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-Leu-Phe, it was possible to detect a time-dependent increase in the hydrolytic (as measured by the production of phosphatidic acid, PA) and the transphosphatidylation (as measured by the production of phosphatidylethanol, PEt) activities of phospholipase D in intact cells prelabeled with a radioactive fatty acid. Both activities were inhibited by preincubation of cells with genistein. Appropriate conditions were developed 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-Phe-treated cells. Conversely, no activity was found in lysates immunoprecipitated with an irrelevant antibody (GTPase-activating protein, GAP) that nevertheless was able to recognize a tyrosylphosphorylated form of GAP, as demonstrated by western blotting. These data suggest that a PCho-PLD, or a tightly bound protein, is tyrosine phosphorylated during cell activation.
Gomez-Cambronero
J
J
Department of Physiology, University of Connecticut Health Center, School of Medicine, Farmington 06030, USA.
D000911:Antibodies, Monoclonal; D002250:Carbon Radioisotopes; D020404:Glycerophospholipids; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D010712:Phosphatidic Acids / Q000096:biosynthesis*; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000276:immunology* / Q000378:metabolism; D019000:Phosphotyrosine / Q000276:immunology*; D011233:Precipitin Tests; D013268:Stimulation, Chemical; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D002250:Carbon Radioisotopes; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C051521:phosphatidylethanol; D019000:Phosphotyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010739:Phospholipase D