Transcript Document

Pathway in early 1992
EGFR
RAS
WT
sevenless
R7 receptor is
required for UV
response
R8
Boss
sev
R7
Strategy: moving from RTK down: discovering SOS
RTK
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
GAP
target protein
Son of Sevenless: hyperactive mutation
Sev/RTK (lf)
Sevenless
Sev (lf) + SOS(gf) R7 fine
Leads to Model:
Sev
A: yes
B: may not.
SOS
R7 fate
+
Roger and Benejee, Cell 1990
The story about Ron Roger
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
Rough eye
+/-
Low T
+/-
Low T
F1 screen
*
*
F1
X TM
Rough eye
SOS encodes an exchange factor for GTPase
Simon et al. 1991 Cell
RTK
SOS
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
GAP
target protein
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Moving from receptor down: biochemistry
RTK
GDP
SOS
RAS
GDP
GTP
RAS
GTP
Inactive
Active state
Pi
GAP
target protein
Discovery of GRB2
Ligand activation
Y
Y-P
EGFR
EGFR
Y-P
Screen lGT11 protein expression library
9 GRB proteins
Which one is one mediated Ras activation?
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 tyrosinephosphorylated epidermal growth factor receptors (EGFRs) and plateletderived 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
Known
Since 1990
LIN-15
EGFR
lin-15 (-)
sem-5(-)
lin-15(-); sem-5(-)
Multivulva
Vulvaless
Vulvaless
ras (gf)
sem-5(-)
ras (gf); sem-5(-)
LIN-15
SEM-5
Vulval induction
Ras
Mutivulva
Vulvaless
Multivulva
Ras
SEM-5 is homologous to GRB-2. Combine the data from the two field:
EGFR
GRB2/SEM-5
Ras
The rest of the story is there in a rush and clash- mid 1993
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.
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.
Many Small GTPases have a conserved C-terminus
Ras is lipid modified for its membrane binding
Y
RAS
GDP
EGFR
SH3
SH2
GRB2
SH2
SH3
SOS
SH3
Y-P SH2 SH3
SH3
EGFR
SOS
RAS
GDP
RAS
GTP
SH3
Half way done
RTK
GRB2
SOS
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
Active state
Pi
GAP
Summary of the original screens
worm
fly
worm
RTK
GRB2
SOS
Ras
LET-23
SEM-5
SOS-1
LET-60
DRK
SOS
Ras1
Sevenless
lin-15(-)
mutagen
sev(lf)
R7 fate
, reduced
biochem
GAP-1
Gap1
lin-15(-); suppressor
V
; ulvaless
Muvulva
,
fly
GAP
mutagen
sev(lf); suppressor/enhancer
R7 ;fate better or worse
binding to phosphorylated tyosine of growth factor: Grb2
Discovery of GAP.
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
GDP
RAS
GDP
GTP
RAS
GTP
Inactive
Effector protein
Active state
Pi
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
12
effector
59
QuickTime™ and a
GIF decompressor
are needed to see this picture.
116
164
QuickTime™ and a
GIF decompressor
are needed to see this picture.
185
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, 61
abolished
+
+++
35, 36, 38
insensitive
-
-
39
Sensitive
+
+
12 is the
effector
GAP
effector.59
116
164
185
A: Convincing. B. Somewhat convincing. C. Not convincing.
GAP binding
Membrane binding
The field was convinced
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
GAP
GAP
Active state
Pi
Cell June 15, 1990
The field was convinced
Annu Rev Cell Biol. 1991;7:601-32.
The field was convinced
Annu Rev Cell Biol. 1991;7:601-32.
The field was convinced
Ann Rev Cell Biol 1991;7:601-32.
Functions
GAP stimulates RAS-GTP hydrolysis
- expected to be a negative factor
GAP also acts as the effector
- expected to be:
A. positive factor
B. negative factor
GDP
GTP
RAS
GDP
RAS
GTP
Inactive
GAP
GAP
Active state
Pi
If you make a knockout of GAP, do you expect the signaling to go
A: up. B: down.
Genetics
Tanaka, Matumoto, and Toh-E: IRA1, an inhibitory regulator of the RAScyclic AMP pathway in S. cerevisiae. Mol Cell Biol. 1989 Feb
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
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
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
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
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 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 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
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
What is the biological function of Ras-Raf binding?
GDP GTP
SOS
Ras
?
Raf
+P
MEK
+P
MAPK
Stokoe D, …..Hancock JF. Activation of Raf as a result of recruitment to the
plasma membrane.Science. 1994 Jun
Ras C-Raf
Raf gf
However: such an experiment did not work for B-Raf, fly and worm Raf
Making a constitutive Raf kinase
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
Activation P sites
Raf
Inhibitory P sites
A
A
Multivulva
- Phosphorylation plays a critical role
Genetics to identify factors downstream of Ras
Y
vulval
functions
X
Ras
gf
Multivulva
WT
sup
SUR-8
SUR-6 SUR-7
RAS
Han lab
Horvitz lab
SUR-5
SUR-10
RAF
1
Vulvaless
2
WT
KSR-1
MEK
SUR-4
SUR-2
LIN-25
LIN-39
MPK
TFs
SUR-9
Genetics to identify factors downstream of Ras
X
Ras
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
C-TAK1/
PAR-1
Cytosolic Zn++
a kinase
?
SUR-6
PP2A
+P
+P
-P
KSR
Raf
Morrison lab
Han + Guan labs
Others
MEK
MPK
Multiple targets of Ras and specificity and cross talks
Ras
SUR8
Raf
PI3K
+P
AKT
+P
+P
+P
MEK
+P
MAPK