Direct Interaction between Endothelial Nitric-oxide
Synthase and Dynamin-2
IMPLICATIONS FOR NITRIC-OXIDE SYNTHASE FUNCTION*
Sheng
Cao
,
Janet
Yao
,
Timothy J.
McCabe§,
Qing
Yao¶,
Zvonimir S.
Katusic
,
William C.
Sessa§, and
Vijay
Shah
¶**
From the
GI Research Unit,
Anesthesia Research
and ¶ Department of Medicine, Mayo Clinic,
Rochester, Minnesota 55905 and the § Department of
Pharmacology, Yale University, New Haven, Connecticut 06520
Received for publication, July 14, 2000, and in revised form, December 15, 2000
 |
ABSTRACT |
Endothelial nitric-oxide synthase (eNOS) is
regulated in part through specific protein interactions. Dynamin-2 is a
large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS.
Additionally, the calcium ionophore, A23187, a reagent that promotes
nitric oxide release, enhances coprecipitation of dynamin with eNOS in
BAEC, suggesting the interaction between the proteins can be regulated
by intracellular signals. In vitro studies demonstrate that
glutathione S-transferase (GST)-dynamin-2 quantitatively
precipitates both purified recombinant eNOS protein as well as in
vitro transcribed 35S-labeled eNOS from solution
indicating a direct interaction between the proteins in
vitro. Scatchard analysis of binding studies demonstrates an
equilibrium dissociation constant (Kd) of 27.6 nM. Incubation of purified recombinant eNOS protein with
GST-dynamin-2 significantly increases eNOS activity as does
overexpression of dynamin-2 in ECV 304 cells stably transfected with
eNOS-green fluorescent protein. These studies demonstrate a direct
protein-protein interaction between eNOS and dynamin-2, thereby
identifying a new NOS-associated protein and providing a novel function
for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.
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INTRODUCTION |
Endothelial nitric-oxide synthase
(eNOS)1 is a
membrane-associated protein that is localized in the Golgi apparatus as
well as within cholesterol-rich plasmalemmal vesicles, termed caveolae (1-3). The ability of eNOS to generate nitric oxide (NO) has traditionally been ascribed to agonist-stimulated
calcium-dependent activation. However, during recent years
several groups have demonstrated that activation of eNOS, both in
conjunction with and independent of intracellular calcium flux, occurs
through the allosteric binding of eNOS with neighboring regulatory
proteins (4-6). In this regard, several eNOS-associated proteins have
been identified, including the caveolae coat protein, caveolin,
calmodulin, Hsp 90, and the bradykinin-2 receptor (6-9).
Dynamin-2 is a large ubiquitously expressed GTP-binding protein that
targets to Golgi membranes and colocalizes with caveolin within
caveolae (10). Although the function of dynamin is best characterized
in membrane scission events (11-14), members of this family of
proteins also modulate signaling pathways by means of distinct protein
interactions (10, 15-17). In support of this concept, dynamin-1 binds
and regulates the calcium-sensitive phosphatase, calcineurin, and the
dynamin family of proteins interact with the SH3 domains of a variety
of signaling proteins by virtue of a proline-rich domain (16-20).
Based on previously published studies indicating that both eNOS and
dynamin-2 colocalize with caveolin and reside within similar membranes
compartments (1, 2, 12-14), we examined whether these two proteins
might interact in a functional manner. Here we demonstrate that pools
of eNOS and dynamin bind within cells in a manner regulated by ionic
stringency and intracellular signals and that in vitro the
proteins bind directly in a stoichiometric and high affinity manner.
Additionally, the interaction of dynamin with eNOS potentiates eNOS
catalysis in cells and in vitro. These studies identify a
new NOS-associated protein and provide a novel function for
dynamin-2.
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EXPERIMENTAL PROCEDURES |
Materials and Antibodies--
All tissue culture and
transfection reagents were obtained from Life Technologies, Inc. eNOS
mAb and pAb were obtained from Transduction Laboratories (Lexington,
KY). Golgi 58-kDa protein was obtained from Sigma. The dynamin-2 pAb
(dyn2T) has been previously described (12). This antibody recognizes
the proline-rich COOH-terminal tail unique to dynamin-2 and
specifically precipitates this dynamin isoform from brain lysates.
Calcium ionophore, A23187, was obtained from Sigma.
3H-Labeled L-arginine and
35S-labeled methionine were obtained from Amersham
Pharmacia Biotech.
