Figure 1

Expression of wild type Gab1 rescues EGF-induced PI-3 kinase and 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 and Gab1 expression. Both WT MEFs and cells expressing exogenous Gab1 show Gab1 tyrosine phosphorylation in response to EGF treatment. B. 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 phosphotyrosine immunoprecipitates were analyzed for PI-3 kinase activity. Gab1 -/- cells fail to show phosphotyrosine-associated PI-3 kinase activity, while cells expressing exogenous Gab1 display phosphotyrosine-associated PI-3 kinase activity that is augmented by EGF treatment. C. 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 the EGF-induced activation of Akt.