Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca^(2+)-signalling induced by Fcy receptor cross-linking in human neutrophils

Abstract

The effect of wortmannin on IgG-receptor (FcyR)-mediated stimulation of human neutrophils was investigated. The Ca^(2+) influx induced by clustering of both Fcy receptors was inhibited by wortmannin, as was the release of Ca^(2+) from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P3 observed after FcyR stimulation, but did not affect the increase in Ins(1,4,5)P3 induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca^(2+) signalling, induced by FcyR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of PtdIns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after FccR cross-linking, even before the onset of the Ca^(2+) response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by cocross- linking of either FcyRIIa and FcyRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of PtdIns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcyR cross-linking in human neutrophils.