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Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechani

Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk (PI3-K) Signaling Scaffold (JNK). Harmatomas. TSC1/TSC2. mTOR.

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Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechani

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  1. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk (PI3-K) Signaling Scaffold (JNK)

  2. Harmatomas TSC1/TSC2 mTOR

  3. Evolutionary Conservation of the MAPK Signaling pathway lin-3 let-23 mammalian C. Elegans sem-5 let-341 let-60 lin-45 mek-2 Chardin et. al. (1993) Science 260: 1338 mpk-1 VanAelst et. al. (1993) PNAS 90: 6213 Han et. al. (1993) Nature 363: 133 Warne et. al. (1993) Nature 364: 352 Vojtek et. al. (1993) Cell 74: 205

  4. pp42 MAP Kinase/MAPK/ErK1/2 Myelin Basic Protein Kinase Microtubule Associated Protein 2 Kinase Mitogen-activated Protein Kinase Rossomando et. al. (1989) PNAS 86: 6940

  5. Raf activates MAPK Kinase (MAPKK) MAPKK Kyriakis et. al. (1992) Nature 358: 417

  6. Activation of MAPK by Oncogenic Ras Ctr Ras-transformed Ctr Ras-transformed Kinase (Total) Kinase (MAPK) Level (MAPK) Leevers & Marshall (1992) EMBO 11: 569

  7. Ras Interacts with Raf Warne et. al. (1993) Nature 364: 352

  8. Mammalian MAP Kinase Cascades Johnson & Lapadat (2002) Science 298: 1911

  9. MAPK Activation Mechanisms/ Concepts of Kinase Cascades S/T-Pro Ras Pathway Perturbation Raf Activation loop MEK MEK MEK TEY MEK MEK MEK ERK ERK ERK MEK/MKK ERK ERK ERK Pathway Amplification Zhang et. al. (1995) Structure 3: 299

  10. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold

  11. Ras Mutations display Tumor Specificity PancreaticCarcinoma K-Ras codon 12 (GGTgly) >GTTval Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser BladderCarcinoma H-Ras codon 12 (GGCgly) >GTCval Melanoma N-Ras codon 61 (CAAgln)>CGAarg

  12. RAS GTP Cycle Ras-( ) GEF/SOS Off-state On-state Ras-GDP Ras-GTP Raf GAP Ras-GDP+Pi High affinity for GDP and GTP (ii) Low intrinsic dissociation constant (iii) Low intrinsic GTPase activity

  13. Functional Domains of Ras

  14. Ras-GDP (Inactive) Pai et. al. (1989) Nature 341: 209 Pai et. al. (1990) EMBO J 9: 2351 Haubruck & McCormick (1991) Biochemica et Biophysica Acta 1072: 215

  15. Ras-GTP (Active) Guanine base Gly12 a b H20 g Gln61 (Raf, PI3K, RalGDS) Pai et. al. (1989) Nature 341: 209 Pai et. al. (1990) EMBO J 9: 2351 Haubruck & McCormick (1991) Biochemica et Biophysica Acta 1072: 215

  16. Oncogenic Ras Guanine base Val12 a b H20 g Leu61 Pai et. al. (1989) Nature 341: 209 Pai et. al. (1990) EMBO J 9: 2351 Haubruck & McCormick (1991) Biochemica et Biophysica Acta 1072: 215

  17. Functional Domains of Ras Exchange factor, SOS Proline-rich DH PH CDC25 Dbl-Homology Catalytic Domain SH3-Binding Membrane- targeting Rho GTPases (Rac)

