WORTMANNIN-SENSITIVE PHOSPHORYLATION, TRANSLOCATION, AND ACTIVATION OF PLC-GAMMA-1, BUT NOT PLC-GAMMA-2, IN ANTIGEN-STIMULATED RBL-2H3 MAST-CELLS

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE recep tor (Fc epsilon RI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P-3 production. Using immune complex phosphol ipase assays, we show that Fc epsilon RI cross-linking activates both PLC gamma 1 and PLC gamma 2. Activation is accompanied by the increase d phosphorylation of both PLC gamma isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLC gamma isoforms h ave distinct subcellular localizations. PLC gamma 1 is primarily cytos olic in resting RBL-2H3 cells, with low levels of plasma membrane asso ciation. After antigen stimulation, PLC gamma 1 translocates to the pl asma membrane where it associates preferentially with membrane ruffles . In contrast, PLC gamma 2 is concentrated in a perinuclear region nea r the Golgi and adjacent to the plasma membrane in resting cells and d oes not redistribute appreciably after Fc epsilon RI cross-linking. Th e activation of PLC gamma 1, but not of PLC gamma 2, is blocked by wor tmannin, a PI 3-kinase inhibitor previously shown to block antigen-sti mulated ruffling and to inhibit Ins(1,4,5)P-3 synthesis. in addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLC gamma 1 with little inhibiti on of PLC gamma 2 phosphorylation. Wortmannin also blocks the antigen- stimulated translocation of PLC gamma 1 to the plasma membrane. Our re sults implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLC gamma 1. Although less abundant than PLC gamma 2, a ctivated PLC gamma 1 may be responsible for the bulk of antigen-stimul ated Ins(1,4,5)P-3 production in RBL-2H3 cells.