Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome

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Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome. Fbio M. Marques Madeira Supervisor: Professor Carol MacKintosh. 1 th February 2013. 14-3-3s dock onto pairs of tandem phosphoSer / Thr. P. P. Kinase 1. 14-3-3. Kinase 2. - PowerPoint PPT Presentation

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Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome

Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinomeFbio M. Marques MadeiraSupervisor: Professor Carol MacKintosh1th February 2013Map the 2R-Ohn protein families of the human kinome that bind to 14-3-3s!!s114-3-3s dock onto pairs of tandem phosphoSer/Thr

PPKinase 1Kinase 2Hundreds of structurally and functionally diverse targets14-3-3113-3-3 proteins are dimers that dock onto specific pairs of phosphorylated sites usually in tandem on the same target. Therefore integrating 2 kinase signalling inputs and exerting a functional change in the target. There are hundreds of diverse targets that bind 14-3-3s.

hundreds2The human 14-3-3 interactome is highly enriched in 2R-ohnologues2R-ohnologues

Invertebrate chordates

Mammals1R-WGD2R-WGDSelection/Loss2A remarkable observation is that the human 14-3-3 interactome is highly enriched in 2R-ohnologues. => 2R-ohnologs are proteins in families of two to four members. And these families were generated by 2 rounds of whole genome duplication at the origin of the vertebrates. So this means that invertebrate chordates animals (namely Brachiostoma and Ciona) only have one protein for each family of 2-4 in humans. Actually, most 2R-ohnologues were lost early after duplications, but several hundred of these families still exist in humans. 3

2R-Ohnologues and the lynchpin phosphositesPP

PP3Lynchpin siteLynchpin siteWe even think that 14-3-3s helped these families to evolve. Suppose that you have a 14-3-3 binding protein which is duplicated. Then if one lynchpin site stays the same, that could give enough regulation for the second site to evolve4

2R-Ohnologues and the lynchpin phosphositesEvolving site(different kinase)PP

PPLynchpin siteLynchpin site314-3-3-binding motif: RXX(pS)XPConserved across family members back to the single pro-orthologue in invertebrate chordates (Branchiostoma and Ciona) and perhaps become a site that is phosphorylated by a different kinase. => These lynchpins can be identified as phosphosites that are conserved across members of the protein family back to the invertebrate chordates. Another import point is that 14-3-3s have strong preferences for certain motifs immediately surrounding the phosphosites. For example, the motif containing a -3 arginine and a +2 proline.5Aims Develop a web resource on the 14-3-3 interactome and a predictor of 14-3-3-binding phosphosites

Use the resource to map experimental/candidate 14-3-3-binding 2R-ohnologue protein families of the human kinome

Biochemically validate high priority candidate 14-3-3 binders4Kinases create 14-3-3 binding sites, and Kinases can also be 14-3-3-binding phosphoproteins.6Why?Huge amount of dispersed data on the 14-3-3 interactomeThe gold standard 14-3-3 binders (>200)High-throughput (HT) 14-3-3 capture experiments (thousands of candidate 14-3-3 binders)No reported resource to store, analyse and display this complex information

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome5ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactomePredictionsDatabase2R-ohnologue human kinase familiesThe gold standard 14-3-3 bindersHT 14-3-3 capture experimentsHT contaminants (in-house)Maps for mouse and rat homologous proteinsUniProt, GO terms and GAD

ConservationPhosphorylationPrediction PSSMPrediction NNDisorderIntracellular8Homepage of ANIA

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome7Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome7Detailed view of each protein queried

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome7Tabular view of results

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome7Detailed analysis of candidate 14-3-3-binding phosphosites

ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome7

82R-ohnologue families of the Human KinomeTotalGDFamilies14220Members35523So this an image of the human kinome that is widely recognised. So notice that many of the branches end with 2 to 4 proteins. each of these are 2R-ohnologue families. 14

TotalLynchpinsTPFamilies201210Members2315138Lynchpin sites for ~65% of the gold-standard 14-3-3 binders87% true-positives2R-ohnologue families of the Human Kinome87% true-positive lynchpin sites 15TotalLynchpinsFamilies14257Members355158Lynchpin sites for ~45% members of the human kinomePAK482R-ohnologue families of the Human Kinome

p21-activated protein kinase 4 (PAK4)PAKs comprise 2R-ohnologue families composed of 2 groups (group I: PAK1-3, and group II: PAK4-6)PAK 4 is a Ser/Thr kinase activated by Rho-family GTPases Cdc42 and Rac, regulators of actin cytoskeleton dynamics

Why?All members are 14-3-3-binding candidates PAK4 was identified in an in-house HT 14-3-3 capture exp. and in several published HT experiments

9and involved in cell migration, morphology, proliferation, and survival

17Candidate 14-3-3-binding phosphosites of PAK4

Ser99Ser162Ser181Ser474...............10With the exception of S47418phosphoSer181 of PAK4 participates in the binding to 14-3-3

S99/162/181/474A14-3-3 Overlay-GFPGFP pull-downsGFP-PAK4S99AS162AS181AS474AS162/181AGFP-PFKFB2S466/483A

14-3-3

GFP-PAK4S99AS162AS181AS474ACalyculin ASecond site that is phosphorylatedDecreased binding11Phorbol esters regulate the phosphorylation of PAK4BI-D1870 + PMASerum StarvedIGF1PI-103 + IGF1EGFPMAForskolinH-89 + ForskolinA769662A23187Calyculin AGFP pull-downsCell lysatespT202/204 ERK1/2pS157 VASPpS473 PKBpT172 AMPK

