Src-mediated Tyrosine Phosphorylation of p47phox in Hyperoxia-induced Activation of NADPH Oxidase and Generation of Reactive Oxygen Species in Lung Endothelial Cells

• Published in: The Journal of biological chemistry Vol. 280; no. 21; pp. 20700 - 20711
• Main Authors: Chowdhury, Ashis K., Watkins, Tonya, Parinandi, Narasimham L., Saatian, Bahman, Kleinberg, Michael E., Usatyuk, Peter V., Natarajan, Viswanathan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.05.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Blotting, Western; Cell Line; CSK Tyrosine-Protein Kinase; Endothelial Cells - enzymology; Endothelial Cells - metabolism; Enzyme Activation - drug effects; Enzyme Inhibitors - pharmacology; Green Fluorescent Proteins - genetics; NADPH Oxidases - metabolism; Oxygen - administration & dosage; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Phosphotyrosine - analysis; Protein-Tyrosine Kinases - analysis; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - metabolism; Pulmonary Artery; Pyrimidines - pharmacology; Reactive Oxygen Species - metabolism; Recombinant Fusion Proteins - metabolism; RNA, Small Interfering - pharmacology; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - metabolism; Superoxides - metabolism; Transfection; Tyrosine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M411722200

Superoxide ( O2−˙) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47phox, the role of tyrosine phosphorylation of p47phox in NADPH oxidase-dependent O2−˙ production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47phox and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O2−˙ and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 μm) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47phox and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47phox in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47phox and p67phox, suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47phox, but not p67phox, with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47phox regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.