Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells.
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.
Natarajan
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Indiana University School of Medicine, Indianapolis, USA. dmagil@welchlink.welch.jhu.edu
Scribner
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al-Hassani
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Vepa
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D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010545:Peroxides / Q000633:toxicity; D010740:Phospholipases / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000633:toxicity
D000975:Antioxidants; D010545:Peroxides; D011506:Proteins; D017382:Reactive Oxygen Species; D017978:Receptors, Growth Factor; C091767:diperoxovanadate; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D010740:Phospholipases; D017027:Protein Tyrosine Phosphatases; D002118:Calcium