Compensatory Phosphorylation and Protein-Protein Interactions Revealed by Loss of Function and Gain of Function Mutants of Multiple Serine Phosphorylation Sites in Endothelial Nitric-oxide Synthase

Abstract

We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative “gain of function” mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions. We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative “gain of function” mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions. endothelial nitric-oxide synthase antibody wild type vascular endothelial growth factor bovine aortic endothelial cells Endothelial-derived nitric oxide (NO) is important in cardiovascular homeostasis, angiogenesis, and vascular remodeling. This has led to a large body of work focused on the regulation of endothelial nitric-oxide synthase (eNOS),1 the enzyme responsible for endothelial-derived NO production. Indeed, the regulation of eNOS activity is remarkably complex. Factors that may affect eNOS activity include post-translational modifications of the enzyme (1Pollock J.S. Klinghofer V. Forstermann U. Murad F. FEBS Lett. 1992; 309: 402-404Crossref PubMed Scopus (81) Google Scholar, 2Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar), protein-protein interactions (3Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 4Garcı́a-Cardeña G. Fan R. Stern D.F. Liu J. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 5Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (865) Google Scholar, 6Fulton D. 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Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar). for serine at 1179, the results in eNOS activity and NO the of site (6Fulton 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 (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphorylation of on bovine eNOS in human) has been B.J. Tiganis T. Stapleton D. F. Power D.A. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is and may be in to B.J. Tiganis T. Stapleton D. F. 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Full Text Full Text PDF PubMed Scopus Google Scholar). Ser-617 and Ser-635 are the and are in to endothelial by and bradykinin, in Ser-635 is in to B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. M. Michell B.J. Kemp B.E. H. H. J. PubMed Scopus Google Scholar). A that mutation of Ser-635 to a 2-fold in activity of the an to B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, that S635D eNOS activity to that of the enzyme in from cells and NO with eNOS (6Fulton 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 (2239) Google S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar). Mutation of Ser-617 to shown to enzyme activity of the enzyme B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of serine phosphorylation sites by eNOS enzyme activity of the enzyme in NOS activity are with and cofactors activity of the enzyme are in the of protein and phosphorylation at other sites not eNOS activity the of NO in cells. that in NO release from cells that eNOS serine phosphorylation site mutants serine to alanine serine to of of phosphorylation In addition, examined mutation of a phosphorylation site the phosphorylation of the other regulatory the of the eNOS phosphorylation site on the of the protein and the to with eNOS. aortic endothelial cells and as (6Fulton 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 (2239) Google Scholar). and cells in bovine and from antibody from Ser-1179 and antibody from and from and Ser-635 as B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. Michell B.J. Kemp B.E. H. H. J. PubMed Scopus Google Scholar). Phosphorylation site mutants of eNOS using the to the bovine eNOS as a for the the mutation to the and for the cells. from for in and the using a least for mutation to the of the of the to the eNOS phosphorylation site mutants and using bovine eNOS by Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google for using bovine eNOS by K. Harrison J.P. M. T.J. J. 1992; 90: PubMed Scopus Google cells to in and with eNOS eNOS phosphorylation site mutants using as by the the growth and with of growth cells for and an of for basal NO as the of NO in cells for in and the and with of an of for and the cells stimulated with and to for an of for and the cells and for protein and using a NO as with on in and to and for at by at for at of protein from by of protein from using and to as (6Fulton 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 (2239) Google Scholar). activity of and eNOS in of cells by the of to as T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We to the of the and cells with eNOS, S617A eNOS and by with the that are for the sites on eNOS, the eNOS but not work has the of Ser-1179 D. Fontana J. G. Gratton J.P. M. Li Michell B. Kemp B.E. D. Sessa W.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We examined the of on phosphorylation of and with for and with to eNOS and by with the of the that the putative Akt phosphorylation Ser-1179 and are with at and to basal of phosphorylation by Ser-116 is and in to a putative phosphorylation site, a in phosphorylation in to phosphorylation at are with eNOS phosphorylation B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 12: 845-848Abstract Full Text Full Text PDF Scopus (412) Google Scholar, R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. Fontana J. G. Gratton J.P. M. Li Michell B. Kemp B.E. D. Sessa W.