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
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m211926200
ISSN1083-351X
AutoresPhilip M. Bauer, David Fulton, Yong Chool Boo, George P. Sorescu, Bruce E. Kemp, Hanjoong Jo, William C. Sessa,
Tópico(s)Ion Transport and Channel Regulation
ResumoWe 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. 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, 7Marrero M.B. Venema V.J. Ju H. He H. Liang H. Caldwell R.B. Venema R.C. Biochem. J. 1999; 343: 335-340Crossref PubMed Scopus (96) Google Scholar), cofactors and prosthetic groups (8Bredt D.S. Snyder S.H. J. Biol. Chem. 1990; 266: 23790-23795Google Scholar, 9Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3128) Google Scholar, 10Stuehr D.J. Cho H.J. Kwon N.S. Weise M. Nathan C.F. Proc. Natl. Sci. U. S. A. 1991; 88: 7773-7777Crossref PubMed Scopus (733) Google Scholar), calcium/calmodulin (11Forstermann U. Pollock J.S. Schmidt H.H. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar), and phosphorylation (12Michel T. Li G.K. Busconi L. Proc. Natl. Acad. Sci. 1993; 90: 6252-6256Crossref PubMed Scopus (305) Google Scholar). Of the known phosphorylation sites on eNOS, serine 1179 (Ser-1179) in bovine eNOS (Ser-1177 in human) has been characterized most extensively. Treatment of bovine aortic endothelial cells with a variety of stimuli, including vascular endothelial growth factor (VEGF), adenosine-3′,5′-triphosphate (ATP), bradykinin, sheer stress, and acetylcholine results in phosphorylation at Ser-1179 and activation of the enzyme. In addition, several kinases that phosphorylate Ser-1179 have been identified, 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 (protein kinase B) (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, 15Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar), and protein kinase A (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). Ser-1179 phosphorylation has been shown to be critical for activation of eNOS. Phosphorylation of eNOS on Ser-1179 results in increased enzyme activity accompanied by an increase in NO production. Mutation of Ser-1179 to alanine prevents phosphorylation at this site resulting in decreased enzyme activity and NO production (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). Additionally, substituting aspartate for serine at 1179, which mimics the phosphorylated state, results in enhanced eNOS activity and NO production further supporting the indispensable role of this 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, 17McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Phosphorylation of threonine 497 (Thr-497) on bovine eNOS (Thr-495 in human) has also been documented (18Michell 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 (475) Google Scholar). Thr-497 is basally phosphorylated and may be dephosphorylated in response to bradykinin stimulation (18Michell 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 (475) Google Scholar, 19Harris 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 (331) Google Scholar, 20Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (609) Google Scholar). Dephosphorylation of Thr-497 results in increased enzyme activity; however, the relative contribution of Thr-497 to the regulation of NO production by eNOS has not been well established. Recently, three additional phosphorylation sites have been identified on eNOS: serine 116 (Ser-116) (21Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar, 22Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), serine 617 (Ser-617) (23Michell 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), and serine 635 (Ser-635) (23Michell 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). Ser-116, like Thr-497, is basally phosphorylated and is dephosphorylated in response to certain stimuli in endothelial cells. Dephosphorylation of this residue on eNOS was reported to result in an increase in eNOS activity in ionophore-stimulated cells (22Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The Ser-617 and Ser-635 residues are located within the autoinhibitory loop and are phosphorylated in response to endothelial cell stimulation by VEGF, ATP, and bradykinin, in addition Ser-635 is phosphorylated in response to shear stress (23Michell 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,24Boo Y.C. Hwang J. Sykes M. Michell B.J. Kemp B.E. Lum H. Jo H. Am. J. Physiol. Heart Circ Physiol. 