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Pathway in early 1992

Pathway in early 1992. RAS. EGFR. WT. sevenless. R7 receptor is required for UV response. R8. Boss. sev. R7. Strategy: moving from RTK down: discovering SOS. RTK. GDP. GTP. RAS GDP. RAS GTP. target protein. Inactive. Active state. GAP. Pi. Leads to Model:. A: yes

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Pathway in early 1992

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  1. Pathway in early 1992 RAS EGFR

  2. WT sevenless R7 receptor is required for UV response R8 Boss sev R7

  3. Strategy: moving from RTK down: discovering SOS RTK GDP GTP RAS GDP RAS GTP target protein Inactive Active state GAP Pi

  4. Leads to Model: A: yes B: may not. R7 fate Sev SOS - + Son of Sevenless: hyperactive mutation Sev/RTK (lf) Sevenless Sev (lf) + SOS(gf) R7 fine Roger and Benejee, Cell 1990 The story about Ron Roger

  5. F1 screen mutagen mutagen F2 screen TM * * X TM X TM * * X F1 F1 * * X * * F3 homozygotes Drosophila: F1 screen vs F2 screen A conversation with Rubin

  6. F1 screen * TM X * F1 Mike Simon : landmark modify screen Simon et al. 1991 Cell R7 Ts allele Sev SOS RAS Rough eye, no R7 Mostly wt, small % defects Rough eye Rough eye High T Low T Low T Low T +/- +/-

  7. SOS encodes an exchange factor for GTPase Simon et al. 1991 Cell RTK SOS GDP GTP RAS GDP RAS GTP target protein Inactive Active state GAP Pi

  8. Moving from receptor down: biochemistry RTK SOS GDP GTP RAS GDP RAS GTP target protein Inactive Active state GAP Pi

  9. Screen lGT11 protein expression library Y-P 9 GRB proteins Discovery of GRB2 Ligand activation Y Y-P EGFR EGFR Which one is one mediated Ras activation?

  10. Discovery of GRB2: complementary works by biochemists and geneticists A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling. Lowenstein et al. Cell. Aug. 1992

  11. Known Since 1990 LIN-15 Vulval induction Ras EGFR LIN-15 SEM-5 Ras SEM-5 is homologous to GRB-2. Combine the data from the two field: GRB2/SEM-5 Ras EGFR The sem-5 story - early 1992 SEM-5 was identified as by a suppressor mutant screen Clark et al. Nature Mar 1992 lin-15 (-)Multivulva sem-5(-) Vulvaless lin-15(-); sem-5(-) Vulvaless ras (gf) Mutivulva sem-5(-) Vulvaless ras (gf); sem-5(-) Multivulva

  12. The rest of the story is there in a rush and clash- mid 1993 Simon MA, Dodson GS, Rubin GM.SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73:169-77. Olivier JP, Raabe T, Henkemeyer M, Dickson B, Mbamalu G, Margolis B, Schlessinger J, Hafen E, Pawson. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos.Cell. 1993 Apr 9;73:179-91. Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA.Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363:45-51. Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D.The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.Nature.1993 May 6;363:83-5. Buday L, Downward J.Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.Cell. 1993 May 7;73:611-20. Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH, Bar-Sagi D.Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260:1338-43. Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J.The function of GRB2 in linking the insulin receptor to Ras signaling pathways.Science. 1993 Jun 25;260:1953-5. Baltensperger K, Kozma LM, Cherniack AD, Klarlund JK, Chawla A, Banerjee U, Czech MP.Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes. Science. 1993 Jun 25;260:1950-2.

  13. Many Small GTPases have a conserved C-terminus

  14. Ras is lipid modified for its membrane binding

  15. SH3 SOS Y-P SH2 RAS GDP RAS GTP SH3 EGFR Y RAS GDP EGFR SH3 SH2 SH3 GRB2 SH3 SOS SH2 SH3

  16. Half way done RTK GRB2 SOS GDP GTP RAS GDP RAS GTP Inactive Active state GAP Pi

  17. GAP GAP-1 Gap1 mutagen lin-15(-); suppressor worm lin-15(-) Vulvaless ; Muvulva , fly mutagen sev(lf); suppressor/enhancer sev(lf) R7 fate reduced ; R7 fate better or worse , binding to phosphorylated tyosine of growth factor: Grb2 Discovery of GAP. biochem Summary of the original screens RTK GRB2 SOS Ras worm LET-23 SEM-5 SOS-1 LET-60 fly Sevenless DRK SOS Ras1