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). examined eNOS phosphorylation as with for and eNOS by and to as of phosphorylation for of the phosphorylation sites from cells. Ser-1179 and Ser-617 and with phosphorylation at and at Ser-116 a at that for the of the Ser-635 and a to that of Ser-617 and Ser-1179 the of eNOS phosphorylation are and are to with R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have examined the of eNOS phosphorylation site Ser-116 to alanine Ser-617 to on in NOS in with for Ser-116 and R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells with S635D eNOS and for NOS activity in the as the of the serine to alanine mutation of phosphorylation sites on NOS of of and of of eNOS both an in NOS activity with of of S617A eNOS of of activity Mutation of on eNOS to of of in an in enzyme activity with eNOS of of NOS activity of S617D eNOS of of 2-fold eNOS, S635D eNOS activity of of to eNOS. Thus, and whereas S617A and S635D to In activity the of to are with and cofactors and not the of NO in cells phosphorylation, and protein-protein interactions eNOS examined the of Ser-635 to phosphorylation at that site, on the of eNOS to NO in cells basal stimulated and eNOS as positive and negative that basal NO release from cells with S617A of of a in NO release with eNOS of of NO release from of of as and of of eNOS NO release from eNOS of of cells to that of cells with eNOS. of mutation of Ser-617 and Ser-635 to opposite in NOS activity NO release assays. stimulated with to NO release. stimulated and S617A eNOS and of of to of NO with eNOS of of whereas NO release from eNOS of of to that of the enzyme and that from eNOS of of as shown (6Fulton 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 (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). that of the eNOS at Mutation of serine phosphorylation sites to but not the of the whereas alanine the site phosphorylation but may cause in as of the of individual phosphorylation site in eNOS, cells with S635D eNOS and basal and NO release as Ser-1179 to has been shown to eNOS activity and NO release with and as a positive in (6Fulton 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 (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Mutation of Ser-116 to on basal NO of of an stimulated of of with eNOS of of stimulated, of of S617D NO both of of and stimulated for with of of cells with the S635D in the most in NO of of the phosphorylation site including NO release from S635D 5-fold of of and stimulated NO release of of that of eNOS. of NO both and stimulated of of stimulated, of of as in by that of of the eNOS in phosphorylation at one site in eNOS phosphorylation at other cells with eNOS serine to alanine mutants and the serine phosphorylation sites by using and to stimulated with for and to of basal and stimulated phosphorylation with the stimulated cells increased phosphorylation with basal on the phosphorylation site phosphorylation at Ser-1179 increased by in both and S617A eNOS, but in the eNOS. phosphorylation at Ser-116 by the of phosphorylation at Ser-617 S617A between the Phosphorylation at Ser-635 2-fold in eNOS and in S617A eNOS. Phosphorylation at Ser-617 by mutation at of the other phosphorylation Thus, phosphorylation, basal and stimulated at other serine and is a of and S617A eNOS, whereas with eNOS. of hsp90 with eNOS to eNOS from J.P. Fontana J. D.S. G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), as a for the interaction of Akt with eNOS J. D. Y. McCabe T.J. T. Sessa W.C. 90: PubMed Scopus Google Scholar), and regulates and from the to the of eNOS T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). in not phosphorylation of eNOS regulate the of the with eNOS. cells with eNOS and from cells cells stimulated with for and eNOS using to the with and by using to eNOS, Akt Mutation of Ser-1179 to alanine on the of hsp90 Akt to with eNOS basal stimulated eNOS hsp90 and Akt with the enzyme basal stimulated in hsp90 Akt and to eNOS. S617A eNOS a in hsp90 and a in Akt with S617A eNOS basal of S617A eNOS a in hsp90 with eNOS in Akt with eNOS, S617A hsp90 and Akt. basal hsp90 to eNOS by mutation of Ser-635 to Akt stimulated eNOS 2-fold hsp90 eNOS whereas Akt to that of the enzyme. of hsp90 and Akt with eNOS hsp90 and Akt not in cells in the as the cells not Thus, the interaction of with and S617A mutants may the phosphorylation of mutants on of are that eNOS is by and multiple phosphorylation events on serines 116, 617, and of phosphorylation of is for activation by kinase Using a and individual in eNOS (serine to and (serine to aspartate) serines 1179 and 635 are the most important positive regulatory sites for basal and NO release and phosphorylation of Ser-116 is In addition, for the an between the phosphorylation sites and protein-protein interactions of eNOS with hsp90 and Akt. Mutation of the known serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site demonstrating cooperation between multiple phosphorylation events and mutation of serines 116 and 617 to alanine promoted a greater interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. Thus, of phosphorylation and protein-protein interactions an to regulate eNOS work in the has focused on the of phosphorylation in regulating eNOS on of the phosphorylation sites in eNOS focused on the individual serines 1179, 116, 617, and In results from multiple groups in and in serine 1179 has been the most 1179 be by multiple kinases including AMP kinase (13Chen 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 (720) Google Scholar), Akt (6Fulton 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 (2239) Google Scholar, 14Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 