title: Vinculin, a cytoskeletal substrate of protein kinase C.
abstract: Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin; D016596:Vinculin
chemical_list: D009124:Muscle Proteins; D010446:Peptide Fragments; D016596:Vinculin; D011494:Protein Kinases; D011493:Protein Kinase C; D014357:Trypsin