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What did genetics say

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. Cell 1989 Aug:

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What did genetics say

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  1. 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

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

  3. 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 The Story of Raf 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.

  4. 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.

  5. 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?

  6. 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?

  7. 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

  8. 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

  9. 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

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

  11. 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)

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

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

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

  15. 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

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

  17. 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

  18. 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 Rough eye High T Low T Low T Low T Low T +/- +/-

  19. 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 b. Reducing activities in two genes acting in parallel pathways

  20. Summary: 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

  21. Modifior screen 2 suppressor screen

  22. Suppressor of ras mutations define regulators Y vulva l X Ras function s Multivulv a gf WT sup Vulvales s 1 2 W T SUR-7 SUR-8 KSR-1 SUR-6 TFs RAS RAF MEK MPK SUR-5 SUR-9 SUR-4

  23. TFs RTK GRB2 SOS RAS RAF MEK MPK 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

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

  25. SUR-8 and KSR do not have redundant biochem functions 100 100 100 100 98 98 % vulval induction 3 0 Ksr-1 mpk mpk-1(rf) sur-8 mpk-1 Ksr-1(lf) mpk-1(rf) sur-8(lf) KSR-1 MAPK SOS RAS RAF MEK SUR-8 Two separate inputs

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

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

  28. Synthetic Muv phenotype define redundant genetic pathways C l a s s A C l a s s B r e p r e s s i o n S y n M u v S y n M u v V u l v a l d i f f e r e n t i a t i o n

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

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

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

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

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

  34. 90 % Multivulva 40 29 0 0 0 0 Ark-1 Unc-101 Ark-1 Sli-1 Ark-1 Gap-1 Unc-101 Ark-1 Sli-1 Gap-1 RTK GRB2 SOS RAS RAF ARK-1 UNC-101 SLI-1 (CBL) GAP-1

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