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*

Philip M. BauerDagger , David FultonDagger , Yong Chool Boo§, George P. Sorescu§, Bruce E. Kemp, Hanjoong Jo§, and William C. SessaDagger ||

From the Dagger  Department of Pharmacology and Vascular Cell Signaling and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, the § Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, 308D WMB, Atlanta, Georgia 30322, and the  St. Vincent's Institute of Medical Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia

Received for publication, November 22, 2002, and in revised form, February 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1, 2), protein-protein interactions (3-7), cofactors and prosthetic groups (8-10), calcium/calmodulin (11), and phosphorylation (12).

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 (13), Akt (protein kinase B) (6, 14, 15), and protein kinase A (16). 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 (6, 15). 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 (6, 15, 17).

Phosphorylation of threonine 497 (Thr-497) on bovine eNOS (Thr-495 in human) has also been documented (18). Thr-497 is basally phosphorylated and may be dephosphorylated in response to bradykinin stimulation (18-20). 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) (21, 22), serine 617 (Ser-617) (23), and serine 635 (Ser-635) (23). 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 (22). 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 (23, 24). 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 (23). 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 (6, 15). Mutation of Ser-617 to aspartate was recently shown to increase calcium sensitivity without changing maximal enzyme activity of the purified enzyme (23).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell Culture and Reagents-- Bovine aortic endothelial cells (BAEC) were isolated and cultured as previously described (6). 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 (23, 24).

Mutagenesis-- 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 by PfuTurbo DNA polymerase. The product was treated with DpnI 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. (2) except for S635D, which was made using bovine eNOS cloned by Nishida et al. (25)

Measurement of NO Release from eNOS-transfected COS-7 Cells-- 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.

Western Blot Analysis-- 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 mM vanadate, 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 (6).

NOS Activity Assay-- 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 Vmax conditions as previously described (17).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Specificity of Phospho-eNOS-specific Antibodies-- 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 (26).


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Fig. 1.   Characterization of phosphospecific eNOS antibodies recognizing phosphoserine 116, 617, or 635. COS-7 cells were transfected with wild-type (WT), S116A, S617A, or S635A eNOS and detergent-solubilized lysates subjected to Western blot analysis with the corresponding phosphospecific antibodies. Membranes were stripped and reprobed with total eNOS antibody. Results are representative of at least three experiments.

VEGF-stimulated Phosphorylation of eNOS-- 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 (14, 22, 23, 26).


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Fig. 2.   Time course of VEGF-stimulated eNOS phosphorylation on serine 1179, 116, 617, and 635. A, BAECs were stimulated with VEGF (50 ng/ml) for the various times indicated and semi-purified eNOS subjected to Western blot analysis for total eNOS, or phosphoserine-eNOS 1179, 116, 617, or 635. B, densitometric ratio of phospho-eNOS to total eNOS. Blots are representative of three separate experiments. Densitometric ratios represent mean ± S.E. (n = 3).

ATP-stimulated Phosphorylation of eNOS-- 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 (22, 23).


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Fig. 3.   Time course of ATP-stimulated eNOS phosphorylation on serine 1179, 116, 617, and 635. A, BAECs were stimulated with ATP (10 µM) for the various times indicated and semipurified eNOS subjected to Western blot analysis for total eNOS, or phosphoserine-eNOS 1179, 116, 617, or 635. B, densitometric ratio of phospho-eNOS to total eNOS. Blots are representative of three separate experiments. Densitometric ratios represent mean ± S.E. (n = 3).

In Vitro NOS Activity of eNOS Phosphorylation Site Mutants-- 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 (22, 23). 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.


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Fig. 4.   NOS activity of eNOS serine to alanine or serine to aspartate mutants. A, NOS activity assays in cell lysates from cells transfected with WT eNOS or S116A, S617A, or S635A eNOS. B, NOS activity in cell lysates from cells transfected with WT eNOS or S116D, S617D, or S635D eNOS. NOS activity was monitored as described under "Experimental Procedures." In A and B cell lysates were analyzed by Western blot using eNOS-specific antibodies, and the inset demonstrates equal expression of WT eNOS and eNOS phosphorylation site mutants from an individual experiments. The values below the blot represent mean densitometric ratios of mutant/WT eNOS proteins (mean ± S.D.). from four individual experiments. In all experiments, background activity was determined by L-citrulline generation from cells transfected with the beta -galactosidase cDNA. This is essentially negligent since COS cells do not express any known NOS isoform. For NOS activity assays, the data represent mean ± S.E. of duplicate determinations of three independent protein lysates (n = 3 separate transfectants per experiment) and are representative of at least three independent experiments. *, significantly different (p < 0.01) from values obtained for WT eNOS.

