Figure 5

FIT-mediated specific phosphorylation of p130Cas leads to activation of Rac1. 293T cells were transfected with vectors expressing Rac1 and other proteins as indicated. Cell lysates were subjected to GST-PBD pull-down assay (top panel) or immunoprecipitation (IP; middle panel) and analyzed by western blot. Membranes were probed with anti-Rac1 (top panel) or anti-pTyr (middle panel). Whole cell lysates (WCL) were also probed by anti-Rac1 to determine the total amount of Rac1 expressed (bottom panel). FIT-mediated phosphorylation of p130Cas and subsequent activation of Rac1 is predicted to occur only in cells co-expressing FIT-compatible Src (ZipA-ΔSrc) and Myc-tagged FIT-compatible p130Cas (Myc-ZipB-p130Cas). ΔSrc = v-Src lacking SH2 and SH3 domains. Approximate position of molecular weight markers (kDa) is shown on left.