IMMUNOPRECIPITATION OF A PHOSPHOLIPASE-D ACTIVITY WITH ANTIPHOSPHOTYROSINE ANTIBODIES

When granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated human neutrophils were challenged with the chemotactic factor fMet-Le u-Phe, it was possible to detect a time-dependent increase in the hydr olytic (as measured by the production of phosphatidic acid, PA) and th e transphosphatidylation (as measured by the production of phosphatidy lethanol, PEt) activities of phospholipase D in intact cells prelabele d with a radioactive fatty acid, Both activities were inhibited by pre incubation of cells with genistein, Appropriate conditions were develo ped to test the PLD transphosphatidylation activity against exogenous phosphatidylcholine (PCho) in an in vitro system, As in intact cells, increased PLD activity could be detected in cell lysates obtained from fMet-Leu-Phe-treated cells compared with controls, When lysates were immunoprecipitated with antiphosphotyrosine antibodies, a PLD activity was found only in immune complexes that were prepared from fMet-Leu-P he-treated cells, Conversely, no activity was found in lysates immunop recipitated with an irrelevant antibody (GTPase-activating protein, GA P) that nevertheless was able to recognize a tyrosylphosphorylated for m of GAP, as demonstrated by western blotting, These data suggest that a PCho-PLD, or a tightly bound protein, is tyrosine phosphorylated du ring cell activation.