线虫发育及基因功能研究2(韩珉)

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).
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.
LIN-15
SEM-5
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
Low T
Mostly wt, small % defects
Low T Low T
+/+/-
Rough eye Rough eye
F1 screen F1
* X TM *
SOS encodes an exchange factor for GTPase
Simon et al. 1991 Cell
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.
sev(lf); suppressor/enhancer R7;fate better or worse
biochem
binding to phosphorylated tyosine of growth factor: Grb2 Discovery of GAP.
The holy grail: what is the effector of Ras?
Many Small GTPases have a conserved C-terified for its membrane binding
Y EGFR
RAS GDP
SH3 SH2
SOS
SH3
SH3 SH2 SH3 GRB2
Y-P SH2 SH3 SH3
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.
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.
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.
EGFR
SOS
RAS GDP
RAS GTP
Half way done
RTK
GRB2 SOS
GDP
GTP
RAS GDP
Inactive
Pi
RAS GTP
Active state
GAP
Summary of the original screens
RTK worm LET-23 fly Sevenless
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.
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
A: Yes, B: no
GDP
RAS GDP
Inactive
Pi
GTP
RAS GTP
Effector protein
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.
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
LIN-15
Since 1990
EGFR
Ras
Vulval induction
lin-15 (-) sem-5(-) lin-15(-); sem-5(-)
Multivulva Vulvaless Vulvaless
ras (gf) sem-5(-) ras (gf); sem-5(-)
Mutivulva Vulvaless Multivulva
GRB2 SEM-5 DRK
SOS SOS-1 SOS
Ras LET-60 Ras1
GAP GAP-1 Gap1
worm
lin-15(-) Muvul,va
mutagen
lin-15(-); suppressor V; ulvaless
fly
sev(lf)
mutagen
R7 fa,te reduced
RTK
SOS
GDP
GTP
RAS GDP
Inactive
Pi
RAS GTP
target protein
Active state
GAP
M acintosh P IC T im age form at
is not supported
Moving from receptor down: biochemistry
RTK
GDP SOS GTP
RAS GDP
Inactive
Pi
RAS GTP
target protein
Active state
GAP
Discovery of GRB2
Y EGFR
Ligand activation
Y-P EGFR
Y-P Screen lGT11 protein expression library
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.
Inactive
Pi
RAS GTP
target protein
Active state
GAP
Mike Simon : landmark modify screen
Simon et al. 1991 Cell
Ts allele Sev
SOS RAS
R7
High T
Rough eye, no R7