WORTMANNIN BLOCKS LIPID AND PROTEIN-KINASE ACTIVITIES ASSOCIATED WITHPI-3-KINASE AND INHIBITS A SUBSET OF RESPONSES INDUCED BY FC-EPSILON-R1 CROSS-LINKING

We have investigated the effects of wortmannin, an inhibitor of phosph atidylinositol 3-kinase (PI S-kinase), on antigen-mediated signaling i n the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cyt oplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secreti on, membrane ruffling, spreading, pinocytosis, and the formation of ac tin plaques implicated in increased cell-substrate adhesion. In additi on, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previous ly shown that wortmannin causes a dose-dependent inhibition of PI S-ki nase activity and also inhibits antigen-stimulated degranulation. We r eport that the antigen-induced synthesis of inositol(1,4,5)P-3 is also markedly inhibited by wortmannin. Consistent with evidence in other c ell systems implicating phosphatidylinositol (3,4,5)P-3 in ruffling, p retreatment of RBL-2H3 cells with wortmannin inhibits membrane rufflin g and fluid pinocytosis in response to Fc epsilon R1 cross-linking. Ho wever, wortmannin does not inhibit antigen-induced actin polymerizatio n, receptor internalization, or the actin-dependent processes of sprea ding and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsi lon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mito gen-activated protein kinase as measured by in vitro kinase assays. St rikingly, there is substantial in vitro serine/threonine kinase activi ty in immunoprecipitates prepared from Fc epsilon R1-activated cells u sing antisera to the p85 subunit of PI 3-kinase. This activity is inhi bited by pretreatment of the cells with wortmannin or by the direct ad dition of wortmannin to the kinase assay, suggesting that PI S-kinase itself is capable of acting as a protein kinase. We conclude that Fc e psilon R1 cross-linking activates both lipid and protein kinase activi ties of PI 3-kinase and that inhibiting these activities with wortmann in results in the selective block of a subset of Fc epsilon R1-mediate d signaling responses.