TY - JOUR
T1 - Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia
AU - Kayyali, Usamah S.
AU - Donaldson, Cameron
AU - Huang, Hailu
AU - Abdelnour, Raja
AU - Hassoun, Paul M.
PY - 2001/4/27
Y1 - 2001/4/27
N2 - The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Post-translational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (∼50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ∼4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.
AB - The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Post-translational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (∼50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ∼4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.
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U2 - 10.1074/jbc.M010100200
DO - 10.1074/jbc.M010100200
M3 - Article
C2 - 11278616
AN - SCOPUS:0035958075
SN - 0021-9258
VL - 276
SP - 14359
EP - 14365
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -