Pathway in early 1992
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Pathway in early 1992. RAS. EGFR. 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. target protein. Inactive. Active state. GAP. Pi. Leads to Model:. A: yes

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Pathway in early 1992

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Pathway in early 1992

Pathway in early 1992

RAS

EGFR


Pathway in early 1992

WT

sevenless

R7 receptor is required for UV response

R8

Boss

sev

R7


Strategy moving from rtk down discovering sos

Strategy: moving from RTK down: discovering SOS

RTK

GDP

GTP

RAS

GDP

RAS

GTP

target protein

Inactive

Active state

GAP

Pi


Son of sevenless hyperactive mutation

Leads to Model:

A: yes

B: may not.

R7 fate

Sev

SOS

-

+

Son of Sevenless: hyperactive mutation

Sev/RTK (lf)Sevenless

Sev (lf) + SOS(gf) R7 fine

Roger and Benejee, Cell 1990

The story about Ron Roger


Drosophila f1 screen vs f2 screen

F1 screen

mutagen

mutagen

F2 screen

TM

*

*

X

TM

X

TM

*

*

X

F1

F1

*

*

X

*

*

F3 homozygotes

Drosophila: F1 screen vs F2 screen

A conversation with Rubin


Mike simon landmark modify screen

F1 screen

*

TM

X

*

F1

Mike Simon : landmark modify screen

Simon et al. 1991 Cell

R7

Ts allele

Sev

SOS

RAS

Rough eye, no R7

Mostly wt, small % defects

Rough eye

Rough eye

High T

Low T

Low T

Low T

+/-

+/-


Sos encodes an exchange factor for gtpase

SOS encodes an exchange factor for GTPase

Simon et al. 1991 Cell

RTK

SOS

GDP

GTP

RAS

GDP

RAS

GTP

target protein

Inactive

Active state

GAP

Pi


Moving from receptor down biochemistry

Moving from receptor down: biochemistry

RTK

SOS

GDP

GTP

RAS

GDP

RAS

GTP

target protein

Inactive

Active state

GAP

Pi


Discovery of grb2

Screen lGT11 protein expression library

Y-P

9 GRB proteins

Discovery of GRB2

Ligand activation

Y

Y-P

EGFR

EGFR

Which one is one mediated Ras activation?


Discovery of grb2 complementary works by biochemists and geneticists

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

Known

Since 1990

LIN-15

Vulval induction

Ras

EGFR

LIN-15

SEM-5

Ras

SEM-5 is homologous to GRB-2. Combine the data from the two field:

GRB2/SEM-5

Ras

EGFR

The sem-5 story - early 1992

SEM-5 was identified as by a suppressor mutant screen

Clark et al. Nature Mar 1992

lin-15 (-)Multivulva

sem-5(-) Vulvaless

lin-15(-); sem-5(-) Vulvaless

ras (gf) Mutivulva

sem-5(-) Vulvaless

ras (gf); sem-5(-) Multivulva


The rest of the story is there in a rush and clash mid 1993

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

Many Small GTPases have a conserved C-terminus


Ras is lipid modified for its membrane binding

Ras is lipid modified for its membrane binding


Pathway in early 1992

SH3

SOS

Y-P

SH2

RAS

GDP

RAS

GTP

SH3

EGFR

Y

RAS

GDP

EGFR

SH3

SH2

SH3

GRB2

SH3

SOS

SH2

SH3


Half way done

Half way done

RTK

GRB2

SOS

GDP

GTP

RAS

GDP

RAS

GTP

Inactive

Active state

GAP

Pi


Pathway in early 1992

GAP

GAP-1

Gap1

mutagen

lin-15(-); suppressor

worm

lin-15(-)

Vulvaless

;

Muvulva

,

fly

mutagen

sev(lf); suppressor/enhancer

sev(lf)

R7 fate reduced

;

R7 fate better or worse

,

binding to phosphorylated tyosine of growth factor: Grb2

Discovery of GAP.

biochem

Summary of the original screens

RTK

GRB2

SOS

Ras

worm

LET-23

SEM-5

SOS-1

LET-60

fly

Sevenless

DRK

SOS

Ras1


Pathway in early 1992

The holy grail: what is the effector of Ras?


