PHOSPHOLIPASE-D ACTIVITY IN PHAGOCYTIC LEUKOCYTES IS SYNERGISTICALLY REGULATED BY G-PROTEIN-BASED AND TYROSINE KINASE-BASED MECHANISMS

The regulation of phospholipase D (PLD)-type effector enzymes by G-pro teins and protein kinases/phosphatases was characterized in the U937 h uman promonocytic leucocyte line. PLD activity was assayed by measurin g (in the presence of 1 % ethanol) the accumulation of phosphatidyleth anol in cells permeabilized with beta-escin, a saponin-like detergent. Basal PLD activity was very low when cells were permeabilized and inc ubated in cytosol-like medium containing micromolar [Ca2+]. When this medium was supplemented with exogenous MgATP or guanosine 5'-[gamma-th io]triphosphate (GTP[S]), PLD activity increased by 9- and 14-fold res pectively. Cells permeabilized in the absence of exogenously added MgA TP, but in the presence of 1 muM vanadate/100 muM H2O2, also exhibited a modest 12-fold increase in PLD activity. However, the simultaneous presence of either GTP[S] plus exogenous MgATP or GTP[S] plus vanadate /H2O2 (and endogenous MgATP) induced similar 60-75-fold increases in t he rate and extent of phosphatidylethanol accumulation. These latter e ffects of vanadate/H2O2 were strongly correlated with the very rapid a ccumulation of multiple tyrosine-phosphorylated proteins. Other studie s utilized cells which were permeabilized in the presence of GTP[S] an d then washed before assay of PLD. These cells retained - 60 % of the MgATP-regulatable PLD activity (EC5O approximately 100 muM MgATP) obse rved in freshly permeabilized non-washed cells. In the absence of GTP[ S] pre-treatment, washed cells retained minimal PLD activity. Genistei n, a tyrosine kinase inhibitor, significantly attenuated the ability o f MgATP to stimulate PLD activity and accumulation of tyrosine-phospho rylated proteins in the washed GTP[S]-treated cells. These data sugges t that PLD activity in myeloid leucocytes involves co-ordinate regulat ion by both G-protein(s) and tyrosine phosphorylation.