Fig. 6. Crk rapidly binds the phopshorylated EphA3 ID and is able to distinguish between ligand-independent and ligand-mediated receptor phosphorylation. (A) Coomassie-stained SDS PAGE (top) and anti-PY western blot (bottom) illustrating the phosphorylated EphA3 ID as purified from the E. coli (1), dephosphorylated by TC45Pase (2) and rephosphorylated by the addition of ATP and Mg²⁺ (3). (B) Ephrin-A5-dependent association of Crk with endogenous EphA3 in LiBr melanoma cells. Adherent cultures of LiBr cells were treated for 10 minutes with preclustered ephrin-A5 Fc (+) or left untreated (-). Endogenous Crk in cell lysates was immunoprecipitated with anti-CrkII antibody and western blots probed with the same antibody to detect total CrkII or with anti-EphA3 antibodies to detect CrkII-associated EphA3. Parallel samples were immunoprecipitated with anti-EphA3 Mab and western blots probed for EphA3 and phosphotyrosine as indicated. (C) Dose-response of ephrin-A5 stimulated CrkII recruitment to EphA3. EphA3-transfected 293T cells were transferred to tissue culture wells containing surface-bound ephrin-A5 Fc at the indicated concentrations for 10 minutes, and endogenous CrkII in the cell lysates was immunoprecipitated with anti-Crk antibody. In parallel samples, the cells left in suspension were left untreated (-) or stimulated with 10 nM preclustered ephrin-A5 in solution (+). Blots of whole-cell lysates and of precipitated proteins were probed with anti-EphA3 and anti-CrkII antibodies as indicated. (D) Time course of CrkII binding to EphA3. EphA3 was (co) immunoprecipitated from lysates of EphA3 293T cells, which had been treated for the indicated times with 1.5 µg/ml of preclustered ephrin-A5 Fc, and analysed by western blot with anti- EphA3 and anti-phosphotyrosine antibodies as indicated. (E) 293T cells were left nontransfected (nil) or were transiently transfected with cDNAs encoding single, double (2x) or triple (3x) Tyr (Y)-Phe(F) mutants of EphA3. Anti-EphA3 immunoprecipitates from lysates of cells stimulated for 10 minutes with preclustered ephrin-A5 Fc (+) or left untreated (-), were analysed by western blot probed with anti-PY antibody. As loading control the total lysate was analysed by anti EphA3 western blot. Endogenously expressed Crk was immunoprecipitated from the same lysates and analysed by immunoblot for associated EphA3. (F) Crk was extracted from lysates of Crk-transfected 293T cells using Sepharose-coupled phosphopeptide Y596, 602P (Y2xP-Seph) or the nonphosphorylated peptide as control (Y2x-Seph) and analysed by western blot using the anti-Crk monoclonal antibody (left panel). The CrkII band in the crude cell lysate is shown in a parallel lane. Lysates from ephrin-A5 Fc stimulated cells were immunoprecipitated with anti-Crk antibody in the absence (nil) or presence of 50 µM of EphA3-derived phosphopeptides as indicated in the figure (right panel). Immunoprecipitates were analysed by western blots probed with antibodies to EphA3.