  18. How SOS activates Ras? Boriack-Sjodin et al., (1998) Nature 394: 337 Wittinghofer (1998) Nature 394: 317

  19. Stimulation of Ras intrinsic GTPase activity by GAP Scheffzek et al., (1996) Nature 384: 591

  20. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold

  21. Ras-induced Transformed phenotypes Ras Ctr Ctr Ras actin Tumor induction (nu/nu) actin depolymerization Apoptosis in IL-3 Deprived 32D Shape Growth Survival Ctr Ras NIH3T3 +H-Ras R12 Onset of senescence (MEFs) Loss of contact inhibition Anchorage-independent Growth Morphology Aging Adhesion Osada et. al. (1999) Mol. Cell. Biol. 19: 6333 Zhang et. al. (1995) Structure 3: 299 Serrano et. al. (1997) Cell 88: 593

  22. Generation of Ras Effector-binding site mutants Random mutagenesis White et. Al., (1995) Cell 80: 533

  23. Ras-Effector Binding Domain (aa32-40) Mutants 12 S35 G37 C40 Residue Substrate- binding Raf RalGDS/JNKPI3-K Biological property DNA Rho-like MembraneSynthesis Foci Ruffling Full Transformation

  24. Ras has multiple downstream substrates Vojtek & Der (1998) J. Biol. Chem 273: 19925

  25. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold

  26. Multi-step activation of Raf

  27. B-Raf mutations in human cancer E B-Raf 586DFGLATVKSRWSGSHQFEQ c-Raf 486 --------------Q-V-- Basal Kinase Activity T491 S494 S338 Y340 1 647 c-Raf + * * * * pS445 D448 T598 S601 1 765 +++ B-Raf * * * Davies et. al., (2002) Nature 417: 949

  28. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold

  29. H-Ras:Raf-RBD Affinity Pull-down Assay R-Ras3 WT in vivo in the lab Ras.GTP bFGF none PMA NGF EGF Ctr WB: a-R-Ras3 R-Ras3 (Pull-down) a-R-Ras3 R-Ras3 (Input) in vitro Ras.GDP Ras.GTP Kimmelman et. al. (2002) Mol. Cell. Biol. 22: 5946

  30. H-Ras/c-Raf FRET Probe YFP H-Ras Raf-RBD CFP CAAX NH2- -COOH

  31. Mochizuki et al., (2001) Nature 411: 1065

  32. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold

  33. Cell fate determination in PC12 cells growth Differentiation Kimmelman et. al. (2002) Mol. Cell. Biol. 22: 5946 Wu et. al. (2001) J. Neuroscience 21: 5406

  34. Cell fate determination by the MAPK Pathway (transient verse sustained activation) Neuronal Differentiation Proliferation York et. al. (1998) Nature 392: 622 Marshall (1998) Nature 392: 553

  35. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk (PI3-K and MAPK) Signaling Scaffold

  36. Activation of the PI3-K pathway Ras > Brazil et. al. (2002) Cell 111: 293

  37. PI3-K pathway and Cancer Syndromes RTK GF Cancer Syndromes PIP3 p PI3-K Lipid Kinase GI, Br, Ov PTEN Lipid PTPase Cowden’s, Multiple Akt1/2 Ser/Thr Kinase PANC Hamartin Tuberin (Tuberous Sclerosis Complex) TSC2 Ras GAP TSC TSC1 (Ras-homology enriched in brain) Small GTPase RheB (Target of rapamycin) mTOR Kinase S6K 4EBP-1 Kinase Inhibitor of eIF4E Protein synthesis Kovich & Cohen (2004) Dematology Online Journal 10: 3. Cell growth/size/survival Perelman (2004) Dematology Online Journal 10: 17.

  38. Pathway Crosstalk MAPK and PI3-K control active Raf +IGF-1 d.n. Raf active Akt +PD98059 >MEK +PD98059+IGF-1 differentiation growth Rommel et. al. (1999) Science 286: 1738

  39. Advanced Signaling Class 2005 (Ras-MAPK) Ras-Raf-MEKK-MAPK Pathway Ras and its Regulators Substrate specificity Mechanisms of activation/B-Raf in cancer Spatial-temporal Regulation Signaling Kinetics Pathway Cross-talk Signaling Scaffold (JNK)

  40. JNK Pathways

  41. Whitmarsh et. al. (1998) Science 281: 1671

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