Protein Kinase Signaling Pathways

1. Jim Woodgett Protein Kinase Signaling Pathways • Cellular Functions • Involvement in Cancer • Targets for Therapeutics?

2. Signal Transduction and Disease • Mammalian MAPK pathways • Mitogenic and stress responses • Phosphatidylinositol 3’ kinase pathway • Importance of suppression of apoptosis in cancer • Potential drug targets and complexities of feedback regulation • Pathways collaborate and integrate

3. Spaghetti Junction

4. RAS RAF MEK ERK

5. Phospho-specific antibodies to MEK and ERK - allow in situ assessment of activation New therapeutics? Selective inhibitors to MEK (Parke-Davis) Inhibitors to Braf for melanoma (Hoffman- La Roche) Bcr-Abl/c-Kit inhibitors - STI571

6. P P P P MAPK signaling PI3’ kinase signaling ErbB3 ErbB2 PIP2 PIP3 PI3K P P PDK1 P SHC P P P P Grb2 P Crk PTEN PKB Ras SOS P P RAF Bad P P MEK mitogenic signal GSK3 P P Afx ERK FKHR TCF Fos FasL p27Kip1

9. Differential activation of MAPKs

10. Scaffold Proteins (JIPs) irradiation ? ER

11. Micro-organization of signaling Signaling pathways often have common components To provide insulation and specificity, as well as tuning specific protein components to a particular signal, a subset of clustering proteins act to scaffold components A caveat of this microcosmic organization is that measuring the entire signaling activity of a cell simply aggregates what may be selective and very local responses

12. PI3’Kinase pathway and cancer • Activation of the pathway in a variety of human cancers (inc. breast, ovary, prostate) • amplification of AKT2/PKBβ and PI3’K • PTEN tumor suppressor acts to repress the pathway • Multiple components are mutated in cancer • Diagnostic/therapeutic target?

13. Nature Reviews Drug Discovery4, 988-1004

14. PI3’ kinase signaling MAPK signaling ErbB3 ErbB2 PIP2 PIP3 P PI3K P P PDK1 P P SHC P P P P P Grb2 P P Crk PKB Ras SOS P PTEN P RAF Bad anti-apoptotic signal GSK3 MEK Afx ERK FKHR Cyclin D FasL p27Kip1

15. Akt1/ Akt2/ Akt3/ Activatory phosphorylation sites

16. Activation of PKB/Akt P P PDK1 PDK2 T308 S473 PKB/Akt Both phosphorylation events required for PKB/Akt activation

17. Identity of PDK2 (1) PKB/Akt P P PKB inactivates the RhebGTPase activating protein TSC1/2 This allows Rheb to be in an active state TSC2 TSC1 RHEB-GTP RHEB-GDP GTP GDP

18. Rheb does what? RHEB-GTP Rheb activates mTOR to induce activation of protein synthesis via 4EBP1 and p70 S6 kinase P P mTORC1 Raptor mTOR Rapamycin p70 S6 kinase 4EBP1

19. A second form of mTOR(C2) P mTORC2 mTORC2 is PDK2 Rictor mTOR S473 PKB/Akt

20. PI3K PIP3 PIP2 PTEN Rictor mTORC2 PDK1 P P PKB/Akt P TSC2 TSC1 All together… RHEB-GTP RHEB-GDP Raptor mTORC1 P P p70 S6 kinase 4EBP1

21. PI3K PIP3 PIP2 PTEN Rictor mTORC2 PDK1 P P PKB/Akt P TSC2 TSC1 All mutated in cancer… RHEB-GTP RHEB-GDP Raptor mTORC1 P P p70 S6 kinase 4EBP1

22. PKB/AKT (direct) targets Several classes of substrates: Apoptosis: Bcl-xL, Bad, XIAP, Mst1, caspase 9 Metabolism: PRAS40, PFKFB2, GSK-3, mTOR, IRS1, eNOS Transcription: Tal1, YB1, PGC1, Nur77, p300, GATA1/2, Foxo proteins, CBP Cell cycle: p21Waf1, Myt1, CDK2, Cdc25b

23. PI3K activation in mouse models • CD2 - directed expression of activated PKBα in T cells • MMTV - directed expression of activated PKBα in mammary gland • Complementation of PI3K-defective polyoma middle T mutant

24. PI3K signaling in cancer Y Y P-PKB PKB Stroma Stroma Tumor Tumor PKB is activated in large fraction of breast cancers Vuk Stambolic

25. Transgenic expression of PKB in T cells B6/PKB5-4 B6/PKB4-1 B6/PKB4-1 B6/PKB5-4 B6/PKB4-1 B6/PKB5-4 B6 B6 B6 kDa 85 anti-PKB 49 anti-P-S473 PKB 85 49 anti-p85 PI3K (control) 85 Thymus Lymph Node T Cells

26. Activated PKB protects thymocytes against death fas ligand dexamethasone g radiation 1 mM 100 ng/ml % Viability Time (hours) PKB Wt B6 Viable Cells B6/PKB5-4 anti Bcl-XL B6/PKB4-1 anti Bcl-2 Time (days)

27. 10 8 6 % apoptotic cells 4 2 0 FVB PKB Reduced apoptosis in PKB tg mammary glands FVB control MMTV-PKB TUNEL staining PKB reduces apoptotic potential of cells

28. PI3’ kinase signaling ErbB3 ErbB2 PIP2 PIP3 P PI3K P P PDK1 P P SHC P P P P P Grb2 P P Crk PKB Ras SOS P PTEN P RAF Bad MEK GSK3 Afx ERK FKHR Cyclin D FasL p27Kip1

29. Polyoma Middle T signaling Middle T antigen Tyr 315/322 PIP2 PIP3 M T PI3K P P PDK1 S R C P P P P P Grb2 PKB Ras SOS SHC P PTEN P P RAF Bad MEK GSK3 Afx ERK FKHR Cyclin D FasL p27Kip1

30. Polyoma Middle T antigen • MMTV-PyMT mice develop multifocal mammary tumors within 6-8 weeks • Mutation of MT at tyrosines 315/322 blocks binding to PI3K. • associated with delay in tumors, cystic hyperplasia • Crossed MT315/322 mutant mice with PKB mice to assess complementation

31. Tumorigenesis in bitransgenic mice PKB MT315/322 PKB + MT315/322 Whole mount Section

32. 1.6 1.2 0.8 0.4 0 MT FVB PKB MT+PKB Progression of tumorigenesis MT315/322F (line 1) MT315/322F (line 1) MT315/322F+ PKB % apoptotic cells % tumor-free animals Days

33. PI3K, PKB and mammary tumorigenesis • Activation of PKB retards involution but does not grossly perturb mammary development • insufficient for tumorigenesis, per se • PKB activation does provide a supportive environment for tumorigenesis by reducing cell death: • allows tolerance of genomic instability? • keeps damaged cells alive? • increases numbers of ‘high risk” cells? • PKB therapeutics? • promising target but selectivity is important (T cell transgenics display auto-immune problems)

34. Summary • Signaling pathways: • Control many cellular processes/responses • Are often dysregulated in human disease • Are highly connected and partially redundant • Are communication lines. The consequences of their activation depends on the agonist and cell type • Due to their regulatory nature, components of signaling pathways are potent therapeutic targets

35. More reading • MAPKs • Science (2002) 298, 1911-1912 • Curr Opin Genet Dev (2002) 12, 14-21 • PI3K signalling • Curr. Opin. Genet. Dev. (2010) 20, 87-90 • J Clin. Oncol. (2010) 28, 1075-1083 • Oncotarget (2011) 2, 435-494