Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxov anadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of D PV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH 2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion . Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or tran sient transfection with a p38 dominant negative mutant mitigated the PLD ac tivation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p3 8 MAPK activity as determined by activated transcription factor-2 phosphory lation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antib odies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-chall enged ECs. Binding assays demonstrated interaction of glutathione S-transfe rase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phospho rylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated reg ulation of PLD in ECs.