Cell Culture--
Bovine aortic endothelial cells (BAEC), Clone
9 cells (rat hepatocyte cell line) (14), and stably transfected
eNOS-GFP ECV 304 cells (21) were used in these studies. The BAEC used
in this study were between P2 and P5 and were
obtained from Clonetics (San Diego, CA). BAEC were cultured in EGM,
Clone 9 cells in Ham's F-12K medium, and ECV 304 cells in Dulbecco's
modified Eagle's medium with 400 µg/ml G418. Media were supplemented
with 10% fetal bovine serum, L-glutamine (1 mM), penicillin (100 IU/ml), and streptomycin (100 µg/ml). For transfections, Clone 9 cells were plated on glass
coverslips in 12-well plates and cotransfected with a bovine eNOS
expression vector (pcDNA3) and a rat dynamin-2 expression vector
(pEGFP-N1) (22). eNOS-GFP ECV 304 cells were plated in 100-mm dishes
and cotransfected with a rat dynamin-2 expression vector (pcDNA3).
Transfections were performed using LipofectAMINE (Life Technologies,
Inc.) as per the manufacturer's protocol. Thirty six hours after
transfection, Clone 9 cells were prepared for immunofluorescence
microscopy, and eNOS-GFP ECV 304 cells (21) were prepared for NOS
activity assay and immunoprecipitation analysis.
Expression and Purification of Recombinant eNOS and GST-Dynamin
Fusion Protein--
Recombinant eNOS protein was purified from
Escherichia coli as described previously (23, 24). In brief,
bovine eNOS in the plasmid, pCW was coexpressed with pGroELS plasmid
into protease-deficient E. coli. eNOS was purified from
extracts using a 2',5'-ADP Sepharose 4B column. Typically 2.5-4 mg of
eNOS was recovered from each preparation, yielding a single major band
as determined by SDS-PAGE. Purified eNOS was stabilized with
L-arginine (0.5 mM) and 5-fold molar excess of
BH4. A cDNA construct encoding full-length
GST-dynamin-2 fusion protein was created by subcloning dynamin-2
cDNA into the GST fusion protein vector, pGEX-1. GST-dynamin and
GST constructs were transformed into BL21 (DE3) and induced with
isopropyl-1-thio-
-D-galactopyranoside (1 mM)
overnight and lysed by sonication with lysozyme 200 µg/ml in a buffer
containing 20 mM Hepes (pH 7.2), 100 mM KCl, 2 mM MgCl, 1 mM dithiothreitol, 2 µM leupeptin, 1 mM PMSF. Samples were
resonicated after addition of Triton X-100 to a final concentration of
1%. Cell debris was removed by centrifugation, and supernatant was
mixed with glutathione-Sepharose beads and agitated for 2 h at
4 °C. Samples were centrifuged at 500 rpm, and pellets were washed
three times in phosphate-buffered saline with 1% Triton X-100.
Specificity and quality of GST-dynamin was assessed by Coomassie
staining of SDS-PAGE gels and Western blotting of transferred proteins
as depicted in Fig. 4A.
In Vitro Translation of [35S]Methionine
eNOS--
[35S]Methionine eNOS was translated in rabbit
reticulocyte lysates using the TnT Coupled Reticulocye Lysate System
(Promega, Madison, WI). The reaction mix, containing bovine eNOS DNA
(or alternatively negative control containing empty vector), SP6 RNA polymerase promoter, and [35S]methionine, was incubated
at 30 °C for 90 min. Translation products were examined by SDS-PAGE
analysis and autoradiography of dried gels. Binding assays using
[35S]methionine eNOS were performed as described below.
Confocal Immunofluorescence Microscopy--
BAEC and Clone 9 cells (48 h after cotransfection with eNOS and GFP-dynamin) were fixed
in 2% paraformaldehyde. Double-labeling immunofluorescence was
performed in BAEC as previously described in liver endothelial cells
(25), by incubating cells with eNOS pAb and Golgi 58-kDa protein mAb,
dynamin pAb and Golgi 58-kDa protein mAb, or alternatively eNOS mAb and
dynamin pAb. Immunofluorescence in Clone 9 cells transfected with
GFP-dynamin was performed by incubating cells in eNOS mAb only. Primary
antibodies were detected using fluorescein isothiocyanate- and Texas
Red-coupled secondary antibodies. Washes were performed with
phosphate-buffered saline, 0.1% bovine serum albumin after both
primary and secondary antibody incubation. Cells were mounted in
Anti-fade (Molecular Probes, Eugene, OR) and visualized using a
confocal microscope (LSM 510, Zeiss, Germany).
