Figure 6

CAP is a substrate of Src kinase. A: Purified CAP-GST or GST was incubated with recombinant, purified Src kinase and [γ-³²P]-ATP, run into SDS-PAGE gel and analyzed by autoradiography. Specific phosphorylation of CAP-GST could be detected after incubation with active Src but not after inhibition of Src activity with PP2. Autophosphorylation of Src was used as an internal control for successful inhibition. B: CAP-EGFP or EGFP were immunoprecipitated from cells treated with vanadate and either Src inhibitors PP1 or PP2 or a non-inhibiting analogue PP3. Inhibition of endogenous Src activity clearly reduced the Tyr phosphorylation of CAP-EGFP. C: Coexpression of active forms of Src kinase (c-Src or Y527F-Src) notably increased the Tyr-phosphorylation of CAP-EGFP as compared to cells coexpressing the inactive Src mutant (Dead-Src). D: Intact SH3 domains of CAP are not necessary for the phosphorylation of CAP by Src. CAP-EGFP or CAP mutants carrying mutations in the SH3 domains were coexpressed and the phosphorylation of CAP was studied after immunoprecipitation. Mutation of the critical tryptophane in the third SH3 domain (W660F-CAP-EGFP) or in all three SH3 domains (3xWF-CAP-EGFP) exhibited no effect on the degree of Tyr phosphorylation of CAP by Src. E: Active forms of Src (c-Src and Y527F-Src) were coprecipitated with WT CAP and to a severely reduced degree with the ΔSH3 mutant of CAP, whereas kinase dead Src did not associate with either the WT or ΔSH3 CAP.