PHOSPHORYLATION OF VITRONECTIN ON SER362 BY PROTEIN-KINASE-C ATTENUATES ITS CLEAVAGE BY PLASMIN

Vitronectin, found in the extracellular matrix and in circulating bloo d, has an important role in the control of plasminogen activation. It was shown to be the major protein substrate in human blood fluid for a protein kinase A (PKA) released from platelets upon their physiologic al stimulation with thrombin. Since vitronectin was shown to have only one PKA phosphorylation site, but to contain 2-3 mol covalently bound phosphate, it was reasonable to assume that other protein kinases mig ht phosphorylate vitronectin at other sites in the protein. We have re ported earlier that human serum contains at least three protein kinase s, one of which was found to be cAMP independent and to phosphorylate a repertoire of plasma proteins that was very similar to that obtained upon phosphorylation of human plasma with protein kinase C (PKC). Sin ce there are now several examples of proteins with extracellular funct ions that are phosphorylated by PKC, we undertook to study the phospho rylation of vitronectin by PKC. Here, we show that vitronectin is a su bstrate for PKC, and characterize the kinetic parameters of this phosp horylation (K-m approximate to tenfold lower than the concentration of vitronectin in blood), indicating that, from the biochemical point of view, this phosphorylation can occur at the locus of a hemostatic eve nt. We also identify Ser362 as the major PKC phosphorylation site in v itronectin, and confirm this localization by means of synthetic peptid es derived from the cluster of basic amino acids in vitronectin surrou nding Ser362. We show that the PKC phosphorylation at Ser362 alters th e functional properties of vitronectin, attenuating its cleavage by pl asmin at Arg361-Ser362. This phosphorylation has the potential to regu late plasmin production from plasminogen by a feedback mechanism invol ving the above-mentioned plasmin cleavage, a loosening of the vitronec tin grip on inhibitor 1 of plasminogen activators, and a subsequent la tency of this regulatory inhibitor.