Phosphorylation and dephosphorylation of Thr497 in eNOS acts as an intrinsic switch determining whether the enzyme generates nitric oxide or superoxide in cells.
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. 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. Endothelial nitric-oxide synthase (eNOS) 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; NO, nitric oxide; HEK, human embryonic kidney; BAEC, bovine aortic endothelial cell; PMA, phorbol 12-myristate 13-acetate; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; HA, hemagglutinin; WT, wild-type; l-NAME, l-nitroarginine methyl ester.1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; NO, nitric oxide; HEK, human embryonic kidney; BAEC, bovine aortic endothelial cell; PMA, phorbol 12-myristate 13-acetate; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; HA, hemagglutinin; WT, wild-type; l-NAME, l-nitroarginine methyl ester. produces the free radical gas, nitric oxide, which has been implicated in the regulation of cardiovascular homeostasis. Due to its importance to overall cardiovascular health, nature has evolved multiple control mechanisms that tightly regulate the enzymatic function of eNOS. eNOS is a prototype for proteins regulated by spatial and temporal signals, namely subcellular targeting to the Golgi complex and lipid rafts/caveolae by protein-protein interactions and phosphorylation events (1Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar). These complex post-translational mechanisms control the state of eNOS activation/inactivation and the fidelity of electron flux through the reductase domain of the protein to the oxygenase domain where the chemistry of NO synthesis occurs. Dysregulation of the synthesis of eNOS cofactors, protein-protein interactions, and phosphorylation could potentially “uncouple eNOS,” i.e. electron transfer reactions result in the production of superoxide, instead of NO. eNOS can be phosphorylated on serines 116, 617, 635, and 1179 and phosphorylated on threonine 497 (bovine amino acids) (2Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 3Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 5Michell B.J. Harris M.B. Chen Z.P. Ju H. Venema V.J. Blackstone M.A. Huang W. Venema R.C. Kemp B.E. J. Biol. Chem. 2002; 277: 42344-42351Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 6Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (705) Google Scholar, 7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar, 8Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2196) Google Scholar, 9Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (2995) Google Scholar). The phosphorylation of serines 617, 635, and 1179 all result in the activation of eNOS function, whereas the phosphorylation of serine 116 and threonine 497 may reduce eNOS function (3Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar, 10Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). There is a good correlation between threonine 497 (bovine)/threonine 495 (human) dephosphorylation and NO production (measured as cGMP) but not Ser1179 phosphorylation when bradykinin is used as an agonist for NO production, suggesting that Thr497 dephosphorylation is sufficient for eNOS activation (3Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar). Using cells transfected with a de-phosphomutant of eNOS (T495A in human eNOS), there is an enhanced interaction of the essential allosteric regulator calmodulin, which correlates well with increased eNOS calcium sensitivity in vitro. In addition, it was suggested that the reciprocal dephosphorylation of threonine 497 and phosphorylation of serine 1179 were essential for eNOS activation and that dephosphorylation may be a prerequisite for serine 1179 phosphorylation (3Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar, 11Greif D.M. Kou R. Michel T. Biochemistry. 2002; 41: 15845-15853Crossref PubMed Scopus (91) Google Scholar). Because the T495A eNOS mutant (de-phospho) demonstrated enhanced calcium sensitivity compared with T495D eNOS (potential phosphomimetic) and the rate of electron flux through the reductase domain of eNOS critically determines eNOS activity and the potential for uncoupling eNOS (12Ortiz de Montellano P.R. Nishida C. Rodriguez-Crespo I. Gerber N. Drug Metab. Dispos. 