title: Cyclin-dependent kinase 5 phosphorylates endothelial nitric oxide synthase at serine 116.
abstract: Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.
authors.affiliation: School of Medicine, Konkuk University, Seoul, South Korea.
authors.forename: Du-Hyong
authors.initials: DH
authors.lastname: Cho
authors.forename: Jungwon
authors.initials: J
authors.lastname: Seo
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Chulman
authors.initials: C
authors.lastname: Jo
authors.forename: Yoon Jung
authors.initials: YJ
authors.lastname: Choi
authors.forename: Jae-Won
authors.initials: JW
authors.lastname: Soh
authors.forename: Inho
authors.initials: I
authors.lastname: Jo
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology*; D011336:Probability; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077546:Roscovitine; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; D051360:Cyclin-Dependent Kinase 5