PHOSPHORYLATION AND SUBCELLULAR REDISTRIBUTION OF PLECKSTRIN IN HUMANNEUTROPHILS

Pleckstrin, originally described as a major substrate of protein kinas e C (PKC) in platelets, was found to be highly expressed in human neut rophils (intracellular concentration, similar to 15 mu M). As PKC isof orms play an important role in mediating neutrophil antimicrobial resp onses, we studied the regulation of pleckstrin phosphorylation in resp onse to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, plec kstrin was rapidly phosphorylated, which resulted in a shift in its el ectrophoretic mobility. Several lines of evidence suggest that pleckst rin is phosphorylated in part by a nonconventional PKC following stimu lation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the d uration of phosphorylation, while the phosphatidic acid phosphohydrola se antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the pho sphorylation of pleckstrin. Both PKC delta and -zeta are expressed in human neutrophils, Increased association of pleckstrin with both micro somes and with the cytoskeleton was observed in stimulated cells. Thes e findings suggest that phosphorylation by nonconventional PKC isoform s induces a conformational change in pleckstrin that promotes its inte raction with membranes and/or with the cytoskeleton. Such a translocat ion may serve to target proteins or lipids recognized by pleckstrin ho mology domains to sites where they can contribute to the microbicidal response.