Research Report 2000 - MDC

Coupling of Gab1 to c-Met, Grb2
and downstream effectors
mediate biological responses
Ute Schaeper, Martin Sachs, Niels
H. Gehring, Renate Franke and
Ingrid Walther. In collaboration with
Bettina Kemkes (GSF Munich) and
Carmen Birchmeier (MDC)
Gab1, like the insulin receptor
substrates (IRS), the FGF receptor
substrate FRS/SNT, and p62dok
family members belongs to a newly
identified group of docking proteins
that function as specific substrates of
tyrosine kinases. Gab1 contains an Nterminal
PH domain and a novel
phosphotyrosine recognition domain
which mediate direct association with
the c-Met receptor. Gab1 binds to two
sites of the cytoplasmic tail of c-Met,
Y14 (Y1349) and to a lesser extent
Y15 (Y1356). Gab1 also forms a
constitutive complex with Grb2 and
this interaction is mediated via the
C-terminal SH3 domain of Grb2.
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We have now mapped the c-Met and
Grb2 interaction sites using reverse
yeast two-hybrid technology. The c-
Met binding site is localized to a 13
amino acid region unique to Gab1.
Insertion of this site into the Gab1related
protein p97/Gab2 was
sufficient to confer c-Met binding
activity. Association with Grb2 was
mapped to two sites: a classical SH3
binding site (PXXP) and a novel Grb2
SH3 consensus binding motif
(PP(V/I)(D/N)RXXKP). To detect
phosphorylation-dependent
interactions of Gab1 with downstream
substrates, we have developed a
modified yeast two-hybrid assay and
identified PI(3)K, Shc, Shp2 and
CRKL as interaction partners of Gab1.
In a trk-met specific branching
morphogenesis assay, association of
Gab1 with Shp2, but not PI(3)K,
CRKL or Shc was essential to induce
branching morphogenesis in MDCK
cells. A fundamental role of Gab1 for
c-Met specific signaling is also
supported by gene ablation
experiments in the mouse: Gab1 -/embryos
produced in our laboratory
are characterized by strongly reduced
and delayed migration of myogenic
precursor cells into the limbs, a
phenotype reminiscent of HGF/SF -/and
c-Met -/- mutant embryos.
Figure 29: Ablation of the β-catenin gene in
mice results in a defect of anterior-posterior
axis formation at embryonal day 6.0. The
marker Cerberus is mislocated in the -/- βcatenin
embryos (arrow at the distal tip in d)
whereas, in the wild-type embryo, Cerberus
marks the anterior side (arrow in c).
Subsequent head development at the anterior
side is abrogated (as shown by the marker
Otx2, compare e and f). Embryonal and
extraembryonal tissues are properly developed
(as shown by expression of BMP4, see a and b).
Work by J. Hülsken, R. Vogel, V. Brinkmann,
B. Erdmann, C. Birchmeier, W. Birchmeier.
J. Cell Biol. 2000.