Figure 2
Expression of a Gab1 mutant protein deficient in p85 binding fails to rescue EGF-induced PI-3 kinase/Akt activation in Gab1 deficient MEFs. 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 co-immunoprecipitation with p85, p85 expression, Gab1 co-immunoprecipitation with Shp2, and Gab1 expression. Both Gab1 and Gab1F446/472/589 are tyrosine phosphorylated in response to EGF treatment, and both form a stable complex with Shp2. However, Gab1F446/472/589 fails to associate with the p85 subunit of PI-3 kinase. Additionally total cell lysates from the indicated cell lines were immunoblotted with anti-Gab1 antibodies, providing independent evidence that Gab1 is expressed at approximately equal levels in all cell lines. 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. 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 = Gab1F446/472/589. 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. Ectopic expression of wild type Gab1 restored EGF-induced PI-3 kinase activity, while expression of Gab1F446/472/589 fails to rescue PI-3 kinase activity in response to EGF treatment. 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. Ectopic expression of Gab1 in Gab1 -/- MEFs rescues activation of Akt in response to EGF treatment, while expression of Gab1F446/472/589 fails to rescue the EGF-induced Akt activation.