FIG. 6.

A dominant-negative form of PYK2 blocks Semaphorin 4D/Plexin-B1-mediated Akt and Src phosphorylation and chemotaxis. (A) Transfected cells show immunoreactivity for PYK2, indicating overexpression of the kinase-dead, dominant-negative version of PYK2 (DN-PYK2), compared to empty vector-transfected controls (pCEFL). (B) Cells transfected with empty vector control DNA (C) or kinase-dead dominant-negative PYK2 (DN-PYK2) were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt and Src. Phospho-Akt [WB: P-Akt (Ser 473); upper panels] and phospho-Src (WB: P-Src; lower panels) were seen in response to Semaphorin 4D treatment of control cells but not in those expressing DN-PYK2. Total Akt and Src were used as loading controls. (C) Endothelial cells transfected with empty vector control DNA (vector) or kinase-dead dominant-negative PYK2 (DN-PYK2) were used in a cell migration assay with 200 ng/ml Semaphorin 4D as the chemoattractant (S4D). Media containing 10% fetal bovine serum (S) were used as positive controls for migration. The bars represent the fold increase of migration as determined by densitometry relative to that seen in negative control wells containing 0.1% BSA (C).