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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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).
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RESULTS |
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.
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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).
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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).
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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
-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.
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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
-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.
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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 -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.
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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.
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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
-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.
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DISCUSSION |
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.