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

Ashis K Chowdhury,Tonya Watkins,Narasimham L Parinandi,Bahman Saatian,Michael E Kleinberg,Peter V Usatyuk,Viswanathan Natarajan

doi:10.1074/jbc.m411722200

Abstract

Superoxide (O(2)(-)) 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 p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) 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 p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) 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 mum) 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 p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.

Highlights

Superoxide (O2.) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system
We addressed the role of the nonreceptor tyrosine kinase, Src, in mediating tyrosine phosphorylation of p47phox and regulation of NADPH oxidase activation in endothelial cells (ECs) exposed to hyperoxia
human pulmonary artery endothelial cells (HPAECs) were exposed to either normoxia or hyperoxia for 2 h, and cell lysates (500 ␮g of protein) were subjected to immunoprecipitation with c-Src or Fyn or Yes or Lyn antibodies, and the immunoprecipitates were analyzed for kinase activity using a synthetic Src peptide substrate and [␥-32P]ATP according to the manufacturer’s instruction

Summary

Introduction

Superoxide (O2.) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Inhibition of Src activation by PP2 or expression of a dominant-negative mutant of Src prevented hyperoxia-mediated phosphorylation of Src, translocation of p47phox to the cell periphery, and generation of ROS mediated by NADPH oxidase.

Results

Conclusion