Multiple roles for PI 3-kinase in the regulation of PLCg activity and Ca 21 mobilization in antigen-stimulated mast cells

Abstract: Cross-linking the IgE-bound FceRI with polyvalent antigen leads to Ca21-dependent de-granulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FceRI-mediated tyro-sine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCg activity and is demonstrated by a dramatic inhibition of FceRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphoryla-tion of PLCg1 and by direct stimulation of both PLCg isoforms. In vitro stimulation of PLCg activ-ity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzy-matic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca21 influx. Compared with control cells, Ca21 re-sponses are markedly diminished in antigen-stimu-lated cells after wortmannin pretreatment. Differ-ences include both a longer lag time to the initial elevation in Ca21 after antigen and an inhibition of the sustained Ca21 influx phase. However, thapsigar-gin challenge during the sustained phase demon-strates no difference in the state of the Ca21 stores in antigen-stimulated cells in the presence or ab-sence of wortmannin. These data suggest that suffi-cient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca21 influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca21 influx in antigen-stimulated RBL-2H3 cells, one that is de-pendent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-ki-nase. J. Leukoc. Biol. 65: 321–329; 1999