12: 845-848Abstract Full Text Full Text PDF Scopus (412) Google Scholar), protein kinase and protein kinase (16Butt E. Bernhardt M. Smolenski A. Kotsonis P. Frohlich L.G. Sickmann A. Meyer H.E. Lohmann S.M. Schmidt H.H. J. Biol. Chem. 2000; 275: 5179-5187Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). of Akt of Akt NO release from negative Akt but not NO release (6Fulton 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 (2239) Google of and negative Akt and Fujio Y. Y. G. D. Sessa W.C. Walsh K. J. 2000; PubMed Scopus Google Scholar). More of a of eNOS the of eNOS the the of and the actions of a for site M. Y. J. D. Gratton J.P. Sessa W.C. 90: PubMed Scopus Google Scholar). for site eNOS is to and phosphorylation at 1179 the in eNOS and of from the to the T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the serine phosphorylation sites regulate eNOS activity and NO R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of phosphorylation in a on mutation of the phosphorylation site to an alanine results in the of phosphorylation a mutation of the phosphorylation site to may the cause an in a of In the of of serine 116 on the eNOS activity in R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), phosphorylation of serines 617 by Akt and 635 by protein kinase A on NOS activity with from and activity of eNOS, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, have shown in eNOS activity in cells to bovine in NO in cells (6Fulton 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 (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar). and the did not the activity of the enzyme with the basal and release of NO that in one may sites of phosphorylation protein-protein interactions. in activity and the release of NO with alanine mutants of S617A is the that basal NO eNOS, whereas and eNOS with to wild with with and S617A NO eNOS. Thus, in using serine to alanine of the phosphorylation the phosphorylation of eNOS at Ser-635 and Ser-1179 are important for basal NO release and phosphorylation at Ser-617 negatively regulates basal NO release. phosphorylation at Ser-1179 NO release, phosphorylation at Ser-116 and Ser-617 negatively regulates NO release. the Ser-116 is important for agonist, but not basal, NO release R. D. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Ser-635 is important for basal, but not stimulated NO Ser-617 negatively regulates basal and stimulated NO release and Ser-1179 phosphorylation is stimulatory for both basal and NO release. In the of putative and S635D increased basal and NO release with S635D being the most efficacious, whereas basal NO release to eNOS and stimulated NO release from in with the alanine the of 1179 and Ser-635 as stimulatory We that at Ser-116 not activity NO release that is not a to the phospho-state is to eNOS. that S617A and S617D both NO did eNOS at least of site is an site and at is to the in to alanine at site phosphorylation at 617 but the in an alanine at site phosphorylation at 617 but regulates phosphorylation phosphorylation is increased and Ser-116 and Ser-635 phosphorylation are in of the that S617D has a greater to basal in is by other that regulate eNOS at This S617A being and S617D being in NOS activity Thus, are with and Ser-1179 as important regulatory sites for NO release from cells and Ser-617 phosphorylation may a regulatory on with eNOS. a of phosphorylation the known sites of eNOS phosphorylation. in with as the of are In addition, the and protein-protein interactions phosphorylation on Ser-1179 and Ser-116 D. Fontana J. G. Gratton J.P. M. Li Michell B. Kemp B.E. D. Sessa W.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. R. Michel T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on is that phosphorylation at one site may be important for at an site, as is the for the phosphorylation of and Ser-1179 sites in the individual alanine mutants basal and the phosphorylation at Ser-1179 increased in both and S617A eNOS, but not eNOS, that Ser-1179 phosphorylation may to the in NO release with and S617A eNOS the co-association of hsp90 and Akt with a for the phosphorylation on Ser-1179 and NO release. between phosphorylation on and Ser-635 is by of phosphorylation on Ser-116 and a in Ser-635 phosphorylation in cells In cells eNOS, basal and stimulated NO but in the interaction of hsp90 and Akt. This accompanied by an in phosphorylation at Ser-635 basal NO release, but did not in the of eNOS. phosphorylation site that not by the individual alanine mutants Thus, that mutation of individual phosphorylation sites may events on This may between the protein-protein is that S635D basal and stimulated NO release from cells with basal NO release being greater that with eNOS. the of NO release from eNOS, negative (6Fulton 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 (2239) Google Scholar, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar). that both S635D and eNOS eNOS (6Fulton 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 (2239) Google Scholar, T.J. D. Sessa W.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, B.J. M.B. Z.P. Ju H. Venema V.J. Venema R.C. Kemp B.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and results NOS activity in greater activity eNOS, whereas S635D is to enzyme is from results NO release. This may be to phosphorylation events in the of S635D protein-protein interactions that are regulatory in cells but are in of the the that multiple sites of serine phosphorylation to NO release and that to an individual site may be the mutants are examined in multiple