title: Calcineurin-mediated dephosphorylation of eNOS at serine 116 affects eNOS enzymatic activity indirectly by facilitating c-Src binding and tyrosine 83 phosphorylation.
abstract: It has been shown previously that phosphorylation of the endothelial nitric oxide synthase (eNOS) at serine 116 (S116) under basal conditions suppresses eNOS enzymatic activity in endothelial cells. It has also been shown that vascular endothelial growth factor (VEGF) treatment of endothelial cells produces a rapid S116 dephosphorylation, which is blocked by the calcineurin inhibitor, cyclosporin A (CsA). In this study, we show that activation of eNOS in response to a variety of other eNOS-activating agonists and the cytosolic calcium-elevating agent, thapsigargin also involves CsA-inhibitable S116 dephosphorylation. Studies with the purified eNOS enzyme also demonstrate that neither mimicking phosphorylation at S116 nor phosphorylation of the purified enzyme at S116 in vitro has any effect on enzymatic activity. Phospho-mimicking, however, does interfere with the interaction of eNOS with c-Src, an interaction which is known to activate eNOS by phosphorylation at tyrosine 83 (Y83). Agonist-stimulated eNOS-Src complex formation, as well as agonist-stimulated Y83 phosphorylation, are blocked by calcineurin inhibition by CsA and by a cell-permeable calcineurin inhibitory peptide. Taken together, these data suggest a mechanism of eNOS regulation whereby calcineurin-mediated dephosphorylation of eNOS at S116 affects eNOS enzymatic activity indirectly, rather than directly, by facilitating c-Src binding and Y83 phosphorylation.
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, GA 30912, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Ruan
authors.forename: Christina M
authors.initials: CM
authors.lastname: Torres
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Buffett
authors.forename: Simone
authors.initials: S
authors.lastname: Kennard
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: Richard C
authors.initials: RC
authors.lastname: Venema
mesh_terms: D000818:Animals; D019556:COS Cells; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D016572:Cyclosporine / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D014443:Tyrosine; D012694:Serine; D019284:Thapsigargin; D016572:Cyclosporine; D052250:Nitric Oxide Synthase Type III; D019703:Calcineurin; D002118:Calcium
keywords: ANG; BAECs; BK; CAIP; Calcineurin; Cyclosporin A; Dephosphorylation; ERK; Endothelial nitric oxide synthase (eNOS); PP1; PP2A; PP2B; PP2C; Phosphorylation; Pin; TG; VEGF; angiopoietin; bovine aortic endothelial cells; bradykinin; calcineurin autoinhibitory peptide; eNOS; endothelial nitric oxide synthase; extracellular signal regulated kinase; protein interacting with never in mitosis A; protein phosphatase-1; protein phosphatase-2A; protein phosphatase-2B; protein phosphatase-2C; thapsigargin; vascular endothelial growth factor