title: Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells.
abstract: Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.
authors.affiliation: Department of Vascular Surgery, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Masanori
authors.initials: M
authors.lastname: Nakayama
authors.forename: Takaaki M
authors.initials: TM
authors.lastname: Goto
authors.forename: Mutsuki
authors.initials: M
authors.lastname: Amano
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Komori
authors.forename: Kozo
authors.initials: K
authors.lastname: Kaibuchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004330:Drosophila; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D013917:Thrombin; D020742:rhoA GTP-Binding Protein