Immunoprecipitation and Western Blotting--
BAEC were
homogenized in a lysis buffer (50 mM Tris-HCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM
leupeptin, 1 mM PMSF, 1% (v/v) Nonidet P-40, 0.1% SDS,
0.1% deoxycholate (pH 7.5)). In some experiments, BAEC were incubated
in media containing 10 µM A23187 or equal volume of
vehicle (Me2SO) for 10 min prior to lysis. Protein
quantification of samples was performed using the Bio-Rad protein
assay. eNOS immunoprecipitation was performed by incubating 1-ml
aliquots of detergent-soluble protein lysate with excess eNOS mAb
overnight (1:200 dilution) or alternatively with equal concentration of mouse IgG after preclearing of samples with Pansorbin as previously described (26). Alternatively, dynamin immunoprecipitation was performed by incubating lysates with excess dynamin pAb (5 µg/ml) overnight. Immunocomplexes were bound by incubating protein samples with protein A beads for 1 h at 4 °C. Triplicate samples of
bound proteins were extensively washed in a buffer (50 mM
Tris-HCl, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM leupeptin, 1 mM PMSF) containing 0 mM NaCl, 100 mM NaCl, or alternatively 1 M NaCl. Bound proteins were eluted by boiling samples in
Laemmli loading buffer. Gel electrophoresis of proteins and Western
blotting were performed as previously described (25), using eNOS mAb
and dynamin pAb. Membranes were stained with Ponceau S or gels with
Coomassie Blue to confirm equal protein loading. Densitometric analysis
of autoradiographs was performed using Scion Image from the National
Institutes of Health.
In Vitro Binding Assays--
Increasing concentrations of
recombinant eNOS proteins (60-300 nM) were incubated
overnight at 4 °C with GST-dynamin beads or, alternatively, GST
beads alone in immunoprecipitation buffer in the absence of detergents.
Bound proteins were washed in a buffer containing 50 mM
Tris (pH 7.7), 200 mM NaCl, and 1 mM EDTA (8).
Bound proteins were eluted with Laemmli buffer and used for gel
electrophoresis. In vitro binding of GST-dynamin with 35S-eNOS was examined by incubating GST-dynamin beads
(60-600 nM) or GST beads alone, with a fixed concentration
of in vitro translated eNOS (3 µl of rabbit reticulocyte
lysate) or, alternatively, by incubating increasing concentrations of
in vitro translated eNOS (1-12 µl of rabbit reticulocyte
lysate) with a fixed concentration of GST-dynamin beads, in 300 µl of
a buffer containing 50 mM Tris-HCl, 0.1 mM EDTA overnight. Bound proteins were washed three times with a buffer containing 50 mM Tris-HCl, 200 mM
NaCl, 1 mM EDTA and then eluted and used for gel
electrophoresis. Binding studies with GST-dynamin and recombinant eNOS
were analyzed by SDS-PAGE and Western blot analysis, whereas binding
studies with GST-dynamin and 35S-eNOS were examined by
SDS-PAGE and autoradiography of dried gels. Quantification of
autoradiographs was performed by densitometric analysis using Scion
Image. Estimation of equilibrium dissociation constant
(Kd) was performed by incubating a fixed
concentration of GST-dynamin beads (5 nM) with purified
recombinant eNOS (0-320 nM) premixed with proportionate
volume of 35S-eNOS (0-16 µl) used as a radiolabel tracer
(the protein concentration of the radiolabel tracer is negligible
compared with the protein concentration of the recombinant protein).
Bound radioactive counts were measured directly by scintillation
counting. Quantitation of bound and free 35S-eNOS allowed
for determination of bound and free recombinant eNOS.
Kd was calculated by Scatchard plot analysis of bound and free levels of recombinant eNOS.
NOS Activity Assays--
NOS activity from recombinant eNOS
protein and eNOS-GFP EVC 304 cell lysates was assessed by measuring the
conversion of 3H-labeled L-arginine to
3H-labeled L-citrulline in the presence and
absence of L-nitroarginine methyl ester
(L-NAME) as described previously (6, 26). GST-dynamin or,
alternatively, GST alone was eluted from glutathione-Sepharose beads
with reduced glutathione, and the eluted protein was incubated with 15 pmol of purified recombinant eNOS protein in molar ratios of 0:1,
0.5:1, 1:1, and 2:1 as described by Ju et al. (4). Cell lysates were prepared from eNOS-GFP ECV 304 cells cotransfected with 3 or 6 µg of dynamin-2 DNA using the buffer described above for
Western blot and separated into 200-µg aliquots for NOS assay. To
determine NOS activity, triplicate samples of recombinant proteins or
duplicate samples of cell lysate were incubated with a buffer containing 1 mM NADPH, 3 µM
tetrahydrobiopterin, 100 nM calmodulin, 2.5 mM
CaCl2, 10 µM L-arginine and
L-[3H]arginine (0.2 µCi) at 37 °C for 20 min in the presence and absence of 1 mM L-NAME.
