Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia

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 posttra nslational levels by agents such as lipopolysaccharide and hypoxia. Post-tr anslational modification of the protein, for example through thiol oxidatio n or proteolysis, has been shown to be important in converting XDH to XO. T he possibility of posttranslational modification of XDH/XO through phosphor ylation has not been adequately investigated in mammalian cells, and studie s have reported conflicting results. The present report demonstrates that X DH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (R PMEC) and that phosphorylation is greatly increased (similar to 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be medi ated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the r esults indicate that p38 kinase, a stress activated kinase, becomes activat ed in response to hypoxia (an similar to4-fold increase after 1 h of exposu re of RPMEC to hypoxia) further supporting a role for this kinase in hypoxi a-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosph orylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows th at XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for h ypoxia-induced enzymatic activation.