2002; 283: H1819-H1828Crossref PubMed Scopus (211) Google Scholar). A recent report showed that mutation of Ser-635 to aspartate causes a 2-fold increase in maximal activity of the purified enzyme, an effect comparable to S1179D (23Michell 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). However, previous studies demonstrated that S635D eNOS activity was similar to that of the wild-type (WT) enzyme when assayed in cell lysates from transfected cells and S635A produced equal or more NO compared with WT 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,15Dimmeler 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 aspartate was recently shown to increase calcium sensitivity without changing maximal enzyme activity of the purified enzyme (23Michell 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). Previous studies describing the role of these serine phosphorylation sites do so by examining eNOS enzyme activity of the purified enzyme or in detergent-solubilized cell lysates. However,in vitro NOS activity assays are performed with optimal calcium and cofactors present. Furthermore, activity assays of the purified enzyme are done in the absence of eNOS-associated proteins (caveolin-1, heat shock protein 90, etc.) and without phosphorylation at other sites and, therefore, do not always accurately reflect eNOS activity or the production of NO in live cells. For that reason, in this study we measured NO release from intact cells that express eNOS serine phosphorylation site mutants (either serine to alanine or serine to aspartate mutants) of each of these phosphorylation sites. In addition, we examined if mutation of a single phosphorylation site influenced the phosphorylation of the other regulatory sites. Finally, we determined the effect of the eNOS phosphorylation site mutations on the ability of the heat shock protein 90 (hsp90) and the kinase, Akt, to co-associate with eNOS. Bovine aortic endothelial cells (BAEC) were isolated and cultured as previously described (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). BAEC and COS-7 cells were cultured in high glucose Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, penicillin, streptomycin, and l-glutamine. 2′-5′ ADP-Sepharose 4B was obtained from Amersham Biosciences. Anti-eNOS antibody was obtained from Zymed Laboratories Inc., anti-phospho-eNOS Ser-1179 and anti-Akt antibody from Cell Signaling Technologies, and anti-hsp90 was from Transduction Laboratories. Anti-phospho-eNOS Ser-116, Ser-617, and Ser-635 antibodies were generated as previously described (23Michell 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, 24Boo Y.C. Hwang J. Sykes M. Michell B.J. Kemp B.E. Lum H. Jo H. Am. J. Physiol. Heart Circ Physiol. 2002; 283: H1819-H1828Crossref PubMed Scopus (211) Google Scholar). Phosphorylation site mutants of eNOS were generated using the QuikChange mutagenesis kit (Stratagene) according to the manufacturer's instructions. Briefly, WT bovine eNOS was used as a template for the mutagenesis reaction. Primers containing the desired mutation were extended during temperature cycling byPfuTurbo DNA polymerase. The product was treated withDpnI to digest the parental DNA template and select for the synthesized DNA-containing mutations. The DNA was then transformed into XL1-Blue cells. Colonies were isolated from agar plates, grown for 8 h in LB-ampicillin and the plasmid isolated using a mini-prep kit (Qiagen). At least 4 clones for each mutation were sequenced to confirm the presence of the mutation. The sequences of the primers used to generate the eNOS phosphorylation site mutants were: S116A, 5′-ctgcagacccggcccGccccgggacctccac-3′; S116D, 5′-ctgcagacccggccgGAtccgggacctccac-3′; S617A, 5′-caagatccgcttcaacGCAgtctcctgctcagac-3′; S617D, 5′-caagatccgcttcaacGAcgtctcctgctcagaccc-3′; S635A, 5′-gcggaagagaaaggagGccagcaacacagacagtgc-3′; S635D, 5′-gcggaagagaaaggagGAcagcaacacagacagtgc-3′; and their respective reverse complements. All mutations were made using bovine eNOS cloned by Sessa et al. (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) except for S635D, which was made using bovine eNOS cloned by Nishida et al. (25Nishida K. Harrison D.G. Navas J.P. Fisher A.A. Dockery S.P. Uematsu M. Nerem R.M. Alexander R.W. Murphy T.J. J. Clin. Invest. 1992; 90: 2092-2096Crossref PubMed Scopus (616) Google Scholar) COS-7 cells were grown to confluence in 6-well plates and then transfected with WT eNOS or eNOS phosphorylation site mutants using LipofectAMINE 2000 (Invitrogen) transfection reagent as described by the manufacturer. The next day the growth medium was aspirated and replaced with 2 ml of fresh growth medium. The cells were incubated for 24 h, and an aliquot of medium was taken for basal NO measurements, assayed as nitrite, the stable breakdown product of NO in aqueous medium. The cells were then incubated for 4 h in serum-free medium, and 30 min before stimulation the medium was aspirated and replaced with 1 ml of fresh serum-free medium. After 30 min an aliquot of medium was taken for background nitrite measurement, and the cells were stimulated with 10 μm ATP and allowed to incubate for an additional 30 min. An aliquot of medium was again taken for nitrite measurement and the cells collected and lysed for protein assay and Western blot analysis. Nitrite levels were then measured using a Sievers NO analyzer as previously described. Cells were washed twice with phosphate-buffered saline, lysed on ice in 50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40 (v/v), 10 mm NaF, 1 