title: TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct, inhibitory interaction of Pin1 with eNOS.
abstract: Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine-proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
authors.forename: Simone
authors.initials: S
authors.lastname: Kennard
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Ruan
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Buffett
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA; Department of Pediatrics, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA. Electronic address: rvenema@gru.edu.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Venema
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D051379:Mice; D000072340:NIMA-Interacting Peptidylprolyl Isomerase / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D012694:Serine; D013045:Species Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases; C000606846:PIN1 protein, human
keywords: Endothelial nitric oxide synthase (eNOS); Pin1; TNFα