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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 kinome. Fábio 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.

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

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  1. Mapping the 14-3-3-binding 2R-ohnologue protein families of the human kinome Fábio M. Marques Madeira Supervisor: Professor Carol MacKintosh 1th February 2013

  2. 14-3-3s dock onto pairs of tandem phosphoSer/Thr P P Kinase 1 14-3-3 Kinase 2 Hundreds of structurally and functionally diverse targets 1

  3. The human 14-3-3 interactome is highly enriched in 2R-ohnologues 1R-WGD 2R-WGD Selection/Loss 2R-ohnologues Invertebrate chordates Mammals 2

  4. 2R-Ohnologues and the ‘lynchpin’ phosphosites P P P P Lynchpin site Lynchpin site 3

  5. 2R-Ohnologues and the ‘lynchpin’ phosphosites P P P P Lynchpin site Lynchpin site Evolving site (different kinase) • Conserved across family members back to the single pro-orthologue in invertebrate chordates (Branchiostomaand Ciona) • 14-3-3-binding motif: RXX(pS)XP 3

  6. Aims • 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 binders 4

  7. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Why? • Huge amount of dispersed data on the 14-3-3 interactome • The 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 5

  8. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome • 2R-ohnologue human kinase families • The gold standard 14-3-3 binders • HT 14-3-3 capture experiments • HT contaminants (in-house) • Maps for mouse and rat homologous proteins • UniProt, GO terms and GAD Database Predictions • Conservation • Phosphorylation • Prediction PSSM • Prediction NN • Disorder • Intracellular

  9. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Homepage of ANIA 7

  10. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Tabular view of results 7

  11. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Detailed view of each protein queried 7

  12. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Tabular view of results 7

  13. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome Detailed analysis of candidate 14-3-3-binding phosphosites 7

  14. 2R-ohnologue families of the Human Kinome 8

  15. 2R-ohnologue families of the Human Kinome Lynchpin sites for ~65% of the gold-standard 14-3-3 binders 87% true-positives 8

  16. 2R-ohnologue families of the Human Kinome Lynchpin sites for ~45% members of the human kinome PAK4 8

  17. 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 9

  18. Candidate 14-3-3-binding phosphosites of PAK4 Ser99 Ser162 Ser181 Ser474 ... ... ... ... ... 10

  19. phosphoSer181 of PAK4 participates in the binding to 14-3-3 Calyculin A S99/162/181/474A S162/181A GFP-PFKFB2 S162A S466/483A S181A S99A S474A S162A S181A S99A S474A GFP-PAK4 GFP-PAK4 14-3-3 Overlay GFP pull-downs 14-3-3 α-GFP Second site that is phosphorylated Decreased binding 11

  20. Phorbol esters regulate the phosphorylation of PAK4 ‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases H-89 + Forskolin BI-D1870 + PMA PI-103 + IGF1 Serum Starved A769662 A23187 Forskolin EGF PMA IGF1 Calyculin A 14-3-3 Overlay GFP pull-downs 14-3-3 α-GFP pT202/204 ERK1/2 Abnormal patterns of phosphorylation pS157 VASP pS473 PKB Cell lysates An outcome of PAK4 overexpression pT172 AMPK 14-3-3 α-GFP 12

  21. Phorbol esters regulate the phosphorylation of PAK4 ‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases H-89 + Forskolin BI-D1870 + PMA PI-103 + IGF1 Serum Starved A769662 A23187 Forskolin EGF PMA IGF1 Calyculin A 14-3-3 Overlay GFP pull-downs 14-3-3 α-GFP Response to phorbol ester stimulation 12

  22. ‘Signalling signatures’ of PAK4 ? S181 PKC, PKD or p90RSK 13

  23. Conclusions • We developed a user friendly web resource for the annotation and prediction of the 14-3-3 interactome • Our projections indicate that 14-3-3s may dock onto ~45% of 2R-ohnologue human kinase family members • We validated PAK4 as a novel 14-3-3-binding target, and pinpointed phosphoSer181 as one of the lynchpin sites • We identified phorbol ester as a stimulus that promotes phosphorylation-dependent binding of 14-3-3 to PAK4 14

  24. Future work • Site-directed mutagenesis of S181A double mutants and loss of Calyculin A-/PMA-stimulated 14-3-3 binding • Stimuli/inhibitor experiments to investigate different patterns of ‘signalling signatures’ of PAK4 • In vivo phosphorylation (using SILAC) of endogenous PAK4 • Further investigate the effects of 14-3-3 binding on PAK4 • Extend studies to all the human 2R-ohnologue families 15

  25. Acknowledgements • Professor Carol MacKintosh • Dr Michele Tinti (Bioinformatics) • Dr GertaHoxhaj and Dr Catherine Johnson (Laboratory) • All members in Carol’s group • MRC PPU and DSST (tissue culture, cloning and sequencing)

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