Effect of eNOS Phosphorylation Site Serine to Alanine Mutations on NO Production-- 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.


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Fig. 5.   Effect of serine to alanine mutations in eNOS on NO production in COS cells. COS cells were transfected with WT eNOS or the respective serine to alanine mutants as indicated. A, basal NO accumulation was measured after 24 h. B, transfected COS cells were serum-starved for 4 h and then stimulated with ATP (10 µM). NO release was measured as nitrite accumulated 30 min after stimulation. C, cell lysates were analyzed by Western blot using eNOS-specific antibodies and demonstrate equal expression of WT eNOS and eNOS phosphorylation site mutants. Values represent mean densitometric ratios of mutant/WT eNOS protein levels ± S.D. from four experiments. In all experiments, nitrite generated from cells transfected with the beta -galactosidase cDNA were subtracted as background since COS cells do not express any known NOS isoform. Data represent mean ± S.E. (n = 5). Experiments shown are representative of 4 independent studies. *, significantly different (p < 0.01) from values obtained for WT eNOS.

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 (6, 15, 17). Fig. 5C demonstrates that each of the eNOS constructs studied was expressed at similar levels.

Effect of eNOS Phosphorylation Site Serine to Aspartate Mutations on NO Production-- 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 (6, 15, 17, 23). 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. 6C demonstrates, by Western blot analysis, that similar amounts of each of the eNOS constructs were expressed in these experiments.


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Fig. 6.   Effect of serine to aspartate mutations in eNOS on NO production in COS cells. COS cells were transfected with WT eNOS or eNOS phosphorylation site serine to aspartate mutants as indicated. A, basal NO release was measured after 24 h of accumulation. B, transfected COS cells were serum-starved for 4 h and then stimulated with ATP (10 µM). NO release was measured as nitrite accumulated 30 min after stimulation. C, cell lysates were analyzed by Western blot using eNOS-specific antibodies and demonstrate equal expression of WT eNOS and eNOS phosphorylation site mutants. Values represent mean densitometric ratios of mutant/WT eNOS protein levels ± S.D. from four experiments. In all experiments, nitrite generated from cells transfected with the beta -galactosidase cDNA were subtracted as background since COS cells do not express any known NOS isoform. Experiments shown are representative of four independent studies. Data represent mean ± S.E. (n = 3-5). *, significantly different (p < 0.01) from values obtained for WT eNOS.

Cooperation of Phosphorylation Sites on eNOS-- To determine whether phosphorylation at one site in eNOS effects phosphorylation at other sites, we transfected COS-7 cells with each eNOS serine to alanine mutants and analyzed the additional serine phosphorylation sites by Western blotting using phosphospecific antibodies (Fig. 7A). Cells were serum-starved overnight and then either subjected to Western analysis or stimulated with 10 µM ATP for 3 min and then subjected to Western analysis. The patterns of basal and stimulated phosphorylation were similar with the stimulated cells exhibiting increased or decreased phosphorylation compared with basal depending on the phosphorylation site examined. For example, phosphorylation at Ser-1179 was increased by about 50% in both S116A and S617A eNOS, but was unchanged in the S635A eNOS. Interestingly, phosphorylation at Ser-116 was markedly reduced by the loss of phosphorylation at Ser-617 (in S617A eNOS) suggesting cooperativity between the sites. Phosphorylation at Ser-635 was almost 2-fold higher in S1179A eNOS and 50% lower in S617A eNOS. Phosphorylation at Ser-617 was unaffected by mutation at any of the other phosphorylation sites. Thus, compensatory phosphorylation, under basal and stimulated conditions, occurs at other serine residues and is a common feature of S1179A, S116A, and S617A eNOS, whereas no compensation takes place with S635A eNOS.


