TYROSINE PHOSPHORYLATION-DEPENDENT ACTIVATION OF PHOSPHATIDYLINOSITIDE 3-KINASE OCCURS UPSTREAM OF CA2-SIGNALING INDUCED BY FC-GAMMA RECEPTOR CROSS-LINKING IN HUMAN NEUTROPHILS()

The effect of wortmannin on IgG-receptor (Fc gamma R)-mediated stimula tion of human neutrophils was investigated. The Ca2+ influx induced by clustering of both Fc gamma receptors was inhibited by wortmannin, as was the release of Ca2+ from intracellular stores. Wortmannin also in hibited, with the same efficacy, the accumulation of Ins(1,4,5)P-3 obs erved after Fc gamma R stimulation, but did not affect the increase in Ins(1,4,5)P-3 induced by the chemotactic peptide, formyl-methionine-l eucine-phenylalanine. Because wortmannin is, in the concentrations;use d here, an inhibitor of PtdIns 3-kinase, these results suggested a rol e for PtdIns 3-kinase upstream of Ca2+ signalling, induced by Fc gamma R cross-linking. Support for this notion was obtained by investigatin g the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and b y studying the kinetics of PtdIns 3-kinase activation. We found transl ocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after Fc gamma R cro ss-linking, even before the onset of the Ca2+ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either Fc gamma RIIa and Fc gamma RIIIb with th e tyrosine phosphatase, CD45, indicating a requirement for protein tyr osine phosphorylation in the recruitment of PtdIns 3-kinase to the pla sma membrane. Taken together, our results suggest a role for PtdIns 3- kinase in early signal transduction events after Fc gamma R cross-link ing in human neutrophils.