title: Cyclin-dependent kinase five mediates activation of lung xanthine oxidoreductase in response to hypoxia.
abstract: BACKGROUND
  Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.
  
  
  RESULTS
  Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.
  
  
  CONCLUSIONS
  These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Bo S
authors.initials: BS
authors.lastname: Kim
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Leonid
authors.initials: L
authors.lastname: Serebreni
authors.affiliation: Environmental Health Sciences, School of Public Health, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Fallica
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Omar
authors.initials: O
authors.lastname: Hamdan
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Lan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Johnston
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Todd
authors.initials: T
authors.lastname: Kolb
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Mahendra
authors.initials: M
authors.lastname: Damarla
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America; Environmental Health Sciences, School of Public Health, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Damico
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, MD 21224, United States of America.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Hassoun
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
chemical_list: C492235:Cdk5r1 protein, rat; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D005182:Flavin-Adenine Dinucleotide; D014968:Xanthine Dehydrogenase; D010770:Phosphotransferases; D051360:Cyclin-Dependent Kinase 5; C495907:Cdk5 protein, rat
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