Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745.
Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B1-kinin receptor (B1R) in human endothelial cells or transfected HEK293 cells to generate 2.5-5-fold higher NO than that stimulated by Arg alone. Increased iNOS activity was dependent on B1R activation of the MAPK ERK. In HEK293 cells transfected with human iNOS and B1R, ERK phosphorylated iNOS on Ser745 as determined by Western analysis using phospho-Ser antibody, in vitro kinase assays with activated ERK, and MALDI-TOF mass spectrometry. Mutation of Ser745 to Ala did not affect basal iNOS activity but eliminated iNOS phosphorylation and activation in response to B1R agonist. Mutation of Ser745 to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B1R agonist. ERK and phospho-ERK (after B1R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases.
Zhang
Yongkang
Y
Department of Pharmacology, Protein Research Laboratory, University of Illinois at Chicago College of Medicine 60612, USA.
Brovkovych
Viktor
V
Brovkovych
Svitlana
S
Tan
Fulong
F
Lee
Bao-Shiang
BS
Sharma
Tiffany
T
Skidgel
Randal A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D043783:Receptor, Bradykinin B1 / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization