Figure 6.

Ligand-independent EGFR activation is unable to recruit the Gab-1/p85/AKT signaling complex. (A) Pre-formed MCAs as in Figure 3 were incubated in DMEM containing either no serum or 10% FBS containing either EGF (10 ng/ml) or 1 μM PD168393. After 6 h, MCAs were collected and lysed, and about 250 μg of protein from each condition were used for immunoprecipitation (IP) with 2 μg control mouse IgG or anti-EGFR. The IP was then analyzed by immunoblotting as indicated. Cell lysates (CL) were analyzed with anti-p-AKT (stripped and reprobed with anti-AKT). (B) HEK293 cells expressing the various indicated constructs were lysed and IP with anti-Flag. The IP was then analyzed by immunoblotting as indicated. FL-wt expressing cells were stimulated with EGF (10 ng/ml) for 10 min (C) Pre-formed HSC-3 MCAs incubated as in (A) were lysed, IP with anti-Gab1 and then analyzed with anti-pY20 (phosphotyrosine). The membrane was stripped and reprobed for total Gab-1. CL was also analyzed for the phospho-EGFR status. (D) HEK293 cells expressing the various constructs as indicated were lysed and IP with anti-Gab1 and analyzed with anti-pY20. The membrane was stripped and reprobed for total Gab1. CL was also analyzed for phospho-EGFR or total EGFR to confirm expression of the constructs. FL-wt expressing cells were stimulated with EGF (10 ng/ml) for 10 min. (E) EGFR-siRNA transfected HSC-3 cells were subjected to MCAs formation in presence of 10% FBS for 24 hr. Cell lysates were prepared and analyzed by IP and Western blotting as indicated. (F) Gab1-siRNA transfected HSC-3 cells were subjected to MCAs formation as above. Cell lysates were prepared and analyzed by IP and Western blotting as indicated. In (E) and (F), lanes 1 are untransfected control; lanes 2 are transfected with control-scrambled siRNA; lanes 3 are transfected with 25 nM and lanes 4 are with 100 nM of either EGFR- or Gab1-siRNA, respectively. Results are representative of at least 2–4 independent experiments.