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Fig. 7.   Mutation of eNOS serine phosphorylation sites modulates phosphorylation of other residues. A, COS cells were transfected with WT, S1179A, S116A, S617A, or S635A eNOS for 24 h and then serum-starved for an additional 24 h. The cells were at that time either lysed or stimulated with 10 µM ATP for 3 min and then lysed. Cell lysates were then partially purified with 2'-5' ADP-Sepharose and analyzed by Western blot using total eNOS or phosphospecific eNOS antibodies for each of the phosphorylation sites. B, quantitative densitometric ratios of phospho-eNOS to total eNOS comparing phosphorylation of each eNOS serine to alanine mutant at each of the phosphorylation sites. The quantitative data are from basal conditions since the data were qualitatively similar to stimulated cells. Data are representative of three independent experiments and results are normalized to WT eNOS and represent mean ± S.E. (n = 3). *, significantly different (p < 0.01) from values obtained for WT eNOS.

Association of hsp90 and Akt with eNOS Phosphorylation Site Serine to Alanine Mutants-- Association of hsp90 with eNOS acts to displace eNOS from caveolin-1 (27), serve as a scaffold for the interaction of Akt with eNOS (28), and regulates CaM sensitivity and electron flux from the reductase to the oxygenase domains of eNOS (17). Therefore, we were interested in whether or not phosphorylation of eNOS can regulate the association of the hsp90/Akt complex with eNOS. COS-7 cells were transfected with WT, S1179A, S116A, S617A, or S635A eNOS cDNAs and serum-starved overnight. Cell lysates were prepared from serum-starved cells or cells stimulated with ATP (10 µM) for 3 min and eNOS partially purified using 2'-5' ADP Sepharose. Protein bound to the Sepharose beads was then eluted with NADPH and analyzed by Western blotting using antibodies to eNOS, hsp90, or Akt (Fig. 8A). Mutation of Ser-1179 to alanine had no effect on the ability of hsp90 or Akt to co-associate with eNOS under basal or stimulated conditions (Fig. 8, A-C). S116A eNOS had ~400% more hsp90 and 50% more Akt associated with it than the WT enzyme under basal conditions. Under stimulated conditions, however, there was no additional increase in hsp90 or Akt association and was similar to WT eNOS. S617A eNOS showed a 400% increase in hsp90 and a 150% increase in Akt was association with S617A eNOS under basal conditions. Upon stimulation of S617A eNOS there was a slight increase in hsp90 association with eNOS although there was no apparent increase in Akt association. When compared with WT eNOS, S617A bound 50% more hsp90 and 75% more Akt. Under basal conditions hsp90 binding to eNOS was unaffected by mutation of Ser-635 to alanine, while Akt association was significantly decreased. Under stimulated conditions, however, S635A eNOS bound nearly 2-fold more hsp90 than WT eNOS whereas Akt binding was similar to that of the WT enzyme. Association of hsp90 and Akt with eNOS was specific since hsp90 and Akt were not detected in beta -galactosidase-transfected cells treated in the same manner as the eNOS-transfected cells (data not shown). Thus, the enhanced interaction of hsp90/Akt with S116A and S617A mutants may explain the enhanced compensatory phosphorylation of these mutants on Ser-1179.


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Fig. 8.   Mutation of eNOS serine phosphorylation sites influences the interaction of eNOS with hsp90 or Akt. A, COS cells were transfected with WT, S1179A, S116A, S617A, or S635A eNOS for 24 h and then serum-starved for an additional 24 h. The cells were at that time either lysed or stimulated with 10 µM ATP for 3 min and then lysed. Cell lysates were then partially purified with 2'-5' ADP-Sepharose and analyzed by Western blot using total eNOS, hsp90, or Akt antibodies. Quantitative densitometric ratio of hsp90 (B) or Akt (C) to total eNOS for each of the eNOS serine to alanine mutants. Blots are representative of three independent experiments. Data are normalized to association of Akt/hsp90 of WT eNOS under basal conditions and represent mean ± S.E. (n = 3). *, significantly different (p < 0.01) from values obtained for WT eNOS under basal conditions. **, stimulated values are significantly different (p < 0.01) from values obtained for unstimulated of the same construct. #, significantly different (p < 0.01) from values obtained from ATP-stimulated WT eNOS.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The central findings of this study are that eNOS is regulated by complex and multiple phosphorylation events on serines 116, 617, 635, and 1179. The relative rate of phosphorylation of these residues is different for G-protein-coupled receptor activation by ATP versus receptor tyrosine kinase signaling via VEGF. Using a reconstituted system and individual point mutations in eNOS reflecting loss-of-function (serine to alanine) and potential gain-of-function (serine to aspartate) mutants, serines 1179 and 635 are the most important positive regulatory sites for basal and ATP-stimulated NO release and phosphorylation of Ser-116 is inhibitory. In addition, we show for the first time, an interrelationship between the four 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, compensatory modulation of phosphorylation and protein-protein interactions provides an interesting, unanticipated mechanism to regulate eNOS function.

