Download Sur-8(lf)

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