Phospho-Y1063 recruits CRK and activates JNK1/2 via MKK4. (A) AKT and ERK1/2 activation by wild-type and mutant E-R3 chimeric receptors as indicated. Immunoblot analysis of HUVEC cell extracts untreated or treated with 10 ng/mL EGF for 20 minutes with specific antibodies as indicated. AKT activation was detected using phospho-S473 AKT-specific antibody, and ERK1/2 activation was revealed with phospho-T202/Y204 ERK-specific antibody. (B) Time-course analysis of CRK binding to ER-3 by coimmunoprecipitation. HUVECs transduced with E-R3 mutants as indicated either untreated or treated with 10 ng/mL EGF at the times indicated were immunoprecipitated with anti-CRK–specific antibodies and revealed with the anti–VEGFR-3 C-terminal domain. (C) CRK-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 CRK-SH2. (D) Time-course analysis of JNK activation in HUVECs transduced with wild-type E-R3 receptor and the receptor mutants as indicated. JNK activation was assessed by phosphorylation of threonine 183 and tyrosine 185 using phosphospecific antibodies. (E) HUVEC extracts assayed for JNK-dependent kinase activity toward recombinant GST–c-JUN 1-79 in vitro by phosphorylation of serine 63 and 73 with phosphospecific antibodies. Ponceau staining of Escherichia coli extracts is shown. (F) Inhibition of JNK activation in HUVECs transduced with E-R3 chimera by dominant-negative (DN) MAPKs as indicated transduced with a retroviral vector (MIGR1).