Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles.
Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.
D000818:Animals; D002746:Chlorpromazine / Q000494:pharmacology; D007525:Isoelectric Focusing; D009124:Muscle Proteins / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src); D010716:Phosphatidylinositols / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D016596:Vinculin; D014764:Viral Proteins / Q000378:metabolism*
D009124:Muscle Proteins; D010716:Phosphatidylinositols; D010743:Phospholipids; D014764:Viral Proteins; D016596:Vinculin; D015688:Oncogene Protein pp60(v-src); D014357:Trypsin; D002746:Chlorpromazine