The reaction mix was terminated by the addition of 1 ml of cold stop
buffer (20 mM Hepes, 2 mM EDTA, 2 mM EGTA (pH 5.5)) and passed over a Dowex AG 50WX-8 resin
column. Radiolabeled counts per min of generated
L-citrulline were measured and used to determine
L-NAME inhibitable NOS activity.
Statistical Analysis--
All data are given as mean ± S.E. Data were analyzed using paired and unpaired Student's
t tests and one-way analysis of variance.
 |
RESULTS |
eNOS Colocalizes with Dynamin within Cells--
Both eNOS and
dynamin appear to reside within similar subcellular compartments based
on previous studies examining subcellular localization of each of the
proteins (2, 12-14). To determine whether eNOS and dynamin colocalize
within cells, we performed confocal immunofluorescence microscopy in
Clone 9 cells heterologously expressing eNOS and dynamin as well as in
BAEC which express both proteins endogenously. As seen in Fig.
1A, in Clone 9 cells
transiently transfected with eNOS and GFP-dynamin, NOS protein is
detected predominantly in a perinuclear distribution. GFP-dynamin is
also detected in a perinuclear distribution with additional fluorescent signal detected within distinct punctate areas within the cytoplasm and
plasma membrane (Fig. 1B). As seen in the merged image (Fig. 1C), there is colocalization of the two proteins in a
perinuclear distribution. To examine the subcellular distribution and
colocalization of endogenously expressed eNOS and dynamin, we next
performed double labeling immunofluorescence microscopy in BAEC. In
BAEC, eNOS is detected in a perinuclear pattern with some protein
detected within distinct regions of plasma membrane (Fig.
1D). In Fig. 1E, immunolocalization of dynamin in
BAEC demonstrates a similar distribution of protein as observed in
Clone 9 cells, and as observed in the merged image in Fig.
1F, colocalization of the two proteins in BAEC is observed
in predominantly a perinuclear pattern (small arrow) as well
as in specific domains of plasma membrane (large arrow).
Next, we further examined the nature of this prominent perinuclear area
of colocalization in BAEC by examining the colocalization of eNOS and
alternatively dynamin, with the Golgi marker, Golgi 58-kDa protein.
eNOS and Golgi 58-kDa protein (Fig. 1, G and H, respectively) are detected in a perinuclear distribution and
colocalization of proteins is detected, indicating that pools of eNOS
protein reside within Golgi membranes as previously noted (Fig.
1I, small arrow) (1, 27). Note that distinct
pools of eNOS in plasma membrane do not colocalize with Golgi 58-kDa
protein, demonstrating the specificity of Golgi 58-kDa protein mAb
(Fig. 1I, large arrow). Next we
examined localization of dynamin (Fig. 1J) and Golgi 58-kDa protein (Fig. 1K) in BAEC. As seen in Fig. 1L,
pools of dynamin also colocalize with Golgi 58-kDa protein. No
fluorescence was detected in negative control slides in which serum was
substituted for the primary antibody or in cells incubated with
secondary antibody alone (data not shown). These studies suggest that
eNOS and dynamin colocalize predominantly within Golgi membranes of cells.

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Fig. 1.
eNOS and dynamin colocalize within
cells. A-C, Clone 9 cells were cotransfected with
vectors encoding GFP-dynamin fusion protein and bovine eNOS. Cells were
fixed in 2% paraformaldehyde, prepared for indirect
immunofluorescence, and visualized with a confocal microscope.
A, immunofluorescent signal for eNOS (red) is
concentrated predominantly in a perinuclear distribution. B
(green), GFP-dynamin immunofluorescence is
detected in a perinuclear pattern. A punctate pattern of fluorescence
is also detected in a plasmalemmal and cytoplasmic distribution. The
merged image in C depicts colocalization (yellow)
between eNOS and dynamin within a perinuclear region of the cell.
