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

Abstract

Protein kinase C (PKC) is a phosphotransferase activated by diacylglycerols, phospholipids and Ca2+, that regulates a wide variety of biological functions by phosphorylating multiple protein substrates such as annexin I. Annexin I is a phospholipid/Ca2+-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 lysine-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 μM. 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 surface-inactive polypeptide bacitracin was less effective. The inhibition by melittin was effectively reversed by the excess addition of phosphatidylserine, but not distinctly by 1-oleoyl-2-acetyl-sn-glycerol or Ca2+, 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.