title: Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells.
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.
authors.affiliation: Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Chowdhury
authors.forename: Tonya
authors.initials: T
authors.lastname: Watkins
authors.forename: Narasimham L
authors.initials: NL
authors.lastname: Parinandi
authors.forename: Bahman
authors.initials: B
authors.lastname: Saatian
authors.forename: Michael E
authors.initials: ME
authors.lastname: Kleinberg
authors.forename: Peter V
authors.initials: PV
authors.lastname: Usatyuk
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Natarajan
mesh_terms: D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D019255:NADPH Oxidases / Q000378:metabolism*; D010100:Oxygen / Q000008:administration & dosage*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D011651:Pulmonary Artery; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C412373:AG 1879; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D011743:Pyrimidines; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D013481:Superoxides; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D010100:Oxygen