D-F, to examine distribution and colocalization
of the endogenous proteins, double labeling immunofluorescence was
performed in BAEC. D, endogenous eNOS (red) is
detected predominantly within a perinuclear distribution with
additional fluorescence detected within distinct regions of the plasma
membrane. E, dynamin (green) immunofluorescent
signal is detected most prominently within a perinuclear distribution
and in regions of plasma membrane. A faint, diffuse cytoplasmic signal
is also detected. The merged images in F demonstrate
prominent colocalization (yellow) in the perinuclear region
of BAEC (small arrow) similar to that noted in Clone 9 cells
as well as overlap within distinct regions of the plasma membrane
(large arrow). G, eNOS (red) is
detected in a perinuclear distribution in BAEC. H, the Golgi
marker, Golgi 58-kDa protein (green) is also detected in a
perinuclear distribution. The merged images in I demonstrate
colocalization of eNOS with Golgi 58-kDa protein in BAEC Golgi membrane
(yellow; small arrow). Also, note the distinct
pools of eNOS in plasma membrane that do not colocalize with Golgi
58-kDa protein (large arrow). J, dynamin
(red) is again detected in a perinuclear distribution as
well as in a diffuse cytoplasmic pattern in BAEC. K, Golgi
58-kDa protein (green) is also detected in a perinuclear
pattern. The merged images in L demonstrate colocalization
of dynamin and Golgi 58-kDa protein in Golgi membrane
(yellow).
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eNOS and Dynamin Bind in a Specific Manner within Cells--
Based
on the prominent colocalization of eNOS and dynamin particularly within
BAEC, we next examined whether the two proteins interact biochemically.
For this purpose, detergent-soluble lysates were prepared from BAEC,
and eNOS protein was immunoprecipitated under nondenaturing conditions.
As seen in Fig. 2A,
immunoprecipitation of eNOS coimmunoprecipitates dynamin
(lane labeled 0 mM NaCl). As seen in
Fig. 2B, immunoprecipitation of dynamin conversely coimmunoprecipitates eNOS from BAEC extracts (lane labeled
0 mM NaCl). As observed in Fig. 2, A
and B, coprecipitation of eNOS and dynamin is maintained in
the presence of an increase in the ionic strength of the wash buffer to
100 mM NaCl (lane labeled 100 mM NaCl). However, when beads are washed in the
presence of 1 M NaCl, the interaction between the two
proteins is markedly diminished suggesting an ionic strength
protein-protein interaction (lane labeled 1 M NaCl). We next examined the specificity of binding and pools of bound protein in cells, by substituting nonimmune sera for
eNOS mAb during the immunoprecipitation and by comparing the pools of
dynamin bound to eNOS with the total pool of dynamin in the lysate,
respectively. As seen in Fig. 2C, ~5% of the dynamin pool contained
in the post-immunoprecipitation lysate (lane labeled post IP that contains 20 µl from the 1 ml of lysate) is
bound to eNOS (lane IP) as assessed by the
similar dynamin Western blot signal intensity in these two lanes.
(Similar results were obtained when the pre-IP lysate (not shown) was
analyzed for dynamin in place of the post-IP lysate as the total pool
of dynamin is not markedly reduced by eNOS immunoprecipitation.) Also,
as seen in lane NIS, neither eNOS nor dynamin is
detected when mouse IgG is substituted for eNOS mAb during the
immunoprecipitation procedure. These studies indicate that fractions of
eNOS and dynamin coexist in a complex within cells.

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Fig. 2.
eNOS and dynamin coimmunoprecipitate in
BAEC. eNOS was immunoprecipitated from BAEC lysate using a
monoclonal antibody or, alternatively, dynamin was immunoprecipitated
using a polyclonal antibody. Samples were prepared for Western blot
(WB) analysis. A, under nondenaturing conditions,
immunoprecipitation of eNOS coprecipitates dynamin. This interaction is
maintained in the presence of 100 mM NaCl wash but markedly
disrupted when bound proteins are washed with 1 M NaCl.
Similar amounts of eNOS are immunoprecipitated in each sample.
B, under identical conditions, immunoprecipitation of
dynamin coprecipitates eNOS when bound proteins are washed with buffer
containing 0 and 100 mM NaCl. Again, the interaction is
predominantly disrupted in the presence of 1 M NaCl wash.
Similar amounts of dynamin are immunoprecipitated in each sample.
C, whereas eNOS mAb precipitates eNOS and dynamin from BAEC
lysate (IP), substitution of a nonimmune serum
(NIS) for eNOS mAb precipitates neither eNOS nor dynamin.
The amount of dynamin bound with eNOS is ~5%, as estimated by the
similar Western blot signal intensity of dynamin bound to eNOS
(IP) as compared with dynamin contained in 20 µl of the
1-ml sample (5% of post-IP sample) of BAEC lysate that was loaded on
the same gel (Post IP). Note that eNOS is almost entirely
immunodepleted in the post-IP. (A similar 5% estimation of binding was
observed when bound dynamin was compared with pre-IP lysate (not shown)
as only a minority of the cellular pool of dynamin binds eNOS.)