  18. The holy grail: what is the effector of Ras?

  19. The Ras effector 1. Must interact with the Ras effector domain 2. The interaction must be required for Ras function 3. Must be required to interact with activated Ras (oncoproteins). A: Yes, B: no GDP GTP RAS GDP RAS GTP Effector protein Inactive Active state Pi

  20. effector 12 116 164 185 59 The Ras effector domain is defined by 1. Mutations in the region affect Ras biological function 2. The mutations have no effect on other biochem properties 3. The domain is proposed to interact with the effector/target

  21. GAP = Ras effector Nature. April 1988

  22. Science. April 1988 GAP = Ras effector

  23. Key data Position of Ras mutations GAP activation (hydrolysis) Interaction With GAP Ras function 12, 59, 61 abolished + +++ 35, 36, 38 insensitive - - 39 Sensitive + + effector 12 116 GAP is the effector. A: Convincing. B. Somewhat convincing. C. Not convincing. 164 185 59 GAP binding Membrane binding Key argument A. The biochemical and genetic criteria for a Ras effector: 1. It should interact with GTP-bound not GDP-bound Ras. 2. It should not interact with an inactive Ras B.Factors interfering with Ras function block the Ras/GAP interaction

  24. GDP GTP RAS GDP RAS GTP GAP Inactive Active state GAP Pi The field was convinced Cell June 15, 1990

  25. The field was convinced Annu Rev Cell Biol. 1991;7:601-32.

  26. The field was convinced Annu Rev Cell Biol. 1991;7:601-32.

  27. The field was convinced Ann Rev Cell Biol 1991;7:601-32.

  28. GAP stimulates RAS-GTP hydrolysis - expected to be a negative factor Functions GAP also acts as the effector - expected to be: A. positive factor B. negative factor GDP GTP RAS GDP RAS GTP GAP Inactive Active state GAP Pi If you make a knockout of GAP, do you expect the signaling to go A: up. B: down.

  29. Genetics Tanaka, Matumoto, and Toh-E: IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in S. cerevisiae. Mol Cell Biol. 1989 Feb Stanaka, …., Matsumoto, Kaziro, Toh-e: S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. March 1990 Buchberg, Cleveland, Jenkins, Copeland: Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway. Nature. 1990 Sep Xu, …., Tamanoi: The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell. 1990 Nov Gaul, Mardon and Rubin., : A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell March 1992 Hajnal…. Kim: Inhibition of C. elegans vulval induction by gap-1 and by let-23 receptor tyrosine kinase. Genes Dev. Oct. 1997

  30. What did genetics say Ras (-) Signaling eliminated GAP(-) Signaling increased Q: Can GAP be the major effector of Ras? A: Yes B: No C: not sure

  31. The end of 1992: GAP was no longer considered a Ras effector

  32. The Story of Raf Cell 1989 Aug: Deborah K. Morrison, David R. Kaplan, Jaime A. Escobedo, Ulf R. Rapp, Thomas M. Roberts and Lewis T. Williams: Direct activation of the serine/threonine kinase activity of raf-1 through tyrosine phosphorylation by the PDGF receptor We have examined the interaction between the serine/threonine kinase proto-oncogene product Raf-1 and the tyrosine kinase PDGF beta-receptor. Raf-1 tyrosine phosphorylation and kinase activity were increased by PDGF treatment of 3T3 cells or CHO cells expressing wild-type PDGF receptors but not mutant receptors defective in transmitting mitogenic signals, suggesting that the increase in Raf-1 kinase activity is a significant event in PDGF-induced mitogenesis. Concurrent with these increases, Raf-1 associated with the ligand-activated PDGF receptor. Furthermore, both mammalian Raf-1 and Raf-1 expressed using a recombinant baculoviral vector, associated in vitro with baculoviral-expressed PDGF receptor. This association was markedly decreased by prior phosphatase treatment of the receptor. Following incubation of partially purified baculoviral-expressed PDGF receptor with partially purified Raf-1, Raf-1 became phosphorylated on tyrosine and its serine/threonine kinase activity increased 4- to 6-fold. This is the first demonstration of the direct modulation of a protein activity by a growth factor receptor tyrosine kinase.

