Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83

David J Fulton, Jarrod E. Church, Ling Ruan, Chunying Li, Sarika G. Sood, Bruce E. Kemp, Ian G. Jennings, Richard C Venema

Research output: Contribution to journalArticle

74 Scopus citations

Abstract

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.

Original languageEnglish (US)
Pages (from-to)35943-35952
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number43
DOIs
StatePublished - Oct 28 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Fulton, D. J., Church, J. E., Ruan, L., Li, C., Sood, S. G., Kemp, B. E., Jennings, I. G., & Venema, R. C. (2005). Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83. Journal of Biological Chemistry, 280(43), 35943-35952. https://doi.org/10.1074/jbc.M504606200