Recent work in the past 3 years has focused on the role of phosphorylation in regulating eNOS function. Initial findings based on mapping of the phosphorylation sites in eNOS focused on the individual residues serines 1179, 116, 617, 635, and threonine 497. In results obtained from multiple groups in vitro and in vivo, serine 1179 has been the most highly studied. Serine 1179 can be phosphorylated by multiple kinases including AMP kinase (13), Akt (6, 14), protein kinase A, and protein kinase G (16). Overexpression of Akt or adenoviral delivery of constitutively active Akt markedly drives NO release from cells; dominant negative Akt attenuates some, but not all agonists promoting NO release (6). In vivo delivery of myr-Akt promotes blood flow and angiogenesis while dominant negative Akt attenuates acetylcholine-induced blood flow changes and endothelium-dependent relaxations (29). More recently, reconstitution of a constitutively active form of eNOS S1179D into the endothelium of eNOS (-/-) mice rescued the vasomotor defect better than the loss of function mutant and prevents the vasconstrictor actions of endothelin-1 strongly supporting a functional role for this site (30). The mechanism for how this site improves eNOS function is due to enhanced calcium/CaM sensitization and phosphorylation at 1179 derepressing the carboxytail autoinhibitory control element in eNOS and thus facilitating of electron transfer from the reductase to the oxygenase domain (17, 31).

Recent studies suggest that the additional serine phosphorylation sites can regulate eNOS activity and perhaps NO production (22, 23). Most studies examining the role of phosphorylation in a given pathway rely on conventional mutagenesis strategies where mutation of the phosphorylation site to an alanine residue results in the lack of phosphorylation or perhaps a structural change. Conversely, mutation of the phosphorylation site to aspartate or glutamate may mimic the activation/inactivation state or cause an untoward structural change resulting in no change or a loss of function. In the context of previous studies, dephosphorylation of serine 116 (based on the S116A mutant) enhances ionophore-stimulated eNOS activity in broken cell lysates (22), while phosphorylation of serines 617 by Akt and 635 by protein kinase A (based on NOS activity assays with purified proteins from baculovirus) influences calcium sensitivity and specific activity of eNOS, respectively (23). However, previous reports have shown no difference in eNOS activity in cells expressing S633D (human equivalent to bovine Ser-635) or no change or slight increase in NO production in cells expressing S633A/S635A (6, 15). Although innovative and interesting, the above studies did not examine the activity of the enzyme with the integrated basal and stimulus-dependent release of NO or consider that mutations in one residue may impact additional sites of phosphorylation or protein-protein interactions.

As seen in our study measuring activity and the release of NO with alanine mutants of these sites, S617A is the only mutant that produces more basal NO than wild-type eNOS, whereas S1179A and S635A eNOS produce less NO, with S116A generating comparable amounts to wild type. Upon challenge with ATP, only S1179A produces less NO, with S116A and S617A producing more NO than wild-type eNOS. Thus, in these paradigms using serine to alanine mutations of the phosphorylation sites, the phosphorylation of eNOS at residues Ser-635 and Ser-1179 are important for promoting basal NO release and phosphorylation at Ser-617 negatively regulates basal NO release. Upon ATP stimulation phosphorylation at Ser-1179 promotes NO release, while phosphorylation at Ser-116 and Ser-617 negatively regulates NO release. These data suggest the following paradigm: Ser-116 is important for agonist, but not basal, NO release (22); Ser-635 is important for basal, but not stimulated NO release; Ser-617 negatively regulates basal and stimulated NO release and Ser-1179 phosphorylation is stimulatory for both basal and agonist induced NO release.

