TEMPORAL REGULATION OF THE IGE-DEPENDENT 1,2-DIACYLGLYCEROL PRODUCTION BY TYROSINE KINASE ACTIVATION IN A RAT (RBL 2H3) MAST-CELL LINE

We explored the possible role of tyrosine kinases in the IgE-dependent regulation of 1,2-diacylglycerol (DAG) production in RBL 2H3 cells. When triggered via their high-affinity IgE receptors (Fc epsilon RI), there was a rapid phosphorylation of tyrosine residues on a number of proteins. The phosphorylation of these proteins and ultimately histamine release were inhibited in a concentration-dependent manner by the tyrosine kinase inhibitor, tyrphostin. In cells labelled with [H-3]myristic acid, we observed a characteristic biphasic increase in [H-3]DAG production. In the presence of tyrosine kinase inhibitor, the initial increase in DAG was still observed, but the secondary increase, which was dependent on phosphatidylcholine-specific phospholipase D (PC-PLD) activation, was completely abolished. Tyrphostin significantly inhibited IgE-dependent activation of PC-PLD, suggesting that PC-PLD activation was regulated by tyrosine phosphorylation. Furthermore, when proteins from RBL 2H3 cells were immunoprecipitated with an antiphosphotyrosine antibody, PC-PLD activity was recovered from the immunoprecipitated fraction. These results demonstrate that the secondary, but not the initial, phase of 1,2-DAG production in response to Fc epsilon RI aggregation is regulated by the initial activation of tyrosine kinases and that PC-PLD may be regulated directly by this mechanism.