Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production

Michelle I. Lin, David Fulton, Roger Babbitt, Ingrid Fleming, Rudi Busse, Kirkwood A. Pritchard, William C. Sessa

Research output: Contribution to journalArticle

189 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)44719-44726
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number45
DOIs
StatePublished - Nov 7 2003

Fingerprint

Phosphorylation
Nitric Oxide Synthase Type III
Threonine
Metabolism
Arginine
Nitric Oxide
Superoxides
Ionophores
Enzyme activity
Immunoprecipitation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production. / Lin, Michelle I.; Fulton, David; Babbitt, Roger; Fleming, Ingrid; Busse, Rudi; Pritchard, Kirkwood A.; Sessa, William C.

In: Journal of Biological Chemistry, Vol. 278, No. 45, 07.11.2003, p. 44719-44726.

Research output: Contribution to journalArticle

Lin, Michelle I. ; Fulton, David ; Babbitt, Roger ; Fleming, Ingrid ; Busse, Rudi ; Pritchard, Kirkwood A. ; Sessa, William C. / Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 45. pp. 44719-44726.
@article{dce71ed2e2474ea0a28ff9779a2a3b3c,
title = "Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production",
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 = "Lin, {Michelle I.} and David Fulton and Roger Babbitt and Ingrid Fleming and Rudi Busse and Pritchard, {Kirkwood A.} and Sessa, {William C.}",
year = "2003",
month = "11",
day = "7",
doi = "10.1074/jbc.M302836200",
language = "English (US)",
volume = "278",
pages = "44719--44726",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "45",

}

TY - JOUR

T1 - Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production

AU - Lin, Michelle I.

AU - Fulton, David

AU - Babbitt, Roger

AU - Fleming, Ingrid

AU - Busse, Rudi

AU - Pritchard, Kirkwood A.

AU - Sessa, William C.

PY - 2003/11/7

Y1 - 2003/11/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0242666181&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0242666181&partnerID=8YFLogxK

U2 - 10.1074/jbc.M302836200

DO - 10.1074/jbc.M302836200

M3 - Article

C2 - 12952971

AN - SCOPUS:0242666181

VL - 278

SP - 44719

EP - 44726

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 45

ER -