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Ionophore Potentiates Binding of Dynamin with eNOS--
To
determine whether binding between eNOS and dynamin within cells is
regulated by intracellular signals, we stimulated BAEC with the calcium
ionophore, A23187 (10 µM), an agonist which promotes NO
release (1). Control cells were treated with vehicle. Detergent-soluble
lysates were prepared, and eNOS protein was quantitatively
immunoprecipitated. As seen in lanes labeled IP, from the
representative blot (Fig. 3A),
eNOS immunoprecipitated from lysates of BAEC incubated with the
ionophore coimmunoprecipitate markedly greater levels of dynamin as
compared with equal amounts of eNOS immunoprecipitated from cells
treated with vehicle (compare the intensity of the dynamin Western blot
signal in lanes labeled IP under vehicle and
A23187). As seen in the lanes labeled Post IP,
after immunoprecipitation, eNOS is barely detected within 20 µl of
the remaining supernatant, whereas the majority of the cellular pool of
dynamin remains unbound to eNOS. In Fig. 3B, when the series
of experiments (n = 3) are examined as a group by
densitometric analysis, a significant increase in dynamin bound to eNOS
is detected after stimulation of cells with A23187 (* indicates
p < 0.05).

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Fig. 3.
Calcium ionophore, A23187, potentiates
binding of dynamin with eNOS. BAEC were incubated in media
containing 10 µM A23187 or, alternatively, vehicle for 10 min. Cell lysates were prepared for eNOS immunoprecipitation and gel
electrophoresis. A, a representative Western blot
(WB) is shown demonstrating that eNOS immunoprecipitated
(IP) from lysates of cells incubated with ionomycin
coprecipitates a quantitatively greater amount of dynamin protein as
compared with equal amounts of eNOS immunoprecipitated (IP)
from lysates of cells incubated with vehicle (compare the intensity of
the dynamin Western blot signal in lanes labeled IP under
vehicle and A23187). (The faint band
above the dynamin signal is the residual eNOS chemiluminescence signal
from the preceding eNOS mAb probe of the blot.) Dynamin protein is
detected in the supernatant recovered after eNOS immunoprecipitation
(post IP), whereas eNOS protein is depleted. B, a
densitometric analysis of three independent experiments demonstrates a
significant enrichment in the amount of dynamin bound to eNOS in
lysates from cells incubated with ionomycin as compared with vehicle
(*, p < 0.05).
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eNOS and Dynamin Interact Directly in Vitro--
Coprecipitation
between eNOS and dynamin from cell lysates may occur directly or
through an adapter protein. To determine whether eNOS and dynamin are
able to bind directly, we next examined whether eNOS in solution binds
to dynamin in the form of a GST fusion protein. Fig.
4A demonstrates the purity and
specificity of the GST dynamin fusion protein. In the left
panel, enrichment of a single 26-kDa protein eluted from GST
beads, and the enrichment of a 130-kDa protein eluted from GST-dynamin
beads is observed by Coomassie staining of an SDS-PAGE gel. The
right panel of Fig. 4A demonstrates the specific
Western blot signal for dynamin that is detected from purified
GST-dynamin beads but not GST beads alone. To determine whether eNOS
and dynamin bind directly in vitro and to quantify the
relative pools of bound protein, we incubated GST dynamin beads or
alternatively GST beads alone with increasing concentrations of
purified recombinant eNOS protein in solution (60-300 nM).
As seen in the representative Western blot in Fig. 4B, left
panel, specific binding of purified eNOS with GST dynamin is
detected, whereas GST beads alone do not coprecipitate eNOS from
solution. Additionally, at a 1:1 molar ratio of GST-dynamin to eNOS
(lane labeled 150 nM eNOS in
Fig. 4B), a majority of the pool of available eNOS protein
is bound to dynamin (compare lane labeled 150 nM eNOS to lane input,
which represents the available eNOS protein in the 150 nM
sample). Further increase in the available concentration of eNOS
results in a smaller increase in eNOS binding (compare lane
labeled 300 nM eNOS to lane 150 nM cm;1 eNOS). These observations are quantified
in Fig. 4B, right panel, in which the densitometric
intensity of the Western blot analysis is depicted graphically. Note
that at a 1:1 molar ratio of eNOS and GST-dynamin (bar
labeled 150 nM), the majority of available eNOS
protein (bar labeled input) binds with dynamin.
