PLATELET-ACTIVATING-FACTOR INDUCES THE TYROSINE PHOSPHORYLATION AND ACTIVATION OF PHOSPHOLIPASE C-GAMMA-1, FYN AND LYN KINASES, AND PHOSPHATIDYLINOSITOL 3-KINASE IN A HUMAN B-CELL LINE

Platelet-activating factor (PAF) is a versatile lipid mediator of infl ammation in a variety of biologic systems. We have previously reported that one of the earliest events in the signal transduction pathway of PAF in a human B lymphoblastoid cell line was the induction of tyrosi ne kinase activity concomitant with the activation of phospholipase C (PLC). We now demonstrate the occurrence of multiple tyrosine phosphor ylation-dependent events which follow the interaction of PAF with its receptor on B cells. Anti-phosphotyrosine immunoprecipitates from lysa tes of PAF-stimulated cells, when fractionated by SDS-PAGE and analyze d by Western blotting with anti-PLC-gamma 1, showed that maximal tyros ine phosphorylation of this enzyme occurred within 2 min of stimulatio n. This phenomenon was verified by immunoprecipitating with anti-PLC-g amma 1 and subsequently probing with anti-phosphotyrosine. Immunopreci pitation of the tyrosine kinases, Fyn and Lyn, from PAF-stimulated cel ls, and use of these immunoprecipitates in kinase assays established t hat the activation of both kinases also occurred within the first 2 mi n of stimulation with phosphorylation occurring on their tyrosine resi dues. Additionally, we also provide evidence for the tyrosine phosphor ylation of the p85 subunit of phosphatidylinositol 3-kinase (Ptdlns 3- kinase) and activation of this kinase by PAF in a dose-dependent manne r, maximal activation occurring within 10 min poststimulation. We have thus demonstrated that the activation of tyrosine kinases is an impor tant proximate step in PAF-mediated signal transduction in B cells, le ading to tyrosine phosphorylation and activation of PLC-yl, Fyn and Ly n kinases, and Ptdlns 3-kinase.