Phospho-Y1230/Y1231 recruits GRB2 and activates ERK1/2 and AKT. (A) Coimmunoprecipitation of chimeric E-R3 with GRB2. HUVECs transduced with wild-type E-R3 or the mutants as indicated either untreated or induced with 10 ng/mL EGF for 10 minutes were immunoprecipitated with anti-EGFR N-terminal–specific antibodies and revealed with anti-GRB2, phosphotyrosine, or VEGFR-3 C-terminal domain as indicated. (B) GBR2-SH2 binds directly to E-R3 chimeric receptor. Far Western blotting of extracts from HUVECs transduced with E-R3 mutants as indicated were immunoprecipitated with anti-EGFR N-terminal–specific antibodies and revealed with biotinylated recombinant GRB2-SH2. (C) Wild-type E-R3, but not mutants Y1230 and Y1231, fully activates AKT and ERK phosphorylation measured with phosphospecific antibodies. (D) Time-course analysis of AKT activation of HUVECs transduced with wild-type ER-3 or with the mutants as indicated. (E)AKT activation is mediated by PI3K. Effect on ER-3–dependent AKT phosphorylation by inhibition of PI3K by wortmanin (WM), or LY294002 (LY), MEK1/2 by U0126 (U), PD98059 (PD). (F) Western blot analysis of GRB2 silencing obtained by transducing HUVECs with lentiviral vectors expressing GRB2 shRNA. (G) PI3K and AKT phosphorylation are dependent upon GRB2 expression. PI3K was immunoprecipitated with antibodies anti-PI3K and P85. The Ip was immunoblotted with anti–P-Tyr. P-AKT and P-JNKs were revealed with phosphospecific antibodies. IP indicates immunoprecipitated.