077.[2] K. V. Chin, W. L. Yang, R. Ravatn, T. Kita, E. Reitman, D. Vettori, M. E.

Cvijic, M. Shin, L. Iacono, Annu. N. Y. Acad. Sci. 2002, 968, 49.[3] R. Kopperud, C. Krakstad, F. Selheim, S. O. Doskeland, FEBS Lett. 2003,

546, 121.[4] S. F. Steinberg, L. L. Brunton, Annu. Rev. Pharmacol. Toxicol. 2001, 41,

751.[5] J. S. Hayes, L. L. Brunton, S. E. Mayer, J. Biol. Chem. 1980, 255, 5113.[6] M. Zaccolo, G. Di Benedetto, V. Lissandron, L. Mancuso, A. Terrin, I. Zam-

paro, Biochem. Soc. Trans. 2006, 34, 495.[7] M. Zaccolo, T. Pozzan, Science 2002, 295, 1711.[8] D. L. Beene, J. D. Scott, Curr. Opin. Cell Biol. 2007, 19, 192.[9] A. J. Laude, A. W. Simpson, FEBS J. 2009, 276, 1800.

[10] M. A. De Matteis, A. Godi, Nat. Cell Biol. 2004, 6, 487.[11] A. Mor, M. R. Philips, Annu. Rev. Immunol. 2006, 24, 771.[12] H. Wu, G. Rossi, P. Brennwald, Trends Cell Biol. 2008, 18, 397.[13] T. Nakamura, K. Aoki, M. Matsuda, Methods 2005, 37, 146.[14] K. J. Herbst, Q. Ni, J. Zhang, IUBMB. Life 2009, 61, 902.[15] M. J. Lohse, M. Bunemann, C. Hoffmann, J. P. Vilardaga, V. O. Nikolaev,

Curr. Opin. Pharmacol. 2007, 7, 547.[16] I. A. Yudushkin, A. Schleifenbaum, A. Kinkhabwala, B. G. Neel, C. Schultz,

P. I. H. Bastiaens, Science 2007, 315, 115.[17] A. Miyawaki, Dev. Cell 2003, 4, 295.[18] S. B. Van Engelenburg, A. E. Palmer, Curr. Opin. Chem. Biol. 2008, 12, 60.[19] T. Balla, Trends Cell Biol. 2009, 19, 575.[20] W. B. Frommer, M. W. Davidson, R. E. Campbell, Chem. Soc. Rev. 2009,

38, 2833.[21] K. Aoki, E. Kiyokawa, T. Nakamura, M. Matsuda, Phil. Trans. R. Soc. B

2008, 363, 2143.[22] A. Y. Ting, K. H. Kain, R. L. Klemke, R. Y. Tsien, Proc. Natl. Acad. Sci. USA

2001, 98, 15003.[23] J. Zhang, Y. Ma, S. S. Taylor, R. Y. Tsien, Proc. Natl. Acad. Sci. USA 2001,

98, 14997.[24] L. M. DiPilato, X. Cheng, J. Zhang, Proc. Natl. Acad. Sci. USA 2004, 101,

16513.[25] V. O. Nikolaev, M. Bunemann, L. Hein, A. Hannawacker, M. J. Lohse, J.

Biol. Chem. 2004, 279, 37215.[26] B. Ponsioen, J. Zhao, J. Riedl, F. Zwartkruis, G. van der Krogt, M. Zaccolo,

W. H. Moolenaar, J. L. Bos, K. Jalink, EMBO Rep. 2004, 5, 1176.[27] L. L. Gallegos, M. T. Kunkel, A. C. Newton, J. Biol. Chem. 2006, 281,

30947.

[28] A. Terrin, G. Di Benedetto, V. Pertegato, Y. F. Cheung, G. Baillie, M. J.Lynch, N. Elvassore, A. Prinz, F. W. Herberg, M. D. Houslay, M. Zaccolo, J.Cell Biol. 2006, 175, 441.

[29] T. Nishioka, K. Aoki, K. Hikake, H. Yoshizaki, E. Kiyokawa, M. Matsuda,Mol. Biol. Cell 2008, 19, 4213.

[30] C. J. Lim, K. H. Kain, E. Tkachenko, L. E. Goldfinger, E. Gutierrez, M. D.Allen, A. Groisman, J. Zhang, M. H. Ginsberg, Mol. Biol. Cell 2008, 19,4930.

[31] L. M. DiPilato, J. Zhang, Mol. Biosyst. 2009, 5, 832.[32] X. Gao, J. Zhang, Mol. Biol. Cell 2008, 19, 4366.[33] J. H. Seong, S. Y. Lu, M. X. Ouyang, H. Huang, J. Zhang, M. C. Frarne, Y. X.

