Multiple roles for PI 3-kinase in the regulation of PLCγ activity and Ca2+ mobilization in antigen-stimulated mast cells

Cross-linking the IgE-bound Fc epsilon RI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating due allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of Fc epsilon RI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLC gamma activity and is demonstrated by a dramatic inhibition of Fc epsilon RI-induced Ins(1,4,5)P-3 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)P-3 production in at least two ways: by promoting translocation and phosphorylation of PLC gamma 1 and by direct stimulation of both PLC gamma isoforms. In vitro stimulation of PLC gamma activity by PtdIns(3,4,5)P-3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin, These data suggest that sufficient Ins(1,4,5)P-3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.