1998; 26: 1185-1189PubMed Google Scholar, 13McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), we sought to examine the relationship between threonine 497 dephosphorylation (mimicked by T497A mutation) and the balance of NO generation versus superoxide anion production in intact cells. Here we show that T497A eNOS increases NOS activity in cell lysates but reduces basal NO production in intact cells. Combining eNOS T497A with a mutation of Ser1179 that renders eNOS constitutively active to produce NO (eNOS S1179D) generates an extremely active enzyme (eNOS T497A/S1179D) but neutralizes the ability of S1179D eNOS to produce basal and stimulated NO and promotes eNOS-dependent superoxide anion release. Thus, the phosphorylation of threonine 497 is a critical residue that may be an intrinsic mechanism to balance whether eNOS generates NO versus superoxide anion in cells. Cell Culture Conditions—Bovine aortic endothelial cells (BAECs, passages 4-8), COS-7, and HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing penicillin (100 units/ml), streptomycin (100 mg/ml), and 10% (v/v) fetal calf serum. All of the experiments using these cells were done when cells reached confluency in 100-mm dishes for BAECs or in 6-well plates for COS-7 and HEK 293 cells. Prior to agonist stimulation, BAECs or transfected COS-7 cells were rendered quiescent in serum-free Dulbecco's modified Eagle's medium for 16 or 6 h, respectively, before the addition of 1 μm ionomycin (Sigma), 1 μm PMA (Calbiochem), 10 μm ATP (Sigma), or 50 ng/ml VEGF (Genentech) at various time points. eNOS Constructs—The mutant bovine eNOS constructs were generated by standard cloning methods. The S1179D, T497A, and T497D mutant eNOS constructs were generated by site-directed mutagenesis (Stratagene). The T497A/S1179D double mutant eNOS construct was generated by subcloning the XhoI/XbaI fragment of S1179D mutant eNOS into the T497A construct. All of the constructs were verified by sequencing. Human eNOS constructs (wild-type, Ala495, and Asp495 also called T495A and T495D in this paper) were generated as described previously (7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar). Transient Transfection and Generation of Stable Cell Line—COS-7 or HEK 293 cells were transfected with the bovine eNOS cDNA (wild-type or mutants) with or without an HA-tagged hsp90 cDNA construct (14Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (292) Google Scholar) using LipofectAMINE 2000 (Invitrogen) as per manufacturer's protocol. Transiently transfected cells were used 48 h post-transfection. Stably transfected HEK 293 cells were used in superoxide anion measurements and were selected in 1 mg/ml G418 for 2-3 weeks. Immunoprecipitation and Immunoblotting—Immediately after agonist stimulation, cells were washed with ice-cold phosphate-buffered saline (without Ca2+ and Mg2+) and subsequently lysed in buffer that contained 50 mm Tris, pH 7.5, 0.1 mm EGTA, 0.1 mm EDTA, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholate, 20 mm NaF, 1 mm Na4P2O7, 0.3 mg/ml Pefabloc SC, 20 mm Na2MoO4, 5 mm ATP, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 2 μg/ml pepstatin A. Cell lysates were Douncehomogenized (50 strokes) and rotated at 4 °C for 15 min before the insoluble materials were by at for 10 In after for protein lysates were or with protein were with an to HA-tagged hsp90 by protein In eNOS and proteins were by to after had been with in both the cell lysates and were in buffer before by transfer of the proteins were using or Ser1179 or Thr497 (3Michell B.J. Chen Z. Tiganis T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: 17625-17628Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). The were and for eNOS NO from h after of eNOS mutants into COS-7 or HEK 293 the were for the of a of NO in by D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2196) Google Scholar). from basal of in were with to proteins by were to to The cells were with serum-free Dulbecco's modified Eagle's medium for 6 h, and medium was to for min at In cells were with 1 mm the the cells were with μm 10 μm ATP or 5 μm for min to The medium was and was using The cells were lysed with 1 or buffer as described to the protein Cell lysates were to to eNOS in all of the NOS activity of the eNOS mutants was by of to COS-7 cells were lysed in the buffer described without or the insoluble by the was to protein of protein was used per and was in a of of activity buffer containing 50 mm Tris, pH 7.5, 0.1 mm EDTA, 0.1 mm EGTA, (Sigma), 1 mm (Sigma), 15 μm μm (Sigma), μm and mm All of the were on at °C for 10 The was by 1 of ice-cold buffer containing 20 mm pH 2 mm EDTA, to The were through and the was on a eNOS COS-7 cells were in for 10 min by cell using 0.1% Prior to with cells were with for 20 of eNOS was using an were with to the were using the and using the of transfected HEK 293 cells were grown to confluency to for NO release or superoxide anion production by D.M. Sessa W.C. M.B. Circ. Res. PubMed Scopus Google Scholar). were with 1 mm for min before with and with 50 μm and 5 μm with or without 1 mm in for The was in the of superoxide of superoxide anion release were from superoxide at using the To the relationship between eNOS phosphorylation on Thr497 and BAECs were with PMA, VEGF, or ionomycin and the time of in phosphorylation was examined using seen in PMA increased the of phosphorylation on a protein phosphorylation but did not phosphorylation on Ser1179. VEGF, an agonist that a stimulated the phosphorylation on Ser1179 that was min