In the context of putative "gain-of-function" mutations, S1179D, S617D, and S635D all increased basal and ATP-stimulated NO release with S635D being the most efficacious, whereas basal S116D NO release was comparable to wild-type eNOS and stimulated NO release from S116D was increased. Collectively, in conjunction with the alanine mutants, these data confirm the roles of 1179 and Ser-635 as stimulatory sites. We also show that substituting aspartate at Ser-116 does not reduce activity or NO release providing evidence that aspartate is not a suitable amino acid substitution to completely mimic the phospho-state since S116D is similar to S116A eNOS. Another unexpected finding was that S617A and S617D both produced more NO than did WT eNOS suggesting at least 4 possible interpretations of these data: 1) this site is an inhibitory site and aspartate at this position is insufficient to produce the "inhibitory state" in cells, akin to S116D; 2) alanine at this site prevents phosphorylation at 617 but changes the conformation in an untoward manner, 3) alanine at this site prevents phosphorylation at 617 but regulates phosphorylation elsewhere (Ser-1179 phosphorylation is increased and Ser-116 and Ser-635 phosphorylation are decreased), or 4) in light of the findings that purified S617D has a greater sensitivity to CaM/calcium, perhaps under basal conditions or after ATP challenge, this change in sensitivity is masked by other mechanisms that also regulate eNOS at steady state. This apparent hysteresis occurred despite S617A being less active and S617D being more active in NOS activity assays under Vmax conditions. Thus, our data are consistent with Ser-116, Ser-635, and Ser-1179 as important regulatory sites for NO release from cells and Ser-617 phosphorylation may exert a regulatory role on CaM/calcium affinity with eNOS.

Clearly, a temporal pattern of phosphorylation exists among the known sites of eNOS phosphorylation. As seen in with VEGF versus ATP as agonists, the rates of phosphorylation/dephosphorylation are different. In addition, the subcellular localization and protein-protein interactions can modulate its phosphorylation on Ser-1179 and Ser-116 residues (26, 32). Based on these studies, it is feasible that phosphorylation at one site may be important for phosphorylation/dephosphorylation at an additional site, as is the case for many proteins. To test this directly, we monitored the phosphorylation of Ser-116, Ser-617, Ser-635, and Ser-1179 sites in the respective individual alanine mutants under basal and ATP-stimulated conditions. Interestingly, the phosphorylation at Ser-1179 increased in both S116A and S617A eNOS, but not S635A eNOS, suggesting that Ser-1179 phosphorylation may contribute to the increase in NO release seen with S116A and S617A eNOS constructs. Also, the co-association of hsp90 and Akt were enhanced with these constructs providing a mechanism for the enhanced phosphorylation on Ser-1179 and NO release. An interrelationship between phosphorylation on Ser-116, Ser-617, and Ser-635 is supported by data showing complete lack of phosphorylation or enhanced dephosphorylation on Ser-116 and a decrease in Ser-635 phosphorylation in cells expressing S617A. In cells expressing S1179A eNOS, basal and stimulated NO production decreased but there were no changes in the interaction of hsp90 and Akt. This was accompanied by an increase in phosphorylation at Ser-635 which can promote basal NO release, but did not occur in the context of S1179A eNOS. The only phosphorylation site that was not modulated by the individual alanine mutants was Ser-617. Thus, these data demonstrate that mutation of individual phosphorylation sites may promote additional phosphorylation/dephosphorylation events on adjacent residues. This may occur through true cooperativity between the sites, protein-protein interactions, or via structural changes.

Another interesting finding is that S635D markedly enhanced basal and stimulated NO release from cells with increase basal NO release being greater than that exhibited with S1179D eNOS. Previous studies examining the activities of S633D or NO release from S633A/S35A eNOS, were essentially negative (6, 15). Recently, it was reported that both S635D and S1179D eNOS were 2-2.5 more active than WT eNOS (6, 17, 23) and S1179D exhibited enhanced calcium/CaM sensitivity. Our results measuring NOS activity in broken cell lysates indicate S635A exhibits greater activity than WT eNOS, whereas S635D is equivalent to WT enzyme is discordant from our results measuring NO release. This apparent discrepancy may be due to additional phosphorylation events in the context of S635D or enhanced protein-protein interactions that are regulatory in intact cells but are minimized in detergent-solubilized lysates. Regardless of the precise mechanism, our data strongly support the idea that multiple sites of serine phosphorylation contribute to NO release and that assigning importance to an individual site may be potentially misleading until the mutants are examined in multiple cell-based systems.

    FOOTNOTES

* This work was supported by Grants RO1 HL57665, HL61371, HL64793 (to W. C. S.), and T32 HL07950-02 (to P. M. B.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Pharmacology, Vascular Cell Signaling & Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536. Tel.: 203-737-2291; E-mail: william.sessa@yale.edu.

Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211926200

    ABBREVIATIONS

The abbreviations used are: eNOS, endothelial nitric-oxide synthase; Ab, antibody; WT, wild type; VEGF, vascular endothelial growth factor; BAEC, bovine aortic endothelial cells.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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