Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D

Abstract

Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H2O2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H2O2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H2O2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 microM vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H2O2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H2O2-induced PLD activation in vascular endothelial cells.

Green Tea Polyphenols Precondition against Cell Death Induced by Oxygen-Glucose Deprivation via Stimulation of Laminin Receptor, Generation of Reactive Oxygen Species, and Activation of Protein Kinase Cϵ

“…The membrane association of PKC presumably is caused by phospholipid-hydrolysis products. ROS activate phospholipid-hydrolyzing enzymes such as phospholipases C and D (81,82). This results in the release of lipid second messengers, such as diacylglycerol, that induce the binding of PKC to the membrane (83).…”

Phospholipase D Activation by Sphingosine 1-Phosphate Regulates Interleukin-8 Secretion in Human Bronchial Epithelial Cells

“…Pretreatments were in MEM alone or with the specified agents for 1 h prior to stimulation, with the exception of PTX, which was 2 h. Cells were incubated in MEM containing 0.1% BSA with or without S1P or other agents at the indicated concentrations and in the presence of 0.05% butanol for the specified lengths of time. Incubations were terminated by the addition of 1 ml of methanol:HCl (100:1 v/v), and lipids were extracted in chloroform:methanol (2:1 v/v) (33). [ 32 P]Phosphatidylbutanol (PBt) formed as a result of PLD activation, and concomitant transphosphatidylation reaction (an index of in vivo PLD activation) was separated by TLC with the upper phase of ethyl acetate:2,2,4-trimethyl pentane:glacial acetic acid:water (65:10: 15:50 by volume) as the developing solvent system.…”

Implication of Phospholipase D2 in Oxidant-induced Phosphoinositide 3-Kinase Signaling via Pyk2 Activation in PC12 Cells

“…The major signaling pathways and/or key mediators to influence survival of cells subjected to oxidant injury are the phosphorylation cascades leading to activation of tyrosine phosphorylation of several growth factor receptors and their subsequent downstream signaling events, including Akt and mitogen-activated protein kinases (4 -9). A number of studies have demonstrated that hydrogen peroxide (H 2 O 2 ) stimulates phospholipase D (PLD), 1 which generates phosphatidic acid (PA), a second messenger in various types of cell, such as endothelial cells (10,11), fibroblasts (12,13), PC12 cells (14 -17), vascular smooth muscle cells (18), and leukemia L1210 cells (19). As the mechanisms underlying the oxidant-induced PLD activation, various factors including protein-tyrosine kinase (10,11,13,14), protein kinase C (PKC) (13,16,19), and mitogen-activated protein kinase (15,17) have been proposed.…”