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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 protooncogene 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 PDGFinduced 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 Y-P Raf-1 P- Raf-1 PDGFR 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. 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 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 Vote: A: Convincing, B: Not convincing However, all above are also true for GAP. Why? What was missing? 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 Ras (lf) Signaling eliminates Raf(lf) Signaling eliminates Ras (gf) Signaling increases (constitutive) Ras (gf); Raf(lf) Signaling eliminates Ras Raf Compare Raf with GAP Ras (lf) Raf (lf) GAP (lf) Signal eliminated Signal eliminated 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 GAP 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? A: Yes. B: No. My answer: sometimes the information is extremely important 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 Linear relationship Gene A On Off Off Gene B Off On Off Function Lack the function Lack the function Lack the function, same as above parallel relationship Gene A On Off Off Gene B Off On Off Function Lack part of the function, weak phenotype Lack part of the function, weak phenotype Lack both, strong phenotype Interaction between partial lf alleles Linear relationship Gene A On Reduced Reduced Gene B Reduced On Reduced Function Function compromised, weak phenotype Function compromised, weak phenotype Lack the function, strong phenotype. parallel relationship Gene A On Reduced Reduced Gene B Reduced On Reduced Function Part of the function reduced Part of the function reduced Both reduced, stronger phenotype Big time example: dauer formaton Pathway A daf-7 TGFb signaling Pathway A On Off Off daf-1 daf-4 daf-8 daf-14 Pathway B daf-11 daf-21 Pathway B Off On Off Dauer formation phenotype Constitutive Dauer at 25°C Constitutive Dauer at 25°C 100% dauer at all temperature Non-allelic noncomplementation Synergistic effect of reducing gene activity in two genes in the same pathway Seeing dominant effect in recessive mutations QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Hartwell et al Genetics Modifior screen 1 Enhancer screen Simon and Rubin, 1991, Cell Drosophila: F1 screen vs F2 screen F2 screen F1 screen mutagen mutagen TM * * X X TM TM * * X F1 F1 * * X * * F3 homozygotes A conversation with Rubin Mike Simon : landmark modify screen Simon et al. 1991 Cell Ts allele Sev SOS RAS R7 High T Rough eye, no R7 Low T Mostly wt, small % defects Low T Rough eye Rough eye +/- Low T +/- Low T F1 screen * * F1 X TM Rough eye 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 function B Summary: 1. Interaction between two null alleles No enhancement: likely linear relationship Drastic enhancement: likely parallel 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 Suppressor of ras mutations define regulators Y vulval functions X Ras gf Multivulva WT sup SUR-8 SUR-6 SUR-7 RAS RAF 1 Vulvaless 2 WT KSR-1 MEK SUR-5 SUR-4 MPK TFs SUR-9 RTK GRB2 SOS RAS RAF MEK MPK TFs SUR-8 KSR-1 SUR-6 SUR-7 SUR-4 SUR-9 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 100 100 100 4 sur-8(lf) sur-8(lf) ksr(lf) ksr(lf) KSR-1 SOS RAS RAF SUR-8 Redundant? MEK MPK SUR-8 and KSR do not have redundant biochem functions 100 100 100 100 98 98 3 0 sur-8(lf) sur-8 mpk-1 mpk-1(rf) Ksr-1(lf) Ksr-1 mpk mpk-1(rf) KSR-1 SOS RAS RAF SUR-8 Two separate inputs MEK MAPK SUR-8 acts in a separate pathway from KSR, while SUR-6 and SUR7 may act in the same pathway as KSR Double mutants Sur-8(lf) + sur-6/7(lf) Ksr(lf) + sur-6/7 (lf) synergistic phenotype? yes no recruiter SUR-8 Ras Raf MEK KSR scaffoldd SUR-5 (novel) SUR-6 PP2A-B MPK SUR-7 pathway Multiple regulatory pathway specify vulval differentiation Induction Repression Ras synMuv _ + Vulval differentiation Synthetic Muv phenotype define redundant genetic pathways Class A SynMuv repression Class B SynMuv Vulval differentiation Genotype Phenotype Class A Class B + + Wild type Class A Class B + - Wild type Class B Class A + - Wild type Class B Class A - Multivulva synMuv screen mutagen lin-8(-) lin-8(-) lin-8(-) synMuvB(-) ; lin-8(-) + Wild type lin-8(-) synMuvB(-) ; lin-8(-) synMuvB(-) Wild type Class A gene Multivulva lin-8 Vulval induction Class B gene lin-35 Screen for synthetic lethal mutations by using an extrachromosomal array mutagen Po gene A (-) gene A (-) Ex gene A(+) F1 Wild type gene A (-) gene A (-) gene A (-) gene B (lf) ; gene A (-) + Wild type Ex gene A(+) F2 Lost the array Wild type Lost the array gene A (-) gene B (lf) ; gene A (-) gene B (lf) gene A (-) gene B (lf) ; gene A (-) gene B (lf) Ex gene A(+) Wild type Wild type mutant Clone individuals With the array F3 Lost the array gene A (-) gene B (lf) ; gene A (-) gene B (lf) Ex gene A(+) gene A (-) gene B (lf) ; gene A (-) gene B (lf) Wild type N-longer see wild-type progeny that have lost the array Dead larva or eggs Due to gene A(-) and gene B(lf) Epistasis with genes in sequential events Event 1 Event 2 Gene A On Off Off Gene B Off On Off Event 2 product Phenotype Event 1 product No event 1 No event 1 Example 1 Vulval development Generation of precursor cells lin-26 On Off Off Induction of vulva fate lin-1 Off On Off Vulval differentiation Phenotype Multivulva No vulva cells No vulva cells Example 2. Cell death in C. elegans Cell killing ced-3 On Off Off Engulfment of killed cells ced-1 Off On Off Completed cell death Phenotype Cell corpes persist Cell lives Cell lives Grwoth medium Mutant Minimal Minmal + Arginine arg 1 - + - - arg 2-3 - + + - arg 4-7 - + + + arg 4-7 Minmal +Ornithine arg 1 arg 2-3 ornithine Minmal +citulline citulline arginine Srb and Horowitz, 1944 90 % Multivulva 40 29 0 0 Ark-1 RTK Sli-1 0 Gap-1 0 Unc-101 GRB2 ARK-1 UNC-101 SLI-1 (CBL) SOS Ark-1 Sli-1 Ark-1 Gap-1 RAS GAP-1 Ark-1 Unc-101 RAF