Multiple roles for PI 3-kinase in the regulation of PLC gamma activity andCa2+ 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 t he regulation of signaling events that lie downstream of Fc epsilon RI-medi ated 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 inh ibition of Fc epsilon RI-induced Ins(1,4,5)P-3 production after treatment o f 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 way s: by promoting translocation and phosphorylation of PLC gamma 1 and by dir ect stimulation of both PLC gamma isoforms. In vitro stimulation of PLC gam ma activity by PtdIns(3,4,5)P-3 synergizes with activation by in vivo tyros ine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compar ed with control cells, Ca2+ responses are markedly diminished in antigen-st imulated cells after wortmannin pretreatment. Differences include both a lo nger lag time to the initial elevation in Ca2+ after antigen and an inhibit ion of the sustained Ca2+ influx phase. However, thapsigargin challenge dur ing the sustained phase demonstrates no difference in the state of the Ca2 stores in antigen-stimulated cells in the presence or absence of wortmanni n, These data suggest that sufficient Ins(1,4,5)P-3 is synthesized in wortm annin-treated cells to mobilize intracellular calcium stores and, furthermo re, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at le ast two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, o ne that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.