To establish further the specificity of this direct interaction, we
next determined whether GST dynamin binds
[35S]methionine-labeled eNOS transcribed in
vitro in reticulocyte lysates by incubating varying concentrations
of GST dynamin beads or GST beads alone, with a fixed volume of
35S-eNOS (3 µl). In the representative autoradiogram
developed from a dried SDS-PAGE gel in Fig. 4C, left panel,
note that whereas GST does not bind 35S-eNOS, GST-dynamin
binds 35S-eNOS in a concentration-dependent
manner. Again note that the majority of the available
35S-eNOS in solution is bound to dynamin (compare the
radiolabel intensity of 35S-eNOS in lanes labeled
300 and 600 nM GST-dynamin,
respectively, with lane labeled input (3 µl of
reticulocyte lysate)). These observations are quantified in Fig.
4C, right panel, in which the densitometric intensity of the
autoradiograph is depicted graphically. Conversely, incubation of
GST-dynamin with increasing concentrations of 35S-eNOS
(1-12 µl of reticulocyte lysate) also results in a
concentration-dependent binding between the proteins (data
not shown). Note that the detected pools of eNOS and dynamin bound
in vitro (Fig. 4, B and C) are significantly greater than that observed from cell lysates (Figs. 2C and 3). We next sought to quantify the binding affinity
between eNOS and dynamin in vitro using radiolabeled tracer
experiments and Scatchard analysis of binding data. 5.0 nM
GST-dynamin was incubated with a logarithmic range of concentrations of
purified recombinant eNOS protein (0-640 nM) premixed with
proportional volumes of 35S-eNOS tracer. Bound
radioactive counts were measured directly by scintillation counting in
duplicate. In Fig. 4D, the Scatchard analysis demonstrates a
Kd of 27.6 nM. To confirm that Kd
estimation using this protocol is independent of the concentration of
the immobilized protein, GST-dynamin, Kd calculations were
performed at three different concentrations of GST-dynamin (5, 15, and
45 nM), each with saturating concentrations of eNOS (0-640
nM), with each experiment yielding similar Kd values. These studies indicate that dynamin-2 binds eNOS in a specific
manner when the two proteins are maintained in solution. Additionally,
these experiments indicate that in vitro, eNOS binds with
dynamin in a direct manner with high affinity.

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Fig. 4.
GST-dynamin and eNOS bind with high
affinity in vitro. A demonstrates the
purity and specificity of GST-dynamin-2. Left, a
representative Coomassie-stained SDS-PAGE gel demonstrates the
enrichment of a single 26-kDa protein from GST beads alone and the
enrichment of a 130-kDa protein from GST-dynamin beads.
Right, Western blot analysis demonstrates a specific signal
for dynamin from GST-dynamin beads but not from GST beads alone.
B, purified recombinant eNOS protein (60-300
nM) was incubated with GST-dynamin beads (150 nM) or, alternatively, with equal amounts of GST beads
alone. Bound proteins were analyzed by Western blot analysis.
Left, the representative blot demonstrates that
GST-dynamin beads coprecipitate recombinant eNOS from solution, whereas
GST beads alone do not coprecipitate eNOS from BAEC lysate. At a 1:1
molar ratio of GST-dynamin to eNOS (lane labeled 150 nM eNOS), a majority of the pool of
available eNOS protein (lane labeled input, which
represents the available recombinant eNOS used in the binding assay at
a 1:1 molar ratio) is bound to dynamin. Further increase in the
available concentration of eNOS (lane labeled 300 nM eNOS) increases binding only minimally
(compare lane labeled 150 nM
eNOS and lane labeled 300 nM
eNOS). The blot is representative of experiments repeated five
separate times with similar results. Right, densitometric
intensity of the Western blot analysis is depicted graphically.
Note that at a 1:1 molar ratio of eNOS and GST-dynamin (bar
labeled 150 nM), the majority of available eNOS
protein (bar labeled input) binds with dynamin.
Results are the mean of duplicate densitometric determinations from
the experiment shown on the left. C, GST-dynamin beads
(60-600 nM) or GST beads alone were incubated with
[35S]methionine-labeled eNOS transcribed in
vitro. Binding of eNOS was analyzed by SDS-PAGE and
autoradiography of dried gels. Left, the representative
autoradiograph demonstrates that GST-dynamin binds 35S-eNOS
from solution in a concentration-dependent manner. The
majority of the available 35S-eNOS in solution
(input, 3 µl of reticulocyte lysate) is bound to
dynamin at concentrations of 300 and 600 nM GST-dynamin.
Similar results were obtained in two independent experiments.