Wang, Chem. Biol. 2009, 16, 48.[34] K. L. Dodge-Kafka, J. Soughayer, G. C. Pare, J. J. Carlisle Michel, L. K. Lan-

geberg, M. S. Kapiloff, J. D. Scott, Nature 2005, 437, 574.[35] G. Di Benedetto, A. Zoccarato, V. Lissandron, A. Terrin, X. Li, M. D. Hous-

lay, G. S. Baillie, M. Zaccolo, Circ. Res. 2008, 103, 836.[36] L. C. Cantley, Science 2002, 296, 1655.[37] R. J. Cain, A. J. Ridley, Biol. Cell 2009, 101, 13.[38] L. F. Chen, C. Janetopoulos, Y. E. Huang, M. Iijima, J. Borleis, P. N. Dev-

reotes, Mol. Biol. Cell 2003, 14, 5028.[39] C. Janetopoulos, P. Devreotes, J. Cell Biol. 2006, 174, 485.[40] A. K. Howe, Biochim. Biophys. Acta Mol. Cell Res. 2004, 1692, 159.[41] P. B. Deming, S. L. Campbell, L. C. Baldor, A. K. Howe, J. Biol. Chem.

2008, 283, 35199.[42] K. Simons, D. Toomre, Nat. Rev. Mol. Cell Biol. 2000, 1, 31.[43] K. Simons, R. Ehehalt, J. Clin. Invest. 2002, 110, 597.[44] P. Sharma, R. Varma, R. C. Sarasij, Ira, K. Gousset, G. Krishnamoorthy, M.

Rao, S. Mayor, Cell 2004, 116, 577.[45] R. Lasserre, X. J. Guo, F. Conchonaud, Y. Hamon, O. Hawchar, A. M. Ber-

nard, S. M. Soudja, P. F. Lenne, H. Rigneault, D. Olive, G. Bismuth, J. A.Nunes, B. Payrastre, D. Marguet, H. T. He, Nat. Chem. Biol. 2008, 4, 538.

[46] A. Bellacosa, J. R. Testa, S. P. Staal, P. N. Tsichlis, Science 1991, 254, 274.[47] P. F. Jones, T. Jakubowicz, F. J. Pitossi, F. Maurer, B. A. Hemmings, Proc.

Natl. Acad. Sci. USA 1991, 88, 4171.[48] D. P. Brazil, Z. Z. Yang, B. A. Hemmings, Trends Biochem. Sci. 2004, 29,

233.[49] M. Aoki, O. Batista, A. Bellacosa, P. Tsichlis, P. K. Vogt, Proc. Natl. Acad.

Sci. USA 1998, 95, 14950.[50] R. M. Adam, N. K. Mukhopadhyay, J. Kim, D. Di Vizio, B. Cinar, K. Boucher,

K. R. Solomon, M. R. Freeman, Cancer Res. 2007, 67, 6238.[51] K. Y. Lee, F. D’Acquisto, M. S. Hayden, J. H. Shim, S. Ghosh, Science 2005,

308, 114.[52] A. Arcaro, M. Aubert, M. E. Espinosa del Hierro, U. K. Khanzada, S. Angel-

idou, T. D. Tetley, A. G. Bittermann, M. C. Frame, M. J. Seckl, Cell. Signal-ling 2007, 19, 1081.

[53] R. Jove, H. Hanafusa, Annu. Rev. Cell Biol. 1987, 3, 31.[54] T. J. Yeatman, Nat. Rev. Cancer 2004, 4, 470.[55] E. Sandilands, C. Cans, V. J. Fincham, V. G. Brunton, H. Mellor, G. C. Pren-

dergast, J. C. Norman, G. Superti-Furga, M. C. Frame, Dev. Cell 2004, 7,855.

[56] T. Y. Jiang, Y. Qiu, J. Biol. Chem. 2003, 278, 15789.[57] R. Peters, Trends Mol. Med. 2006, 12, 83.[58] W. Wong, J. D. Scott, Nat. Rev. Mol. Cell Biol. 2004, 5, 959.[59] M. Colledge, R. A. Dean, G. K. Scott, L. K. Langeberg, R. L. Huganir, J. D.

Scott, Neuron 2000, 27, 107.[60] G. R. Crabtree, S. L. Schreiber, Trends Biochem. Sci. 1996, 21, 418.[61] T. Inoue, W. D. Heo, J. S. Grimley, T. J. Wandless, T. Meyer, Nat. Methods

2005, 2, 415.[62] B. C. Suh, T. Inoue, T. Meyer, B. Hille, Science 2006, 314, 1454.[63] R. H. Kramer, J. J. Chambers, D. Trauner, Nat. Chem. Biol. 2005, 1, 360.[64] G. Mayer. A. Heckel, Angew. Chem. 2006, 118, 5020; Angew. Chem. Int.

Ed. 2006, 45, 4900.[65] Y. I. Wu, D. Frey, O. I. Lungu, A. Jaehrig, I. Schlichting, B. Kuhlman, K. M.

Hahn, Science 2009, 461, 104.[66] A. Levskaya, O. D. Weiner, W. A. Lim, C. A. Voigt, Science 2009, 461, 997.

Received: September 24, 2009

Published online on December 11, 2009

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