Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production.
There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.
Lin
Michelle I
MI
Department of Pharmacology and Program in Vascular Cell Signaling and Therapeutics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Fulton
David
D
Babbitt
Roger
R
Fleming
Ingrid
I
Busse
Rudi
R
Pritchard
Kirkwood A
KA
Sessa
William C
WC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004622:Embryo, Mammalian; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D013912:Threonine; D009569:Nitric Oxide; D015759:Ionomycin; D000255:Adenosine Triphosphate; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013755:Tetradecanoylphorbol Acetate