Role of Src kinase in diperoxovanadate-mediated activation of phospholipase D in endothelial cells

N. L. Parinandi, S. Roy, S. Shi, R. J. Cummings, A. J. Morris, J. G.N. Garcia, V. Natarajan

Research output: Contribution to journalArticle

24 Scopus citations

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 μM 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 μM) 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 μM) treatment. Also, Western blots of Src immunoprecipitates of control cells revealed the presence of PLD1 and PLD2, suggesting the association of PLD with Src kinase under basal conditions. However, exposure of cells to DPV (5μM) for 2 min enhanced the association of PLD2 but not PLD1 with Src. Western blotting of immunoprecipitates of PLD1 and PLD2 isoforms of control BPAECs revealed the presence of Src under basal conditions and exposure of cells to DPV (5 μM) for 2 min enhanced the association of PLD2 with Src in PLD2 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 PLD1 or PLD2 compared to protein kinase Cα 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.

Original languageEnglish (US)
Pages (from-to)231-243
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume396
Issue number2
DOIs
StatePublished - Dec 15 2001
Externally publishedYes

Keywords

  • Peroxovanadium
  • Phospholipase D
  • Protein tyrosine kinase
  • Src kinase
  • Vascular endothelium

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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