Reactive Oxygen Species Signaling through Regulation of Protein Tyrosine Phosphorylation in Endothelial Cells

Abstract

Tyrosine phosphorylation of proteins, controlled by tyrosine kinases and protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and cytokines modulate cellular tyrosine phosphorylation to transmit signals across the plasma membrane to the nucleus. Recent studies suggest that reactive oxygen species (ROS) also induce cellular protein tyrosine phosphorylation through receptor or nonreceptor tyrosine kinases. To determine whether protein tyrosine phosphorylation by ROS regulates endothelial cell (EC) metabolism and function, we exposed vascular ECs to H2O2 or H2O2 plus vanadate. This resulted in a time- and dose-dependent increase in protein tyrosine phosphorylation of several proteins (M(r) 21-200 kDa), as determined by immunoprecipitation and Western blot analysis with antiphosphotyrosine antibody. Immunoprecipitation with specific antibodies identified increased tyrosine phosphorylation of mitogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and FAK (125 kDa) by ROS. An immediate signaling response to increased protein tyrosine phosphorylation by ROS was activation of phospholipases such as A2, C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptors in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is critical to ROS-mediated signal transduction. These results provide evidence that ROS modulate EC signal transduction via a protein tyrosine phosphorylation-dependent mechanism.

Src Tyrosine Kinase Inhibitor PP2 Markedly Enhances Ras-independent Activation of Raf-1 Protein Kinase by Phorbol Myristate Acetate and H2O2

“…Since tyrosine phosphorylation has been shown to be an important event in Raf-1 activation (6, 7), and because one mechanism by which reactive oxygen species has been reported to modulate signal transduction pathway is via alteration in protein tyrosine phosphorylation (26,45), several tyrosine kinase inhibitors were tested to determine their relative effects on Raf-1 kinase activation by the combination of PMA and H 2 O 2 . In these studies v-Ha-ras-transformed cells were used because these cells were found to be more susceptible to PMA and H 2 O 2 activation than parental NIH 3T3 cells.…”

NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction

“…However, the mechanism underlying junctional protein phosphorylation is undetermined. Oxidant-dependent intracellular phosphorylation events are important in growth factor receptor activation and epithelial barrier function, 22,[34][35][36] but few studies have examined the response of individual junctional components to cytokine-induced oxidant activation. Our findings suggest that phosphorylation of VE-cadherin, the predominant component of endothelial adherens junctions, [37][38][39][40] proceeds through the production of endogenous oxidants.…”

Interactions of Oxidants With Vascular Signaling Systems

“…H 2 O 2 has been reported to stimulate multiple forms of vascular phospholipases (PLs), including PLA 2 , PLC, and PLD, 87,88 and ROS and RNS modulate the activity of arachidonic acid-metabolizing enzymes and directly modify lipids to species that are vasoactive. The signaling pathways through which ROS control the activity of PLs are rather poorly understood.…”