Inhibition by melittin of phosphorylation by protein kinase C of annexin I from cow mammary gland

Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phospholipids and Ca**2+, that regulates a wide variety of biological functions by phosphorylating multiple protein substrates such as annexin I. Annexin I is a phospholipid/Ca**2+ -binding protein distributed in various tissues, including the mammary gland, and is thought to mediate the anti-inflammatory actions of glucocorticoids by inhibiting phospholipase A2. Melittin, a phospholipase A2 activator in bee venom, is known to inhibit PKC activity when Iysine-rich histone is used as the substrate. The purpose of the present study was to examine whether phosphorylation by PKC of annexin I from cow mammary gland was inhibited by melittin. Melittin inhibited annexin I phosphorylation by PKC in a dose-dependent manner, and its IC50 value (concentration causing 50% inhibition) was 0.8 microM. The phosphorylation of annexin I was also inhibited by the amphiphilic polypeptides mastoparan and polymyxin B, and their inhibitory effects were comparable to that of melittin. The surfaceinactive polypeptide bacitracin was less effective. The inhibition by melittin was effectively reversed by the excess addition of phosphatidylserine, but not distinctly by 1-oleoy1-2-acetyl-sn-glycerol or Ca**2+, suggesting that melittin inhibited the phosphorylation of annexin I by interacting with phosphatidylserine. The inhibition by melittin of PKC phosphorylation of annexin I seems to be pathophysiologically important, because a melittin-like phospholipase A2- stimulatory protein is present in bovine endothelial cells. "