14-3-3 Overlay-GFP14-3-3

14-3-3-GFPAbnormal patterns of phosphorylationPanel of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases An outcome of PAK4 overexpression12These abnormal pattern of phosphorylation indicates a complex interplay between signalling networks, which might be an outcome of PAK4 overexpresssion.20Phorbol esters regulate the phosphorylation of PAK4BI-D1870 + PMASerum StarvedIGF1PI-103 + IGF1EGFPMAForskolinH-89 + ForskolinA769662A23187Calyculin AGFP pull-downs

14-3-3 Overlay-GFP14-3-3Response to phorbol ester stimulationPanel of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases 12Panel of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

21Signalling signatures of PAK4PKC, PKD or p90RSK

13

?S181Kinase specificities of PAK4 (AGC and CAMK; NetPhorest)Phorbol esters activate PKC/PKD which ultimately activates p90RSKInterplay between Rho GTPases and 14-3-3s in the regulation of cytoskeleton regulation, coordinated by PKD and PKC (through cofilin and Cdc42/Rac)

Therefore, it is possible that in PMA-stimulated cells, one 14-3-3 binding site is phosphorylated by p90RSK and the other by a PKC or PKD. PKD regulates cofilin activity through PAK4 (48). In addition, downstream effects of Cdc42 and Rac1 on cell polarity, migration and morphology, involve coordinated signalling by PKC. Herein, we hypothesize that PKC/PKD and p90RSK might each phosphorylate one of two 14-3-3-binding phosphosites of PAK4, which points the way to discovering a new regulatory mechanism for PAK4 in control of cell morphology.

22ConclusionsWe developed a user friendly web resource for the annotation and prediction of the 14-3-3 interactomeOur projections indicate that 14-3-3s may dock onto ~45% of 2R-ohnologue human kinase family membersWe validated PAK4 as a novel 14-3-3-binding target, and pinpointed phosphoSer181 as one of the lynchpin sitesWe identified phorbol ester as a stimulus that promotes phosphorylation-dependent binding of 14-3-3 to PAK414Future workSite-directed mutagenesis of S181A double mutants and loss of Calyculin A-/PMA-stimulated 14-3-3 bindingStimuli/inhibitor experiments to investigate different patterns of signalling signatures of PAK4In vivo phosphorylation (using SILAC) of endogenous PAK4Further investigate the effects of 14-3-3 binding on PAK4Extend studies to all the human 2R-ohnologue families15Extend studies to PAK5 and PAK6Investigate the effects of 14-3-3 binding on PAK4 activityIn vitro dephosphorylation of GFP-PAK4In vivo phosphorylation (using SILAC) of endogenous PAK4

24AcknowledgementsProfessor Carol MacKintoshDr Michele Tinti (Bioinformatics)Dr Gerta Hoxhaj and Dr Catherine Johnson (Laboratory)All members in Carols groupMRC PPU and DSST (tissue culture, cloning and sequencing)

14-3-3s

Family of regulatory proteins found in all eukaryotesMammals have 7 isoforms ubiquitously expressed in all tissuesSmall proteins (~30 kDa) that form homo- or heterodimersPresenting an highly helical structure

Human kinome

Set of proteins kinases in the human genomeExtensively studiedPK create 14-3-3-binding phosphosites and are also 14-3-3 binders

Human kinome

Why?Discover new 14-3-3-based kinase cascades and mechanisms of signalling cross talkUnderstand 14-3-3-based regulatory interplay between members of 2R-ohnologue familiesExpertise and resources for biochemical validation of candidate 14-3-3 binders

Manageable size; cDNA clones for many human kinases

28PredictionSearchPredictorAlignmentWeb InterfaceIntracellularDisorderPrediction NNPrediction PSSMPhosphorylationDatabaseFeatureConservationUser InputOutputDiagram of the Predictor integrated in ANIAPhorbol esters regulate the phosphorylation of PAK4BI-D1870 + PMASerum StarvedIGF1PI-103 + IGF1EGFPMAForskolinH-89 + ForskolinA769662A23187Calyculin AGFP pull-downs

14-3-3 Overlay-GFP14-3-3

Cell lysates by Dr Gerta Hoxhaj-GFPpS473 PKBpS79 ACCpS157 VASPpT202/204 ERK1/2Response to phorbol ester stimulationPanel of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases 30

Olsson O.pS473 PKB ** pS157 VASPpT172 AMPKpS79 ACC * pT202/204 ERK1/2Signalling pathways activated/inhibited in the panel exp.PKA inhibits AMPK, and CaMKII activates AMPK.31

Sluchanko, N. N., and Gusev, N. B. (2010) 14-3-3 Proteins and regulation of cytoskeleton. Biochemistry (Moscow) 75, 15281546.PK CPAK4 and 14-3-3s are involved in the regulation of cytoskeleton

Koh, W., Mahan, R. D., and Davis, G. E. (2008) Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling. Journal of cell science 121, 9891001.

Johnson, C., Tinti, M., Wood, N. T., Campbell, D. G., Toth, R., Dubois, F., Geraghty, K. M., Wong, B. H. C., Brown, L. J., Tyler, J., Gernez, A., Chen, S., Synowsky, S., and MacKintosh, C. (2011) Visualization and biochemical analyses of the emerging mammalian 14-3-3-phosphoproteome. Molecular & cellular proteomics 10, M110.005751.Mode I: RSX(pS)XPMode II: RXXX(pS)XP Basic residues in positions -3 to -5 and never a +1 P34