Expression of the protein kinase C substrate pleckstrin in macrophages: Association with phagosomal membranes

Despite evidence suggesting that protein kinase C (PKC) isoforms are import ant in phagocytosis by Fc gamma receptors, the mechanisms by which the subs trates of these kinases act are largely unknown. We have investigated the r ole of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage l ineage, Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma, In unstimulated cells, the prot ein was largely confined to the cytosol, Upon ingestion of IgG-opsonized zy mosan particles (OPZ,), however, pleckstrin accumulated on the phagosomal m embrane, This association was transient, being maximal after 15 min and dec lining thereafter. Similar kinetics of association was also seen for both f ilamentous actin and the delta isoform of PRC, Ingestion of OPZ was found t o induce phosphorylation of pleckstrin, To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-II A cells that stably express the Fc gamma RIIA receptor and are competent fo r phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutant s in which the phosphoacceptor sites had been mutated to either alanine (no nphosphurylatable) or glutamine (pseudophosphorylated) were found to accumu late on OPZ phagosomes, Thus, association of pleckstrin with phagosomes is independent of its phosphorylation, Our findings suggest that pleckstrin ma y serve as an intracellular adaptor/targeting protein in response to partic ulate stimuli. By targeting interacting ligands to the phagosomal compartme nt, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome.