Figure 4

Expression of ErbB3 in Gab1-deficient MEFs partially rescues 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 analyzed for EGFR tyrosine phosphorylation and EGFR expression, and for ErbB3 tyrosine phosphorylation and ErbB3 expression. Cells were additionally stimulated with 10 ng/ml NRG and cell extracts analyzed for ErbB3 tyrosine phosphorylation. The endogenous EGFR is tyrosine phosphorylated in response to EGF in all cell lines. ErbB3 exhibits weak constitutive tyrosine phosphorylation that is enhanced by NRG treatment, but is not significantly enhanced by treatment with EGF. Selected bands were quantitated by densitometry to determine relative increase in growth factor-induced tyrosine phosphorylation. B. 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 analyzed for Gab1 tyrosine phosphorylation and Gab1 expression. Cells rescued with wild type Gab1 exhibit Gab1 tyrosine phosphorylation in response to EGF treatment, while Gab1 -/- cells and Gab1-deficient cells expressing ErbB3 do not. 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 phosphotyrosine immunoprecipitates were analyzed for PI-3 kinase activity. Expression of wild type Gab1 in Gab1-deficient MEFs rescues the EGF-induced PI-3 kinase activity. Gab1 deficient MEFs exogenously expressing ErbB3 exhibit PI-3 kinase activity that is largely EGF-independent. 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°. Cell extracts were analyzed for activation of Akt by using antibodies that specifically recognize the phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Ectopic expression of ErbB3 in Gab1 deficient cells results in a partial rescue of EGF-induced Akt activation relative to cells expressing wild type Gab1. E. The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml NRG for varying periods of time at 37°C. Cell extracts were analyzed for activation of Akt by using antibodies that specifically recognize the phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Treatment of Gab1-deficient cells exogenously expressing ErbB3 with NRG results in a robust and sustained activation of Akt, while cells expressing exogenous Gab1 do not exhibit Akt activation in response to NRG treatment.