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

  1. Viswanathan Natarajan,**
  1. Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, the Department of Medicine, Division of Pulmonary, Critical Care and Sleep, Ohio State University, Columbus, Ohio 43210, and Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21201
  1. ** To whom correspondence should be addressed: Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Mason F. Lord Bldg., Center Tower, Rm. 675, 5200 Eastern Ave., Baltimore, MD 21224. Tel.: 410-550-7748; Fax: 410-550-8571; E-mail: vnataraj{at}jhmi.edu.

Abstract

Superoxide (Formula) 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 Formula 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 Formula 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.

  • Received October 14, 2004.
  • Revision received February 28, 2005.
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This Article

  1. The Journal of Biological Chemistry 280, 20700-20711.
  1. All Versions of this Article:
    1. M411722200v1
    2. 280/21/20700 (most recent)

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