Pathway in early 1992

RASEGFR

R7

R8

WT sevenless

R7 receptor is required for UV response

sev

Boss

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

QuickTime™ and aGIF decompressor

are needed to see this picture.

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.

Many Small GTPases have a conserved C-terminus

Ras is lipid modified for its membrane binding

RASGDP

SH2SH3

SH3SOS

SH3 SH3SH2

GRB2

EGFR

Y

RASGDP

SH2SH3

SH3SOS

EGFR

Y-PRASGTP

Half way done

RASGDP

RASGTP

Pi

GTPGDP

GAPActive stateInactive

RTK

SOSGRB2

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 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

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

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)

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.

The field was convinced

Cell June 15, 1990

RASGDP

RASGTP

Pi

GTPGDP

GAP

Active stateInactiveGAP

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 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 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 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

Multiple targets of Ras and specificity and cross talks

Ras

Raf

AKT

PI3K

MEK

+ P + P+ P

MAPK

+ P+ P

SUR8