Lipocortin I is not accessible for protein kinase C bound to the cytoplasmic surface of the plasma membrane in streptolysin-O-permeabilized pig granulocytes.

We previously observed a 38 kDa protein that was a major protein component of the cytosolic extract of pig granulocytes and the dominant substrate of protein kinase C at supra-physiological Ca2+ concentrations. The purified 38 kDa protein itself required Ca2+ to be phosphorylated by protein kinase C. Now we demonstrate that this protein, which is also present in human granulocytes, is identical to lipocortin I. The identification is based on the chromatographic properties and immunoblot of the purified protein which is also a good substrate for tissue transglutaminase. Phosphorylation of lipocortin I by protein kinase C was investigated in granulocytes permeabilized with streptolysin-O. At physiological intracellular Ca2+ concentrations lipocortin I was not phosphorylated at all. At supra-physiological Ca2+ concentrations (0.5 mM), lipocortin I was also not phosphorylated when protein kinase C was translocated to the membrane by treatment of the cells with phorbol myristate acetate. Its phosphorylation was detectable only in control experiments when protein kinase C was activated in the cytosol by the addition of dioleoylglycerol and phosphatidylserine to the permeabilized cells. The data presented show that, in permeabilized granulocytes, Ca(2+)-lipocortin is not formed at physiological Ca2+ concentrations, and at supra-physiological Ca2+ concentrations the Ca(2+)-lipocortin I is not accessible to protein kinase C bound to the cytoplasmic surface of the plasma membrane.