Mutation of human Src-S51 to alanine reduces Src catalytic activity. (A) Chk1 kinase assay. Top: Autoradiography analysis (32P) of phosphorylated Src using purified WT or S51A GST-Src (1–256) substrate. Bottom: Western blot (WB) analysis of total GST. Top and bottom panels are from the same gel. (B) Alignment of Src protein sequences. Human serine 51 is marked by an asterisk. (C) Chk1 in vitro kinase assay. Western blot analysis of phosphorylated (pSrc-S51) by using the anti-pS51 antiserum along with total GST-Src. (D) In vitro kinase assay using recombinant GST-Chk1, His-Src, and purified GST-FAK (871–1020) as substrate. Western blot analysis of phosphorylated FAK-Y925 (pFAK-Y925) and pSrc-S51 using phosphospecific antibodies, and Ponceau staining of GST-FAK (871–1020) and His-Src. Relative band intensity values are shown, and values in the second lane from left (His-Src) were set to 1. (E) Immunoprecipitation (IP) kinase assay using GST-FAK (871–1020) as substrate. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation and immunoprecipitated GFP, and Ponceau staining of GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT were set to 1. Ab, antibody. (F) Immunoprecipitation-kinase assay in the absence or presence of siChk1. Top: Western blot analysis of GFP-associated FAK-Y925 phosphorylation, immunoprecipitated GFP, and GST-FAK (871–1020). Bottom: Western blot analysis of total GFP and actin. Relative band intensity values are shown, and values in GFP–Src-WT without siChk1 were set to 1.