  33. The Story of Raf Cell 1989 Aug: Deborah K. Morrison, David R. Kaplan, Jaime A. Escobedo, Ulf R. Rapp, Thomas M. Roberts and Lewis T. Williams: Direct activation of the serine/threonine kinase activity of raf-1 through tyrosine phosphorylation by the PDGF receptor Raf-1 Raf-1 Y-P P- PDGFR Is this model convincing? A: There is no convincing data to support it. B: The data is good, the proposal is reasonable.

  34. The Story of Raf: summer 1993 Moodie SA, Willumsen BM, Weber MJ, Wolfman A.Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.Science. 1993 Jun Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul ***** Zhang XF,……, Rapp UR, Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul Warne PH, Viciana PR, Downward J.Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul Hughes DA, Ashworth A, Marshall CJ. Complementation of byr1 in fission yeast by mammalian MAP kinase kinase requires coexpression of Raf kinase. Nature. 1993 Jul. Van Aelst L, Barr M, Marcus S, Polverino A, Wigler M.Complex formation between RAS and RAF and other protein kinases. PNAS. 1993 Jul Raf had been around for a long time, why did everyone all of a sudden think it was the Ras effector?

  35. However, all above are also true for GAP. Main data in these six papers 1. Raf directly binds to Ras effector domain 2. Oncogenic Ras still interacts with Raf for the function 3. Ras effector domain mutations disrupt binding to Raf 4. Raf’s ability to bind Ras correlates to its function Vote: A: Convincing, B: Not convincing Why? What was missing?

  36. Ras (lf) Signaling eliminates Raf(lf) Signaling eliminates Ras (gf) Signaling increases (constitutive) Ras (gf); Raf(lf) Signaling eliminates Raf Ras Late 1992 and early 1993 Dickson B, Sprenger F, Morrison D, Hafen E. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature. 1992 Dec Han M, Golden A, Han Y, Sternberg PW. C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation. Nature. 1993 May

  37. Compare Raf with GAP Ras (lf) Signal eliminated Raf (lf) Signal eliminated GAP (lf) Signal increases Raf(lf) suppress activated Ras Ras(lf) suppress GAP(lf) Mammalian cells: Ras directly binds to Raf EGFR GRB2 SOS RAS GDP RAS GTP RAF GAP

  38. Ras C-Raf Raf gf What is the biological function of Ras-Raf binding? GDP GTP ? + P + P MAPK Ras SOS MEK Raf Stokoe D, …..Hancock JF. Activation of Raf as a result of recruitment to the plasma membrane.Science. 1994 Jun However: such an experiment did not work for B-Raf, fly and worm Raf

  39. Activation P sites Raf Inhibitory P sites A A Multivulva Making a constitutive Raf kinase Chong H, Lee J, Guan KL. Positive and negative regulation of Raf kinase activity and function by phosphorylation. EMBO J. 2001 Jul Yoder JH, Chong H, Guan KL, Han M. Modulation of KSR activity in C. elegans by Zn ions, PAR-1 kinase and PP2A phosphatase.EMBO J. 2004 Jan • Phosphorylation plays a critical role

  40. sup Vulvaless 1 2 W T SUR-7 SUR-8 KSR-1 SUR-6 SUR-2 LIN-25 LIN-39 TFs RAS RAF MEK MPK SUR-5 SUR-10 SUR-9 SUR-4 Genetics to identify factors downstream of Ras Y vulva l X Ras function s Multivulv a gf WT Han lab Horvitz lab

  41. Genetics to identify factors downstream of Ras X Ras R7 differentiation Ras (v12) Rough eye Ras (v12); suppressors smooth eye Screened for 1 million F1 fly Get hundreds of mutations. KSR, CNK, PP2A, Mek, MAPK etc. KSR story with Rubin Gerry Rubin’ lab

  42. Genetics to identify factors downstream of Ras SUR-6 Cytosolic Zn++ C-TAK1/ PAR-1 PP2A + P a kinase ? + P - P KSR MPK Raf MEK Morrison lab Han + Guan labs Others

  43. Multiple targets of Ras and specificity and cross talks Ras SUR8 Raf PI3K + P + P + P MEK AKT + P + P MAPK

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