Pathway in early 1992 RAS EGFR

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

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

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.

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

Mike Simon : landmark modify screen Simon et al 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 +/-

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

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

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

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

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

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 Apr 9;73: 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 May 6;363: 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 May 7;73: 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 May 28;260: 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 Jun 25;260: 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 Jun 25;260: 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 Apr 9;73:

Many Small GTPases have a conserved C-terminus

Ras is lipid modified for its membrane binding

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

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

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

The holy grail: what is the effector of Ras?

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

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 effector

GAP = Ras effector Nature. April 1988

GAP = Ras effector Science. April 1988

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 , 36, 38insensitive-- 39Sensitive effector GAP bindingMembrane binding GAP is the effector. A: Convincing. B. Somewhat convincing. C. Not convincing.

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

The field was convinced Annu Rev Cell Biol. 1991;7:

The field was convinced Annu Rev Cell Biol. 1991;7:

The field was convinced Ann Rev Cell Biol 1991;7:

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.

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

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

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

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.

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.

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 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 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 Jul Vojtek AB, Hollenberg SM, Cooper JA.Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 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 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 Jul Raf had been around for a long time, why did everyone all of a sudden think it was the Ras effector?

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

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 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 Dec Ras (gf) Signaling increases (constitutive) Raf(lf) Signaling eliminates Ras (lf) Signaling eliminates Ras (gf); Raf(lf) Signaling eliminates Ras Raf

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

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 Jun Raf gf However: such an experiment did not work for B-Raf, fly and worm Raf

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 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 Jan Raf A Inhibitory P sites Activation P sites Multivulva

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

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

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

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