Early College Schools: A New Pathway From High School through College
Pathway in early 1992
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Transcript of Pathway in early 1992
Strategy: moving from RTK down: discovering SOS
RASGDP
RASGTP
Pi
GTPGDP
GAP
target protein
Active stateInactive
RTK
Son of Sevenless: hyperactive mutation
Roger and Benejee, Cell 1990The story about Ron Roger
-
+
Sev/RTK (lf) Sevenless
Sev (lf) + SOS(gf) R7 fine
Sev SOS R7 fate
Leads to Model: A: yesB: may not.
Drosophila: F1 screen vs F2 screen
X
* *X
**
F3 homozygotes
mutagen
X
TM
TM
*
*F1
F2 screen mutagen
X*
*F1
F1 screen
TM
A conversation with Rubin
Mike Simon : landmark modify screenSimon et al. 1991 Cell
Sev RAS SOS R7
Rough eye, no R7
Mostly wt, small % defects
Rough eye
Rough eye
High T
Low T
Low T
Low T
Ts allele
X*
*F1
F1 screen
TM
+/-
+/-
SOS encodes an exchange factor for GTPaseSimon et al. 1991 Cell
RASGDP
RASGTP
Pi
GTPGDPSOS
GAP
target protein
Active stateInactive
RTK
Moving from receptor down: biochemistry
RASGDP
RASGTP
Pi
GTPGDP
GAP
target protein
Active stateInactive
RTK
SOS
Discovery of GRB2
EGFR
Y
EGFR
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 screenClark et al. Nature Mar 1992
lin-15 (-) Multivulvasem-5(-) Vulvalesslin-15(-); sem-5(-) Vulvaless
RasSEM-5LIN-15
EGFR
LIN-15
Ras Vulval induction
KnownSince 1990
SEM-5 is homologous to GRB-2. Combine the data from the two field:
RasGRB2/SEM-5EGFR
ras (gf) Mutivulvasem-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. 1993 Apr 9;73:179-91.
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.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.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.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.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.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. 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.
Summary of the original screens
lin-15(-)
, Muvulva
lin-15(-); suppressor
; Vulvaless
mutagenworm
biochem binding to phosphorylated tyosine of growth factor: Grb2Discovery of GAP.
RTK GRB2 SOS Ras
worm LET-23 SEM-5 SOS-1 LET-60
fly Sevenless DRK SOS Ras1
sev(lf)
,
sev(lf); suppressor/enhancer
;
fly
R7 fate reduced
mutagen
R7 fate better or worse
GAP
GAP-1
Gap1
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
RASGDP
RASGTP
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
QuickTime™ and aGIF decompressor
are needed to see this picture.
QuickTime™ and aGIF decompressor
are needed to see this picture.
12 59 116effector 185164
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)
InteractionWith GAP
Rasfunction
12, 59, 61 abolished + +++
35, 36, 38 insensitive - -
39 Sensitive + +
12 59 116effector
GAP binding Membrane binding
185164GAP is the effector.
A: Convincing. B. Somewhat convincing. C. Not convincing.
Functions
GAP also acts as the effector
RASGDP
RASGTP
Pi
GTPGDP
GAP
Active stateInactive 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. 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
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 Story of RafCell 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-PRaf-1 Raf-1P-
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
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. 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. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 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 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. 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. 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
Compare Raf with GAP
Ras (lf) Signal eliminatedRaf (lf) Signal eliminatedGAP (lf) Signal increases
Raf(lf) suppress activated Ras
Ras(lf) suppress GAP(lf)
RASGDP
GAP
RASGTP
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?
GDP GTP
Ras C-Raf
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
Making a constitutive Raf kinase
- Phosphorylation plays a critical role
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. Modulation of KSR activity in C. elegans by Zn ions, PAR-1 kinase and PP2A phosphatase.EMBO J. 2004 Jan
Raf
A A
Inhibitory P sites
Activation P sites
Multivulva
Genetics to identify factors downstream of Ras
Ras X vulvalfunctions
gf MultivulvaWT
WT2
Y
Vulvaless1sup
SUR-6 KSR-1SUR-8 SUR-7
SUR-5SUR-10 SUR-4 SUR-9
RAS MPKMEKRAF TFs
SUR-2LIN-25LIN-39
Han labHorvitz 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 labHan + Guan labsOthers