Phosphorylation at tyrosine 733 of ACAP4 induces its association with Grb2. A, EGF stimulation strengthens the ACAP4-Grb2 association. HeLa cells overexpressing FLAG-ACAP4 and GFP-Grb2 were subjected or not to EGF stimulation for 5 min. Extracts of the cells were absorbed by FLAG-M2 antibody, and co-precipitated GFP-Grb2 was detected by immunoblotting (upper panel, GFP blot; lower panel, FLAG blot). B, tyrosine 733 on ACAP4 is phosphorylated after EGF stimulation in HeLa cells. FLAG-ACAP4 and FLAG-ACAP4Y733F were immunoprecipitated from cells with or without EGF stimulation. Samples were then immunoblotted with antibody against phosphotyrosine. C, Y733F mutation of ACAP4 reduces its association with Grb2. GST-Grb2-bound affinity matrix was incubated with lysates of cells transfected with GFP, GFP-ACAP4, and GFP-ACAP4Y733F, respectively. After washing five times, the affinity matrix was boiled in SDS-PAGE buffer and subjected to SDS-polyacrylamide gel followed by Western blotting with GFP antibody (upper panel, GFP blot; lower panel, Ponceau staining).