1. 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

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

3. The human 14-3-3 interactome is highly enriched in 2R-ohnologues 2R-ohnologues Invertebrate chordates Mammals 1R-WGD2R-WGD Selection/Loss 2

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

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

6. Aims 1. Develop a web resource on the 14-3-3 interactome and a predictor of 14-3-3-binding phosphosites 2.Use the resource to map experimental/candidate 14-3-3-binding 2R- ohnologue protein families of the human kinome 3.Biochemically validate high priority candidate 14-3-3 binders 4

7. Why? 1.Huge amount of dispersed data on the 14-3-3 interactome 2.The gold standard 14-3-3 binders (>200) 3.High-throughput (HT) 14-3-3 capture experiments (thousands of candidate 14-3-3 binders) 4.No reported resource to store, analyse and display this complex information ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome 5

8. Predictions Database  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  Conservation  Phosphorylation  Prediction PSSM  Prediction NN  Disorder  Intracellular

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

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

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

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

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

14. 8 2R-ohnologue families of the Human Kinome TotalGD Families14220 Members35523

15. TotalLynchpinsTP Families201210 Members231513 8 Lynchpin sites for ~65% of the gold-standard 14-3-3 binders 87% true-positives 2R-ohnologue families of the Human Kinome

16. TotalLynchpins Families14257 Members355158 Lynchpin sites for ~45% members of the human kinome PAK4 8 2R-ohnologue families of the Human Kinome

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? 1.All members are 14-3-3-binding candidates 2.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 Ser99Ser162Ser181Ser474... 10

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

20. Phorbol esters regulate the phosphorylation of PAK4 BI-D1870 + PMA Serum Starved IGF1 PI-103 + IGF1 EGF PMA Forskolin H-89 + Forskolin A769662A23187 Calyculin A GFP pull-downs Cell lysates pT202/204 ERK1/2 pS157 VASP pS473 PKB pT172 AMPK 14-3-3 Overlay α-GFP 14-3-3 α-GFP Abnormal patterns of phosphorylation ‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases An outcome of PAK4 overexpression 12

21. Phorbol esters regulate the phosphorylation of PAK4 BI-D1870 + PMA Serum Starved IGF1 PI-103 + IGF1 EGF PMA Forskolin H-89 + Forskolin A769662A23187 Calyculin A GFP pull-downs 14-3-3 Overlay α-GFP 14-3-3 Response to phorbol ester stimulation ‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases 12

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

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 1.Site-directed mutagenesis of S181A double mutants and loss of Calyculin A-/PMA-stimulated 14-3-3 binding 2.Stimuli/inhibitor experiments to investigate different patterns of ‘signalling signatures’ of PAK4 3.In vivo phosphorylation (using SILAC) of endogenous PAK4 4.Further investigate the effects of 14-3-3 binding on PAK4 5.Extend studies to all the human 2R-ohnologue families 15

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

26. 14-3-3s  Family of regulatory proteins found in all eukaryotes  Mammals have 7 isoforms ubiquitously expressed in all tissues  Small proteins (~30 kDa) that form homo- or heterodimers  Presenting an highly helical structure

27. Human kinome  Set of proteins kinases in the human genome  Extensively studied  PK create 14-3-3-binding phosphosites and are also 14-3-3 binders

28. Human kinome Why? 1.Discover new 14-3-3-based kinase cascades and mechanisms of signalling ‘cross talk’ 2.Understand 14-3-3-based regulatory interplay between members of 2R- ohnologue families 3.Expertise and resources for biochemical validation of candidate 14-3-3 binders

29. Prediction Search Predictor Alignment Web Interface Intracellular Disorder Prediction NN Prediction PSSM Phosphorylation Database Feature Conservation User InputOutput Diagram of the Predictor integrated in ANIA

30. Phorbol esters regulate the phosphorylation of PAK4 BI-D1870 + PMA Serum Starved IGF1 PI-103 + IGF1 EGF PMA Forskolin H-89 + Forskolin A769662A23187 Calyculin A GFP pull-downs 14-3-3 Overlay α-GFP 14-3-3 Cell lysates by Dr Gerta Hoxhaj α-GFP pS473 PKB pS79 ACC pS157 VASP pT202/204 ERK1/2 Response to phorbol ester stimulation ‘Panel’ of stimulli/inhibitors that activate or inhibit AGC and CAMK kinases

31. Olsson O. pS473 PKB * * pS157 VASP pT172 AMPK pS79 ACC * pT202/204 ERK1/2 Signalling pathways activated/inhibited in the ‘panel’ exp.

32. Sluchanko, N. N., and Gusev, N. B. (2010) 14-3-3 Proteins and regulation of cytoskeleton. Biochemistry (Moscow) 75, 1528–1546. PK C PAK4 and 14-3-3s are involved in the regulation of cytoskeleton

33. 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, 989–1001.

34. 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)XP Mode II: RXXX(pS)XP