The ras effector

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

GTP

RAS

GDP

RAS

GTP

Effector protein

Inactive

Active state

Pi


The ras effector domain is defined by

effector

12

116

164

185

59

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


Gap ras effector

GAP = Ras effector

Nature. April 1988


Gap ras effector1

Science. April 1988

GAP = Ras effector


Key argument

Key data

Position of Ras mutations

GAP activation

(hydrolysis)

Interaction

With GAP

Ras

function

12, 59, 61

abolished

+

+++

35, 36, 38

insensitive

-

-

39

Sensitive

+

+

effector

12

116

GAP is the effector.

A: Convincing. B. Somewhat convincing. C. Not convincing.

164

185

59

GAP binding

Membrane binding

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


The field was convinced

GDP

GTP

RAS

GDP

RAS

GTP

GAP

Inactive

Active state

GAP

Pi

The field was convinced

Cell June 15, 1990


The field was convinced1

The field was convinced

Annu Rev Cell Biol. 1991;7:601-32.


The field was convinced2

The field was convinced

Annu Rev Cell Biol. 1991;7:601-32.


The field was convinced3

The field was convinced

Ann Rev Cell Biol 1991;7:601-32.


Functions

GAP stimulates RAS-GTP hydrolysis

- expected to be a negative factor

Functions

GAP also acts as the effector

- expected to be: A. positive factor

B. negative factor

GDP

GTP

RAS

GDP

RAS

GTP

GAP

Inactive

Active state

GAP

Pi

If you make a knockout of GAP, do you expect the signaling to go

A: up. B: down.


Genetics

Genetics

Tanaka, Matumoto, and Toh-E: IRA1, an inhibitory regulator of the RAS-cyclic 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

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 end of 1992: GAP was no longer considered a Ras effector


The story of raf

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 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 raf1

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

Raf-1

Raf-1

Y-P

P-

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

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

However, all above are also true for GAP.

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

Why? What was missing?


Late 1992 and early 1993

Ras (lf) Signaling eliminates

Raf(lf) Signaling eliminates

Ras (gf) Signaling increases (constitutive)

Ras (gf); Raf(lf) Signaling eliminates

Raf

Ras

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


Compare raf with gap

Compare Raf with GAP

Ras (lf)Signal eliminated

Raf (lf)Signal eliminated

GAP (lf)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

RAF

GAP


What is the biological function of ras raf binding

Ras

C-Raf

Raf gf

What is the biological function of Ras-Raf binding?

GDP

GTP

?

+ P

+ P

MAPK

Ras

SOS

MEK

Raf

Stokoe D, …..Hancock JF. Activation of Raf as a result of recruitment to the plasma membrane.Science. 1994 Jun

However: such an experiment did not work for B-Raf, fly and worm Raf


Making a constitutive raf kinase

Activation P sites

Raf

Inhibitory P sites

A A

Multivulva

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

  • Phosphorylation plays a critical role


Genetics to identify factors downstream of ras

sup

Vulvaless

1

2

W

T

SUR-7

SUR-8

KSR-1

SUR-6

SUR-2

LIN-25

LIN-39

TFs

RAS

RAF

MEK

MPK

SUR-5

SUR-10

SUR-9

SUR-4

Genetics to identify factors downstream of Ras

Y

vulva

l

X

Ras

function

s

Multivulv

a

gf

WT

Han lab

Horvitz lab


Genetics to identify factors downstream of ras1

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 ras2

Genetics to identify factors downstream of Ras

SUR-6

Cytosolic Zn++

C-TAK1/

PAR-1

PP2A

+ P

a kinase

?

+ P

- P

KSR

MPK

Raf

MEK

Morrison lab

Han + Guan labs

Others


Multiple targets of ras and specificity and cross talks

Multiple targets of Ras and specificity and cross talks

Ras

SUR8

Raf

PI3K

+ P

+ P

+ P

MEK

AKT

+ P

+ P

MAPK


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