Src activity is required for Pyk2 phosphorylation in cells plated on fibronectin.

A, serum-starved HeLa cells were detached and kept in suspension in DMEM containing 0.5% BSA for 1 hr. Cells were then seeded on fibronectin- or poly-l-lysine (PLL)-coated dishes, and allowed to attach for the indicated times. Phosphorylation of Pyk2 and FAK was examined by immunoblot, as indicated. B, HeLa cells were kept in suspension with or without indicated Src inhibitors for 1 hr before the attachment to fibronectin-coated dishes for 15 min, and phosphorylation of Pyk2 and FAK was examined as above. C, HeLa cells transfected with mock, wild-type Src (Src-WT) or kinase-dead mutant (Src-KD) were kept in suspension for 1 hr then were attached to fibronectin-coated dishes for 15 min, and the phosphorylation of Pyk2 and FAK was examined by immunoblot as indicated. D, serum-starved SYF and Src++ MEFs were kept in suspension for 1 hr, then were attached to fibronectin-coated dishes for 15 or 30 min as indicated, and the phosphorylation of Pyk2 and FAK was examined as in above.