Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis1,2,3,4. eNOS is phosphorylated in response to various forms of cellular stimulation5,6,7 but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.
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We thank P. Martasek and B. S. Masters for recombinant eNOS; J. Liu for constructing and characterizing the myr-nNOS construct; D. Bredt and T. Billiar for NOS cDNAs; T.Zioncheck for human VEGF; Y. Chen for generating the eNOS S1179A E. Coli expression construct; and K. Williams, M. LoPresti and K. Stone in the Keck Facility for their help with identification of the eNOS phosphopeptides. This work was supported by grants from the NIH and American Heart Association. W.C.S. is an Established Investigator of the American Heart Association. J.P.G. is supported by fellowships from the Heart and Stroke Foundation of Canada and from FCAR.
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