Cyclin-Dependent Kinase 5 Phosphorylates Endothelial Nitric Oxide Synthase at Serine 116

• Published in: Hypertension (Dallas, Tex. 1979) Vol. 55; no. 2; pp. 345 - 352
• Main Authors: Cho, Du-Hyong, Seo, Jungwon, Park, Jung-Hyun, Jo, Chulman, Choi, Yoon Jung, Soh, Jae-Won, Jo, Inho
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD American Heart Association, Inc 01.02.2010; Lippincott Williams & Wilkins
• Subjects: Animals; Arterial hypertension. Arterial hypotension; Biological and medical sciences; Blood and lymphatic vessels; Blotting, Western; Cardiology. Vascular system; Cattle; Cells, Cultured; Chemical and industrial products toxicology. Toxic occupational diseases; Cyclin-Dependent Kinase 5 - genetics; Cyclin-Dependent Kinase 5 - metabolism; DNA, Complementary; Endothelial Cells - cytology; Endothelial Cells - drug effects; Fluorescent Antibody Technique; Gene Expression Regulation; Medical sciences; Metals and various inorganic compounds; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - drug effects; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Phosphorylation - drug effects; Phosphorylation - physiology; Probability; Protein Kinase Inhibitors - pharmacology; Purines - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; Serine - genetics; Serine - metabolism; Signal Transduction; Toxicology; Transfection; Hypertension; Phosphorylation; Enzyme; Serine; Cardiovascular disease; Nitric-oxide synthase; Endothelium; Signal transduction; Aminoacid; Nitric oxide; endothelial nitric oxide synthase; Oxidoreductases; cyclin-dependent kinase 5
• ISSN: 0194-911X; 1524-4563
• DOI: 10.1161/HYPERTENSIONAHA.109.140210

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 3β, 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.