Src Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83

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

doi:10.1074/jbc.m504606200

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.

Highlights

The endothelial nitric-oxide synthase,4 which catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), is posttranslationally regulated by diverse protein-protein interactions and by covalent modification with fatty acylation and phosphorylation. eNOS, like the other two NOS isoforms termed neuronal NOS and inducible NOS, is a homodimer, with each of the two subunits having a bidomain structure consisting of an N-terminal oxygenase domain containing a heme moiety and binding sites for zinc, arginine, and the cofactor, tetrahydrobiopterin, and a C-terminal reductase domain that contains binding sites for FAD, FMN, and NADPH [1]
To further investigate whether Src tyrosine kinase might have a role in regulation of eNOS in endothelial cells, we carried out coimmunoprecipitation experiments to determine whether the two proteins form a complex in bovine aortic endothelial cells (BAECs)
We have identified the site of tyrosine phosphorylation, Tyr-83, and gone on to show that other oxidants such as PV stimulate the tyrosine phosphorylation of eNOS

Summary

Introduction

The endothelial nitric-oxide synthase (eNOS),4 which catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), is posttranslationally regulated by diverse protein-protein interactions and by covalent modification with fatty acylation and phosphorylation. eNOS, like the other two NOS isoforms termed neuronal NOS and inducible NOS, is a homodimer, with each of the two subunits having a bidomain structure consisting of an N-terminal oxygenase domain containing a heme moiety and binding sites for zinc, arginine, and the cofactor, tetrahydrobiopterin, and a C-terminal reductase domain that contains binding sites for FAD, FMN, and NADPH [1]. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site.

Results

Conclusion