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 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 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

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

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?

My answer: sometimes the information is extremely important

A: Yes. B: No.

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

Function

OnOffOff

OffOnOff

Gene A Gene BLack the functionLack the functionLack the function, same as above

Linear relationship

Function

OnOffOff

OffOnOff

Gene A

Gene BLack part of the function, weak phenotypeLack part of the function, weak phenotypeLack both, strong phenotype

parallel relationship

Interaction between partial lf alleles

Function

OnReducedReduced

ReducedOnReduced

Gene A Gene BFunction compromised, weak phenotypeFunction compromised, weak phenotypeLack the function, strong phenotype.

Function

OnReducedReduced

ReducedOnReduced

Gene A

Gene BPart of the function reducedPart of the function reducedBoth reduced, stronger phenotype

Linear relationship

parallel relationship

Big time example: dauer formaton

Dauer formation

On

Off

Off

Off

On

Off

daf-11daf-21

Constitutive Dauer at 25°C

Constitutive Dauer at 25°C

100% dauer at all temperature

daf-7 daf-1daf-4

daf-8daf-14

Pathway ATGF signaling

Pathway B

Pathway A Pathway B phenotype

Non-allelic noncomplementation

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

Synergistic effect of reducing gene activity in two genes in the same pathwaySeeing dominant effect in recessive mutations

Hartwell et alGenetics

Modifior screen 1

Enhancer screen

Simon and Rubin, 1991, Cell

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

Rough eye

High T

Low T

Low T

Low T

Low T

Ts allele

X*

*F1

F1 screen

TM

+/-

+/-

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

Bfunction

1. Interaction between two null allelesNo enhancement: likely linear relationshipDrastic enhancement: likely parallel

Summary:

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

SUR-6 KSR-1SUR-8 SUR-7

SUR-5 SUR-4 SUR-9

Ras X vulvalfunctions

gf Multivulva

Vulvaless

WT

Suppressor of ras mutations define regulators

1

WT2

Y

sup

RAS MPKMEKRAF TFs

SUR-6

KSR-1

SUR-8

SUR-7

SUR-4

SUR-9

RTK RAS MPKMEKGRB2 SOS RAF TFs

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

% v

ulv

al i

nd

uct

ion

100

100 100

ksr(lf)sur-8(lf)

4

sur-8(lf)ksr(lf)

RAS RAF MEKSOS

KSR-1

SUR-8 MPK

Redundant?

SUR-8 and KSR do not have redundant biochem functions

% v

ulv

al i

nd

uct

ion

100

100

mpk-1(rf)Ksr-1(lf) Ksr-1mpk

100

98100

3

RAS RAF MEKSOS

KSR-1

SUR-8 MAPK

98

mpk-1(rf)sur-8(lf) sur-8mpk-1

0

Two separate inputs

Ras Raf

SUR-8

MEK

KSR

MPK

SUR-6PP2A-B

recruiter

pathway

SUR-5 (novel)

SUR-8 acts in a separate pathway from KSR, while SUR-6 and SUR7 may act in the same pathway as KSR

SUR-7

scaffoldd

Double mutants synergistic phenotype?Sur-8(lf) + sur-6/7(lf) yesKsr(lf) + sur-6/7 (lf) no

Vulval differentiation

_ +

InductionRepression

RassynMuv

Multiple regulatory pathway specify vulval differentiation

Vulval differentiation

Class BSynMuv

Class ASynMuvrepression

Wild type

Phenotype

Multivulva

Wild type

Wild type

Class A +Class B +

Class B -Class A +

Genotype

Class A -Class B +

Class B -Class A -

Synthetic Muv phenotype define redundant genetic pathways

synMuv screen

lin-8(-)lin-8(-)

MultivulvaWild type

mutagen

lin-8(-)lin-8(-)

Wild type

;synMuvB(-) +

lin-8(-)lin-8(-)

;synMuvB(-)synMuvB(-)

Vulval induction

lin-8

lin-35

Class A gene

Class B gene

Screen for synthetic lethal mutations by using an extrachromosomal array

gene A (-)gene A (-)

Ex gene A(+) Wild type

mutagen

Po

Wild type

gene A (-)gene A (-)

Ex gene A(+)

gene B (lf) +

;Wild type

Lost the array

F1

gene A (-)gene A (-)

gene A (-)gene A (-)

Ex gene A(+)

gene B (lf)gene B (lf)

; gene A (-)gene A (-)

gene B (lf)gene B (lf)

;

Lost the arrayWith the array

Dead larva or eggs Due to gene A(-) and gene B(lf)

Wild type

F3

N-longer see wild-type progeny that have lost the array

gene A (-)gene A (-)

Ex gene A(+)

gene B (lf)gene B (lf)

;

Wild type

F2

Lost the array

Wild type mutant

Clone individuals

gene A (-)gene A (-)

gene B (lf)gene B (lf)

;

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

Vulval differentiation

Generation of precursor cells

lin-26

On

Off

Off

lin-1

Off

On

Off

Phenotype

Multivulva

No vulva cells

No vulva cells

Induction of vulva fate

Example 1 Vulval development

Example 2. Cell death in C. elegans

Completed cell death

Cell killing

ced-3

On

Off

Off

ced-1

Off

On

Off

Phenotype

Cell corpes persist

Cell lives

Cell lives

Engulfment of killed cells

Mutant

arg 1

arg 2-3

arg 4-7

Grwoth medium

Minimal

-

-

-

Minmal+ Arginine

+

+

+

Minmal+citulline

-

+

+

Minmal+Ornithine

-

-

+

ornithine citulline argininearg 1arg 2-3arg 4-7

Srb and Horowitz, 1944

RTK RAS GRB2 SOS RAF

UNC-101ARK-1 SLI-1(CBL)

GAP-1

Ark-1 Sli-1 Gap-1 Unc-101 Ark-1Sli-1

Ark-1Gap-1

Ark-1Unc-101

0 0 0 0

40

29

90% Multivulva