REACTIVE OXYGEN SPECIES SIGNALING THROUGH REGULATION OF PROTEIN-TYROSINE PHOSPHORYLATION IN ENDOTHELIAL-CELLS

Tyrosine phosphorylation of proteins, controlled by tyrosine kinases a nd protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and c ytokines modulate cellular tyrosine phosphorylation to transmit signal s across the plasma membrane to the nucleus. Recent studies suggest th at 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 en dothelial cell (EC) metabolism and function, we exposed vascular ECs t o H2O2 Or H2O2 plus vanadate. This resulted in a time- and dose-depend ent 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 m itogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and F AK (125 kDa) by ROS. An immediate signaling response to increased prot ein tyrosine phosphorylation by ROS was activation of phospholipases s uch as A(2), C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptor s in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is criti cal to ROS-mediated signal transduction. These results provide evidenc e that ROS modulate EC signal transduction via a protein tyrosine phos phorylation-dependent mechanism.