Figure 3
Expression of a Gab1 mutant protein deficient in Shp2 binding enhances EGF-induced activation of the PI-3 kinase/Akt signaling pathway. A. The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and analyzed for Gab1 tyrosine phosphorylation, Gab1-Shp2 co-immunoprecipitation and Gab1 expression. Gab1F627/659 becomes tyrosine phosphorylated in response to EGF treatment to levels approximately 1.5-fold higher than Gab1 as determined by densitometry, while Gab1F446/472/589/627/659 does not show EGF-induced tyrosine phosphorylation in this assay. Wild type Gab1 forms a stable complex with Shp2 in response to EGF treatment, while Gab1F627/659 and Gab1F446/472/589/627/659 do not. B. The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for EGFR tyrosine phosphorylation and EGFR expression. All of the cell lines examined show similar kinetics of EGF-induced EGFR activation. Immunoblots were quantitated by densitometry, normalized for EGFR expression, and represented linearly. Diamonds = Gab1 -/-, squares = Gab1, triangles = Gab1F627/659, circles = Gab1F446/472/589/627/659. C. The indicated cell lines were serum-starved for 24 hours and stimulated with 100 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and Gab1 immunoprecipitates were analyzed for PI-3 kinase activity. Cells expressing exogenous Gab1F627/659 display enhanced PI-3 kinase activity relative to cells expressing wild type Gab1. Expression of exogenous Gab1F446/472/589/627/659 fails to rescue EGF-induced PI-3 kinase activity in Gab1 deficient MEFs. D. The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for activation of Akt by using antibodies that specifically recognize the serine473-phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Cells expressing exogenous Gab1F627/659 display enhanced activation of Akt with sustained kinetics relative to cells expressing wild type Gab1. Expression of exogenous Gab1F446/472/589/627/659 fails to rescue EGF-induced Akt activation in Gab1 deficient MEFs.