Src is required to initiate adhesion-induced Pyk2 phosphorylation.

A, 293T cells were transfected with GFP-tagged Pyk2-WT, Pyk2-Y402F or Pyk2-KD, with or without Src, serum-starved, detached, and then reattached to fibronectin-coated dishes for 30 min. Cell lysates were collected and assayed by immunoblot to examine Pyk2 phosphorylation. B, SYF cells were transfected and assayed similarly as in A. C, SYF cells were co-transfected with Src and GFP-tagged Pyk2-WT, or Pyk2-Y402F, or Pyk2-KD as indicated. The expressed Src was immunoprecipitated, and the associated Pyk2 was examined by immunoblot. D, GST-Pyk2-WT and GST-Pyk2-Y402F were expressed in E. coli, purified, and employed in an in vitro kinase assay as described in Materials and Methods. Pyk2 phosphorylation by recombinant Src was examined by immunoblot analysis by using anti-phospho-Pyk2 (Y402) antibody.