CAP is phosphorylated by and coimmunoprecipitates with c-Abl. A: Hep3B cells were starved, treated with insulin or vanadate, and CAP was immunoprecipitated with specific antibodies. Tyr phosphorylation of CAP was analyzed by Western blot. Endogenous CAP was phosphorylated after vanadate treatment but not in starved cells or after insulin stimulation. B: Schematic presentation of the CAP constructs based on the isoform CAP1 used in this study. See text for details. C: HeLa cells were cotransfected with CAP-EGFP or EGFP and either c-Abl WT, kinase-dead mutant (Abl KD) or constitutively active Abl (Abl PP). CAP was immunoprecipitated with GFP antibodies and coprecipitation of Abl was detected by means of Western blot (uppermost panel). Abl WT and PP were coprecipitated with CAP whereas Abl KD was not. D: Coprecipitation of Abl WT with various mutants of CAP. Abl WT was found to coprecipitate with full-length CAP, and somewhat less with the third SH3 domain mutant W660F and the 3 × WF mutant in which all three SH3 domains of CAP have been mutated. No coprecipitation was observed with CAPΔSH3 or EGFP only.