What did genetics say Ras (-) Signaling eliminated GAP(-) Signaling increased Q: Can GAP be the 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 is 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

Genetic interaction and interpretation of genetic interactions - Biosynthetic pathway/ genes acting in different steps. -Order genes in a genetic pathway - studies on yeast mating -pheromone response - Epistasis analysis using null mutations- The GAP story - Epistasis analysis using gf mutations - The Ras suppressors -Enhancer and synergistic effect between two alleles - -The Ras pathway. -Understanding at molecular level/biochemical level. -Limitation of genetics

Can we learn from double mutants with two mutations with similar phenotypes? My answer: sometimes the information is extremely important A: Yes. B: No.

Enhancer effect and synergistic effect What are the biological base for such effects? What can we learn from such effects? How can we use such effects to identify genes? How do we design screens to deal with the problem of genetic redundancy (see next lecture)

Interaction between null or severe lf alleles Function On Off On Off Gene AGene B Lack the function Lack the function, same as above Linear relationship Function On Off On Off Gene A Gene B Lack part of the function, weak phenotype Lack both, strong phenotype parallel relationship

Interaction between partial lf alleles Function On Reduced On Reduced Gene AGene B Function compromised, weak phenotype Lack the function, strong phenotype. Function On Reduced On Reduced Gene A Gene B Part of the function reduced Both reduced, stronger phenotype Linear relationship parallel relationship

Big time example: dauer formaton Dauer formation On Off On Off daf-11 daf-21 Constitutive Dauer at 25°C 100% dauer at all temperature daf-7 daf-1 daf-4 daf-8 daf-14 Pathway A TGF  signaling Pathway B Pathway APathway B phenotype

Non-allelic noncomplementation Synergistic effect of reducing gene activity in two genes in the same pathway Seeing dominant effect in recessive mutations Hartwell et al Genetics

Modifior screen 1 Enhancer screen Simon and Rubin, 1991, Cell

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 +/-

b. Reducing activities in two genes acting in parallel pathways Question: If both Sev and Ras were knockout in R7 cells, would you see a severer phenotype in R7 than that in flies with either gene knocked out? Sev Ras Question: What if I ask the same question about two genes acting in parallel pathways? A B function

1. Interaction between two null alleles No enhancement: likely linear relationship Drastic enhancement: likely parallel Summary: 2. Interaction between two partial loss-of function alleles Genes in the same pathway more likely to have drastic enhancement Genes in parallel pathway may have some effects

Modifior screen 2 suppressor screen

SUR-6 KSR-1 SUR-8 SUR-7 SUR-5 SUR-4 SUR-9 Ras X vulval functions gf Multivulva Vulvaless WT Suppressor of ras mutations define regulators 1 WT 2 Y sup RAS MPKMEKRAF TFs

SUR-6 KSR-1 SUR-8 SUR-7 SUR-4 SUR-9 RTKRAS MPKMEKGRB2SOSRAF TFs Question: All sur genes may act after Ras since mutations in these genes suppress activated Ras

SUR-8 & KSR-1 are required for Ras signaling % vulval induction 100 ksr(lf) sur-8(lf) 4 ksr(lf) RAS RAF MEKSOS KSR-1 SUR-8 MPK Redundant?

SUR-8 and KSR do not have redundant biochem functions % vulval induction 100 mpk-1(rf) Ksr-1(lf) Ksr-1 mpk RAS RAF MEKSOS KSR-1 SUR-8 MAPK 98 mpk-1(rf) sur-8(lf) sur-8 mpk-1 0 Two separate inputs

Ras Raf SUR-8 MEK KSR MPK SUR-6 PP2A-B recruiter pathway SUR-5 (novel) SUR-8 acts in a separate pathway from KSR, while SUR-6 and SUR7 may act in the same pathway as KSR SUR-7 scaffoldd Double mutantssynergistic phenotype? Sur-8(lf) + sur-6/7(lf) yes Ksr(lf) + sur-6/7 (lf) no

Vulval differentiation _ + Induction Repression Ras synMuv Multiple regulatory pathway specify vulval differentiation

Vulval differentiation Class B SynMuv Class A SynMuv repression Synthetic Muv phenotype define redundant genetic pathways

synMuv screen lin-8(-) MultivulvaWild type mutagen lin-8(-) Wild type ; synMuvB(-) + lin-8(-) ; synMuvB(-) Vulval induction lin-8 lin-35 Class A gene Class B gene

Screen for synthetic lethal mutations by using an extrachromosomal array gene A (-) Ex gene A(+) Wild type mutagen Po Wild type gene A (-) Ex gene A(+) gene B (lf) + ; Wild type Lost the array F1 gene A (-) Ex gene A(+) gene B (lf) ; gene A (-) gene B (lf) ; Lost the array With the array Dead larva or eggs Due to gene A(-) and gene B(lf) Wild type F3 N-longer see wild-type progeny that have lost the array gene A (-) Ex gene A(+) gene B (lf) ; Wild type F2 Lost the array Wild type mutant Clone individuals gene A (-) gene B (lf) ;

Epistasis with genes in sequential events Event 1Event 2Event 2 product Gene A On Off Gene B Off On Off Phenotype Event 1 product No event 1 Vulval differentiation Generation of precursor cells lin-26 On Off lin-1 Off On Off Phenotype Multivulva No vulva cells Induction of vulva fate Example 1 Vulval development

Example 2. Cell death in C. elegans Completed cell death Cell killing ced-3 On Off ced-1 Off On Off Phenotype Cell corpes persist Cell lives Engulfment of killed cells

Mutant arg 1 arg 2-3 arg 4-7 Grwoth medium Minimal - Minmal + Arginine + Minmal +citulline - + Minmal +Ornithine - + ornithinecitullinearginine arg 1 arg 2-3arg 4-7 Srb and Horowitz, 1944

RTKRAS GRB2SOSRAF UNC-101ARK-1SLI-1 (CBL) GAP-1 Ark-1Sli-1Gap-1 Unc-101 Ark-1 Sli-1 Ark-1 Gap-1 Ark-1 Unc % Multivulva