Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis.
Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFalpha (tumour necrosis factor alpha), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis.
Ritchie
Stuart A
SA
The Henry Wellcome Laboratory for Cell Biology, Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK.
Kohlhaas
Christine F
CF
Boyd
Alasdair R
AR
Yalla
Krishna C
KC
Walsh
Kenneth
K
Connell
John M C
JM
Salt
Ian P
IP
D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects*; D012694:Serine / Q000378:metabolism*
D007004:Hypoglycemic Agents; D007328:Insulin; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III