after agonist but did not Thr497 phosphorylation/dephosphorylation of with ionomycin to the release of calcium and calcium into cells stimulated the phosphorylation on Ser1179 and dephosphorylation of Thus, regulation of Thr497 versus Ser1179. has been suggested that the dephosphorylation of Thr497 is a prerequisite for the phosphorylation of Ser1179. To examine the interrelationship between these in a cells were transfected with WT eNOS and the T497A, S1179D, and T497A/S1179D eNOS and stimulated with ionomycin to examine the time of phosphorylation/dephosphorylation on Ser1179 and The was that T497A enhanced calcium sensitivity and (7Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (596) Google Scholar) and S1179D is a constitutively active of eNOS that also the for activation T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), the mutants between the seen in the basal phosphorylation on Ser1179 is at time cells transfected with WT and T497A eNOS), whereas the basal phosphorylation on Thr497 is cells transfected with WT and S1179D of cells transfected with WT eNOS in a in phosphorylation on Ser1179 and a in phosphorylation on with the in similar in phosphorylation on Ser1179 was seen in cells transfected with T497A eNOS that the of Thr497 phosphorylation has on the of Ser1179 phosphorylation in this The constitutively active eNOS S1179D was phosphorylated on Thr497 and was with In cells transfected with T497A/S1179D eNOS, was the of the Thus, both WT and S1179D eNOS phosphorylated on examined NOS enzymatic activity of the eNOS Transfection of cells with WT eNOS increased NOS activity in cell lysates T497A and S1179D eNOS exhibited greater enzyme activity than WT eNOS, whereas the activity of T497D eNOS was with that of WT Transfection with the double eNOS in a in enzymatic activity compared with WT eNOS and the Thus, T497A and S1179D mutations generates a active of the Because the rate of eNOS activity correlates well with the production of NO for WT and S1179D eNOS, we examined basal and NO release as from cells transfected with the eNOS Transfection with WT eNOS increased basal and release from cells. Transfection with constitutively active S1179D eNOS enhanced both basal and stimulated release from cells compared with WT eNOS. with T497A, T497D, and T497A/S1179D all in reduced basal of In with ATP, of were from T497A eNOS compared with WT whereas NO release from T497D eNOS cells T497A/S1179D eNOS or S1179D eNOS with ATP in similar of release. the experiments using ionomycin as an agonist for NO release and similar and of was reduced in cells transfected with T497A, T497D and T497A/S1179D eNOS and the in NO production was or These that the of phosphorylation on Thr497 in eNOS the ability of eNOS to produce NO in the of an agonist but is by with also compared basal and stimulated release from cells transfected with human eNOS constructs to the were to bovine eNOS. seen in and both T495A and T495D human eNOS exhibited reduced basal of an by the of cells with Thus, the enhanced NOS activity observed with T497A and T497A/S1179D eNOS and between WT and T497D eNOS not with basal or stimulated NO generation of as is seen with WT and S1179D eNOS. for the of Thr497 mutants to produce NO synthesis may be of localization of eNOS. eNOS localization is for the fidelity of NO release W.C. J. A. J. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. Fontana J. Gratton J.P. Michell B. Kemp B.E. D. Sessa W.C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). To examine the localization of eNOS, WT, T497A, T497D, S1179D, and T497A/S1179D eNOS constructs were transfected into cells and eNOS localization was examined by using an eNOS seen in all of the eNOS constructs to the of cells and the Thus, all of the constructs in a similar to WT eNOS. that the in NOS activity versus NO release may be by a in the of to NO synthesis. the rate of electron flux from the reductase domain of eNOS is tightly to NO synthesis in the oxygenase of electron flux by or by a of Ser1179 with in an in electron flux and NO synthesis. both WT and S1179D eNOS superoxide of D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2196) Google Scholar, 13McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) of the serine residue to in a to the phosphorylation Ser1179 in eNOS, electron flux in the NOS reductase domain and in NO suggesting an of electron flux to NO generation S. J. