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Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem 2003 Nov 07;278(45):44719-26

Date

09/04/2003

Pubmed ID

12952971

DOI

10.1074/jbc.M302836200

Scopus ID

2-s2.0-0242666181   195 Citations

Abstract

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.

Author List

Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, Sessa WC

Author

Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphate
Animals
Arginine
COS Cells
Cattle
Cell Line
Cell Membrane
Embryo, Mammalian
Golgi Apparatus
Humans
Ionomycin
Kidney
Kinetics
Mutagenesis, Site-Directed
Nitric Oxide
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Phosphorylation
Structure-Activity Relationship
Superoxides
Tetradecanoylphorbol Acetate
Threonine
Transfection
Vascular Endothelial Growth Factor A
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a