1. Pathway in early 1992 RAS EGFR

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

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

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

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

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

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

9. Moving from receptor down: biochemistry RAS GDP RAS GTP Pi GTPGDP GAP target protein Active stateInactive RTK SOS

10. Discovery of GRB2 EGFR Y Ligand activation Y-P Screen GT11 protein expression library 9 GRB proteins Which one is one mediated Ras activation? Y-P

11. 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

12. 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 RasSEM-5LIN-15 EGFR LIN-15 Ras Vulval induction Known Since 1990 SEM-5 is homologous to GRB-2. Combine the data from the two field: RasGRB2/SEM-5 EGFR ras (gf) Mutivulva sem-5(-) Vulvaless ras (gf); sem-5(-) Multivulva

13. The rest of the story is there in a rush and clash- mid 1993 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. 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.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.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.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.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.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. Simon MA, Dodson GS, Rubin GM. 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.

14. Many Small GTPases have a conserved C-terminus

15. Ras is lipid modified for its membrane binding

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

17. Half way done RAS GDP RAS GTP Pi GTPGDP GAP Active stateInactive RTK SOS GRB2

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

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

20. 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 RAS GDP RAS GTP Pi GTPGDP Effector protein Active stateInactive

21. 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 12 59 116 effector 185164

22. GAP = Ras effector Nature. April 1988

23. GAP = Ras effector Science. April 1988

24. 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 Key data Position of Ras mutations GAP activation (hydrolysis) Interaction With GAP Ras function 12, 59, 61abolished++++ 35, 36, 38insensitive-- 39Sensitive++ 12 59 116 effector GAP bindingMembrane binding 185164 GAP is the effector. A: Convincing. B. Somewhat convincing. C. Not convincing.

25. The field was convinced Cell June 15, 1990 RAS GDP RAS GTP Pi GTPGDP GAP Active stateInactive GAP

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

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

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

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

30. Genetics 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 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 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 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 Tanaka, Matumoto, and Toh-E: IRA1, an inhibitory regulator of the RAS- cyclic AMP pathway in S. cerevisiae. Mol Cell Biol. 1989 Feb Hajnal…. Kim: Inhibition of C. elegans vulval induction by gap-1 and by let-23 receptor tyrosine kinase. Genes Dev. Oct. 1997

31. 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

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

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 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.

34. 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 PDGFR Y-P Raf-1 P-P- Is this model convincing? A: There is no convincing data to support it. B: The data is good, the proposal is reasonable.

35. The Story of Raf: summer 1993 Moodie SA, Willumsen BM, Weber MJ, Wolfman A.Moodie SA, Willumsen BM, Weber MJ, Wolfman A. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.Science. 1993 Jun Warne PH, Viciana PR, Downward J.Warne PH, Viciana PR, Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul Zhang XF,……, Rapp UR, Avruch J.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 Vojtek AB, Hollenberg SM, Cooper JA.Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul ***** Hughes DA, Ashworth A, Marshall CJ.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.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?

36. 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 However, all above are also true for GAP. Why? What was missing? Vote: A: Convincing, B: Not convincing

37. Late 1992 and early 1993 Han M, Golden A, Han Y, Sternberg PW.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 Dickson B, Sprenger F, Morrison D, Hafen E.Dickson B, Sprenger F, Morrison D, Hafen E. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature. 1992 Dec Ras (gf) Signaling increases (constitutive) Raf(lf) Signaling eliminates Ras (lf) Signaling eliminates Ras (gf); Raf(lf) Signaling eliminates Ras Raf

38. 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) RAS GDP GAP RAS GTP SOS GRB2EGFR Mammalian cells: Ras directly binds to Raf RAF

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

40. Making a constitutive Raf kinase - Phosphorylation plays a critical role Chong H, Lee J, Guan KL.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.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 Raf A Inhibitory P sites Activation P sites Multivulva

41. Genetics to identify factors downstream of Ras Ras X vulval functions gf Multivulva WT WT 2 Y Vulvaless 1 sup SUR-6 KSR-1 SUR-8 SUR-7 SUR-5 SUR-10 SUR-4 SUR-9 RAS MPKMEKRAF TFs SUR-2 LIN-25 LIN-39 Han lab Horvitz lab

42. Genetics to identify factors downstream of Ras Ras X 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

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

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