mmvanadate, 1 mm pefabloc, 10 mg/ml leupeptin, and lysates were transferred to microcentrifuge tubes and rotated for 45 min at 4 °C. Insoluble material was removed by centrifugation at 12,000 × g for 10 min at 4 °C. 20 μg of protein from cell lysates were then analyzed by Western blot analysis, or 750 μg of protein from cell lysates were partially purified using 2′-5′ ADP-Sepharose 4B and subjected to Western analysis as described previously (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). The activity of WT and mutant eNOS was determined in detergent solubilized lysates of transfected COS-7 cells by measuring the conversion of [14C]arginine to [14C]citrulline under Vmaxconditions as previously described (17McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). We first set out to determine the specificity of the antibodies generated against phospho-eNOS Ser-116, Ser-617, and Ser-635. COS-7 cells were transfected with WT eNOS, S116A, S617A or S635A eNOS and then analyzed by Western blotting with the corresponding phosphospecific antibodies. Fig. 1 demonstrates that indeed these antibodies are specific for the phosphorylated sites on eNOS, since the antibodies recognized WT eNOS but not their corresponding phosphodeficient mutants. Previous work has documented the specificity of Ser-1179 phospho-Ab (26Fulton D. Fontana J. Sowa G. Gratton J.P. Lin M. Li K.X. Michell B. Kemp B.E. Rodman D. Sessa W.C. J. Biol. Chem. 2002; 277: 4277-4284Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). We next examined the effects of VEGF stimulation on phosphorylation of Ser-1179, Ser-116, Ser-617, and Ser-635. Serum-starved BAECs were treated with VEGF (50 ng/ml) for 1, 3, 5, 15, or 30 min and detergent-solubilized lysates incubated with 2′-5′ ADP-Sepharose 4B to partially purify eNOS and samples analyzed by Western blotting with the each of the phospho-Abs. Fig. 2 demonstrates that the putative Akt phosphorylation sites, Ser-1179 and Ser-617, are phosphorylated with similar kinetics, peaking at ∼5 min and returning to basal levels of phosphorylation by 30 min. Ser-116 is basally phosphorylated and rapidly dephosphorylated in response to VEGF, which recovered over time. Ser-635, a putative PKA phosphorylation site, displayed a more delayed increase in phosphorylation in response to VEGF reaching maximal phosphorylation at ∼30 min (the latest time point tested). These data are consistent with recent studies examining VEGF-induced eNOS phosphorylation (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, 22Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23Michell 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, 26Fulton D. Fontana J. Sowa G. Gratton J.P. Lin M. Li K.X. Michell B. Kemp B.E. Rodman D. Sessa W.C. J. Biol. Chem. 2002; 277: 4277-4284Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Next we examined ATP-mediated eNOS phosphorylation as prototypical G-protein-coupled receptor agonist. BAECs were treated with ATP (10 μm) for various times. Lysates were prepared and eNOS was partially purified by ADP Sepharose and subjected to Western blot analysis as before (Fig.3A). The pattern of phosphorylation for each of the phosphorylation sites varied temporally from VEGF-stimulated cells. Ser-1179 and Ser-617 were rapid and transiently phosphorylated with maximal phosphorylation at 3 min and dephosphorylation at 15 min. Ser-116 exhibited a more delayed dephosphorylation, first noticeable at 5 min, that continued decreasing for the duration of the experiment (30 min). Ser-635 was rapidly and transiently phosphorylated following a similar pattern to that of Ser-617 and Ser-1179 (Fig. 3B). Thus the kinetics of VEGF- versus ATP-stimulated eNOS phosphorylation are different, and ATP responses are similar to those obtained with bradykinin (22Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23Michell 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). Recent studies have examined the effect of mutating eNOS phosphorylation site Ser-116 to alanine or Ser-617 or S635 to aspartate on in vitro NOS activity, either in cell lysates or with purified proteins, respectively for Ser-116 and Ser-617/635 (22Kou R. Greif D. Michel T. J. Biol. Chem. 2002; 277: 29669-29673Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 23Michell 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). In this study, COS-7 cells were transfected with WT, S116A, S617A, S635A, S116D, S617D, or S635D eNOS and assayed for NOS activity (Vmax conditions) in the cell lysates as described under "Experimental Procedures." Fig.4A shows the effect of the serine to alanine mutation of these phosphorylation sites on NOS activity. S116A (36.46 ± 0.84 pmol of citrulline/min/mg of protein) and S635A (41.99 ± 1.56 pmol of citrulline/min/mg of protein) eNOS both showed an increase in NOS activity compared with WT (29.28 ± 1.33 pmol of citrulline/min/mg of protein). S617A eNOS (19.66 ± 0.20 pmol of citrulline/min/mg of protein) activity was significantly