title: Role of Src kinase in diperoxovanadate-mediated activation of phospholipase D in endothelial cells.
abstract: We have shown earlier that oxidant-induced activation of phospholipase D (PLD) in vascular endothelial cells (ECs) is regulated by protein tyrosine kinases. To further understand the regulation of oxidant-induced PLD activation, we investigated the role of Src kinase. Treatment of bovine pulmonary artery ECs (BPAECs) with a model oxidant, diperoxovanadate (DPV), at 5 microM concentration, for 30 min, stimulated PLD activity (four- to eightfold), which was attenuated by tyrosine kinase inhibitors and by Src kinase-specific inhibitors PP-1 and PP-2, in a dose- and time-dependent fashion. Furthermore, BPAECs exposed to DPV (5 microM) for 2 min showed activation of Src kinase as observed by increased tyrosine phosphorylation and autophosphorylation in Src immunoprecipitates, which was attenuated by PP-2. Src immunoprecipitates of cell lysates from control BPAECs exhibited PLD activity in cell-free preparations, which was Arf- and Rho-sensitive and was enhanced at 2 min of DPV (5 microM) treatment. Also, Western blots of Src immunoprecipitates of control cells revealed the presence of PLD(1) and PLD(2), suggesting the association of PLD with Src kinase under basal conditions. However, exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) but not PLD(1) with Src. Western blotting of immunoprecipitates of PLD(1) and PLD(2) isoforms of control BPAECs revealed the presence of Src under basal conditions and exposure of cells to DPV (5 microM) for 2 min enhanced the association of PLD(2) with Src in PLD(2) immunoprecipitates. Transient expression of a dominant negative mutant of Src in BPAECs attenuated DPV- but not TPA-induced PLD activation. In cell-free preparations, Src did not phosphorylate either PLD(1) or PLD(2) compared to protein kinase Calpha or p38 mitogen-activated protein kinase. These data show for the first time a direct association of Src with PLD in ECs and regulation of PLD in intact cells.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Asthma and Allergy Center, Johns Hopkins University, 5501 Bayview Circle, Baltimore, MD 21224, USA. nparinam@mail.jhmi.edu
authors.forename: N L
authors.initials: NL
authors.lastname: Parinandi
authors.forename: S
authors.initials: S
authors.lastname: Roy
authors.forename: S
authors.initials: S
authors.lastname: Shi
authors.forename: R J
authors.initials: RJ
authors.lastname: Cummings
authors.forename: A J
authors.initials: AJ
authors.lastname: Morris
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
authors.forename: V
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002474:Cell-Free System; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D006873:Hydroquinones / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007313:Insecta; D007527:Isoenzymes / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D010545:Peroxides / Q000378:metabolism*; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011651:Pulmonary Artery / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014638:Vanadates / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D006873:Hydroquinones; D007004:Hypoglycemic Agents; D007527:Isoenzymes; D010545:Peroxides; D020033:Protein Isoforms; C091767:diperoxovanadate; C069276:peroxovanadate; D014638:Vanadates; D019061:src-Family Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C048212:erbstatin