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). These that the rate of electron flux into the of NOS the of to NO synthesis. Thr497 phosphorylation the fidelity of electron transfer to NO mutations the phosphorylation of Thr497 may eNOS to superoxide To this cell were generated in HEK 293 cells of WT, T497A, and T497A/S1179D eNOS and the production of superoxide anion was by superoxide and was seen in the cell similar levels of eNOS protein by The basal levels of superoxide by the cells were than the of for this of cells with ionophore in superoxide anion production from cells WT eNOS the addition of enhanced the of superoxide by the of NO on superoxide anion generated from In cells T497A eNOS and T497A/S1179D eNOS more superoxide, an reduced by of the cells with to cells transfected with these basal release was for cells transfected with T497A or T497A/S1179D eNOS and protein for cells WT, T497A, and T497A/S1179D eNOS, compared with WT and compared with T497A was with WT in cells T497A eNOS or T497A/S1179D eNOS Thus, the mutation of Thr497 to a residue that be the the ability of eNOS to as a superoxide anion at the of NO synthesis. The ability of T497A and T497A/S1179D eNOS to produce superoxide anion instead of NO synthesis may in the between the NOS activity in cell lysates and reduced NO production from cells transfected with these T497A eNOS and T497A/S1179D eNOS were not the production of NO basal and stimulated the levels from cells WT or S1179D eNOS, evidence that the of eNOS with the may of eNOS to NO Y. Fontana Sessa W.C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. J. 2001; PubMed Google Scholar, Y. Y. J. 2001; PubMed Scopus Google Scholar, Y. C. J. Circ. Res. 2002; PubMed Scopus Google Scholar, J. Z. Biol. PubMed Scopus Google Scholar). To examine the interaction of eNOS with WT eNOS and eNOS mutants were with HA-tagged hsp90 and the levels of phosphorylation and interaction with hsp90 were seen in the levels of eNOS and in lysates were with the in phosphorylation similar to in Immunoprecipitation of in cells in of eNOS from cells WT and T497A eNOS. the interaction of eNOS with hsp90 was increased in cells transfected with S1179D eNOS and with cells T497A/S1179D eNOS with the interaction of WT and T497A eNOS with hsp90 were whereas the enhanced interaction of S1179D with hsp90 was In both basal and stimulated interaction of T497A/S1179D eNOS with hsp90 was than that seen with the Thus, an interaction with hsp90 may be an mechanism to for the of T497A/S1179D eNOS to produce superoxide the that the phosphorylation/dephosphorylation of Thr497 in bovine eNOS or in human eNOS not eNOS subcellular localization but may regulate NO production and the generation of NO versus superoxide anion of cells. the T497A mutation with the well mutant S1179D eNOS in a active enzyme in vitro. The basal release of NO from cells T497A/S1179D eNOS is than WT eNOS and markedly than that observed with S1179D eNOS. agonist both T497A eNOS and T497A/S1179D eNOS to WT and S1179D eNOS, respectively, higher in cell that at in the of a in the S1179D mutation the in the of eNOS T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), electron the calcium sensitivity of the enzyme and its interaction with and in an enhanced basal NO release. the that Thr497 is phosphorylated in WT and S1179D eNOS. stimulation, WT and S1179D eNOS on Thr497 and the of NO release increases as T497A and T497A/S1179D eNOS, the of NOS activity versus NO release is complex and to the activity of T497A eNOS with of is greater than WT eNOS and with that of S1179D eNOS. the activity of the mutant T497A/S1179D eNOS is greater than T497A and S1179D eNOS. Despite the in NOS of the mutant in to WT and S1179D eNOS, T497A and T497A/S1179D eNOS produce basal NO levels of Ser1179 phosphorylation for The in basal NO release may be by the at Thr497 the of a NOS from its function in a or the of the reductase and oxygenase by agonist is on the that eNOS is in the cells and the that the levels of and similar in the an protein-protein interaction the enhanced of hsp90 with S1179D eNOS and reduced of hsp90 with T497A/S1179D eNOS basal the interaction of T497A with hsp90 and phosphorylation on Ser1179 similar to WT eNOS. Thus, in the interaction of hsp90 with eNOS this is that Thr497 phosphorylation the fidelity of electron flux to NO synthesis basal levels of calcium in the and agonist enhanced protein-protein interactions, and phosphorylation of NO release the to produce superoxide by Thr497 These with eNOS with a (14Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (292) Google Scholar) that of endothelial cells with hsp90 increases both basal and stimulated Ser1179 and Thr497 phosphorylation with enhanced basal and stimulated NO suggesting that phosphorylation of Thr497 promotes the of eNOS and NO synthesis. In that the phosphorylation of Thr497 in eNOS may be for the of to NO synthesis. is that dephosphorylation of eNOS may a of both superoxide and NO. the ability of eNOS to its ability to produce NO in superoxide anion production may in the of eNOS protein and the of the of the that the importance of Thr497 in eNOS is to be by to experiments the phosphorylation of Thr497 electron flux through the reductase domain to the oxygenase domain and the of eNOS Kemp for and for with superoxide anion
Lin et al. (Sat,) studied this question.
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