lower than WT. Mutation of S116 on eNOS to aspartate (Fig. 4B; 52.145 ± 3.285 pmol of citrulline/min/mg of protein) resulted in an increase in enzyme activity compared with WT eNOS (29.28 ± 1.33 pmol of citrulline/min/mg of protein). NOS activity of S617D eNOS (59.610 ± 0.33 pmol of citrulline/min/mg of protein) was 2-fold higher than WT eNOS, while S635D eNOS activity (32.56 ± 0.91 pmol of citrulline/min/mg of protein) was similar to WT eNOS. Thus, under Vmax conditions, S116A, S116D, S617D, and S635A were more active than WT, whereas S617A was less active, and S635D was comparable to WT. In vitro activity assays measuring the conversion of arginine to citrulline are performed with optimal calcium and cofactors present and do not always accurately reflect the production rate of NO in living cells since phosphorylation, subcellular localization, and regulated protein-protein interactions can all impinge upon eNOS activation/inactivation. Therefore, we examined the effect of mutating Ser-116, Ser-617, or Ser-635 to alanine, preventing phosphorylation at that site, on the ability of eNOS to produce NO in intact cells under basal (24-h accumulation) or stimulated (10 μm ATP, 30-min accumulation) conditions. WT and S1179A eNOS were used as positive and negative controls, respectively. Fig. 5A shows that basal NO release from cells transfected with S617A (6.18 ± 0.22 nmol of nitrite/mg of protein) showed a modest increase in NO release compared with WT eNOS (4.02 ± 0.163 nmol of nitrite/mg of protein) while NO release from S1179A (0.631 ± 0.081 nmol of nitrite/mg of protein, as previously shown), and S635A (2.43 ± 0.35 nmol of nitrite/mg of protein) eNOS were decreased. NO release from S116A eNOS (4.127 ± 0.237 nmol of nitrite/mg of protein) transfected cells was similar to that of cells transfected with WT eNOS. The effects of mutation of Ser-617 and Ser-635 to Ala, were diametrically opposite in NOS activity versus NO release assays. Cell were then stimulated with ATP to examine agonist evoked NO release. ATP (10 μm) stimulated S116A and S617A eNOS (1.422 ± 0.143 and 2.265 ± 0.030 nmol of nitrite/mg of protein) to produce moderately higher amounts of NO compared with WT eNOS (1.36 ± 0.12 nmol of nitrite/mg of protein), whereas NO release from S635A eNOS (1.42 ± 0.14 nmol of nitrite/mg of protein) was similar to that of the WT enzyme (Fig. 5B) and that from S1179A eNOS (0.83 ± 0.08 nmol of nitrite/mg of protein) diminished as previously 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, 17McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Fig. 5Cdemonstrates that each of the eNOS constructs studied was expressed at similar levels. Mutation of serine or threonine phosphorylation sites to aspartate can sometimes, but not always, mimics the phosphorylated state of the protein, whereas alanine mutations clearly render the site phosphorylation defective but may also cause untoward conformational changes in proteins. Therefore, as another means of examining the role of each individual phosphorylation site in eNOS, we transfected COS-7 cells with WT, S1179D, S116D, S617D, or S635D eNOS and measured basal and ATP-stimulated NO release as before. Mutating Ser-1179 to aspartate has been previously shown to increase eNOS activity and NO release 2–4 fold when compared with WT and was used as a positive control in this study (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, 17McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 23Michell 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). Mutation of Ser-116 to aspartate (S116D eNOS) had no effect on basal NO production (3.552 ± 0.083 nmol of nitrite/mg of protein; Fig.6A), while exhibiting an increase under stimulated conditions (0.75 ± 0.015 nmol of nitrite/mg of protein; Fig. 6B) when compared with WT eNOS (basal, 3.05 ± 0.08 nmol of nitrite/mg of protein; stimulated, 0.52 ± 0.032 nmol of nitrite/mg of protein). The S617D mutant produced ∼50% more NO both basally (4.85 ± 0.162 nmol of nitrite/mg of protein) and when stimulated for 30 min with ATP (0.87 ± 0.037 nmol of nitrite/mg of protein). Transfecting cells with the S635D mutant resulted in the most robust increase in NO production of any of the phosphorylation site mutants, including S1179D. Basal NO release from S635D was 5-fold higher (14.24 ± 0.28 nmol of nitrite/mg of protein) and stimulated NO release was nearly double (0.99 ± 0.048 nmol of nitrite/mg of protein) that of WT eNOS. The S1179D mutant produced significantly higher amounts of NO both basally and under stimulated conditions (basal, 6.28 ± 0.35 nmol of nitrite/mg of protein; stimulated, 1.32 ± 0.18 nmol of nitrite/mg of protein) as in earlier studies. Fig. 6Cdemonstrates, by Western blot analysis, that similar amounts of each of the eNOS constructs were expressed in these experiments. To determine whether phosphorylation at one site in eNOS effects phosphorylation at other sites, we transfected COS-7 cells with each eNOS
Referência(s)