Right, densitometric intensity of the autoradiograph is
depicted graphically. Note that the majority of available eNOS
(bar labeled input) is bound with GST-dynamin
when present at a concentration of 300-600 nM. Results are
the mean of duplicate densitometric determinations from the
experiment shown on the left. D, a series of
concentrations (0-320 nM) of purified recombinant eNOS
protein, pre-mixed with proportionate amounts of 35S-eNOS
tracer (0-16 µl), were incubated with 5.0 nM
GST-dynamin. Bound 35S-eNOS and an 35S-eNOS
standard curve were analyzed directly by scintillation counting.
Scatchard plot analysis is shown. Results represent an average of
duplicate determinations from the same experiment, and the
experiment shown is representative of two experiments that yielded very
similar binding.
|
|
Dynamin Potentiates eNOS Catalysis--
We next explored the
biological significance of this novel protein-protein interaction
between eNOS and dynamin-2. As ionophore stimulation of cells is
associated with enhanced binding of dynamin and eNOS and, additionally,
is known to increase NO release from cells (1), we next sought to
determine whether dynamin binding impacts eNOS activity in a positive
manner. As preliminary experiments demonstrated that GST has no
appreciable effect on the catalytic activity of recombinant eNOS (eNOS,
51.2 ± 24 pmol/min/mg protein; eNOS + GST, 55.4 ± 21 pmol/min/mg protein, n = 3; not significant), we
incubated recombinant eNOS protein with eluted GST or with increasing
molar ratios of eluted GST-dynamin, and then we assessed samples for
NOS activity by measuring the L-NAME-inhibited conversion of radiolabeled L-arginine to L-citrulline. As
seen in Fig. 5A, provision of
dynamin potentiates L-NAME-inhibited NOS activity in a
statistically significant manner (p < 0.05, analysis
of variance; n = 3, each experiment performed in
triplicate in the presence and absence of L-NAME). A
similar potentiation of L-NAME-inhibited NOS activity was
detected when purified eNOS was incubated with GST-dynamin still bound
with glutathione-Sepharose beads as compared with GST beads alone.
Additionally, GST-dynamin in the absence of eNOS does not increase the
measured amounts of radiolabeled L-citrulline or
L-arginine detected in the NOS assay (data not shown).
Next, to determine whether dynamin activates eNOS within cells,
eNOS-GFP ECV 304 cells were cotransfected with 6 µg of pcDNA3
alone, 6 µg of dynamin DNA, or alternatively 3 µg of pcDNA3 and
3 µg of dynamin DNA. Cell lysates were prepared for
immunoprecipitation studies or, alternatively, for NOS assay and
Western blot analysis. As seen in Fig. 5B (left),
transfection of dynamin DNA results in an increase in coprecipitation
of dynamin with eNOS. Note that equal amounts of eNOS are
immunoprecipitated from each sample. In Fig. 5B
(right), overexpression of dynamin increases
L-NAME-inhibited NOS activity in cell lysates and reaches a
statistically significant level of potentiation with transfection of 6 µg of dynamin DNA (p < 0.05; n = 5).
Thus, overexpression of dynamin-2 in cells results in an increase in
dynamin bound to eNOS and NOS catalytic activity. These studies in
conjunction with the NOS activity assays utilizing recombinant proteins
indicate that dynamin-2 binding may facilitate eNOS activation.

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|
Fig. 5.
Dynamin potentiates eNOS catalysis in
vitro and in cells. A, the catalytic
activity of purified recombinant eNOS protein was examined after
incubation with eluted GST protein, or after incubation with increasing
molar concentrations of eluted GST-dynamin protein as described under
"Experimental Procedures." GST-dynamin significantly increases the
enzymatic activity of purified recombinant eNOS as assessed by the
L-NAME inhibited conversion of radiolabeled
L-arginine to L-citrulline. (*,
p < 0.05 versus GST + eNOS;
#, p < 0.05 versus
GST-dynamin + eNOS (0.5:1); **, p < 0.05 one-way
analysis of variance; GST-dynamin + eNOS (all concentrations)
versus GST + eNOS; n = 3, each individual
experiment performed in triplicate in the presence and absence of
L-NAME). B, dynamin overexpression in cells
potentiates NOS activity. eNOS-GFP ECV 304 cells were cotransfected
with 6 µg of dynamin DNA, 6 µg of empty plasmid
(pcDNA3) or, alternatively, 3 µg of each, and after
36 h, cell lysates were prepared for eNOS immunoprecipitation
studies or, alternatively, NOS activity assay. Left panel,
transfection of dynamin-2 DNA results in increased coprecipitation of
dynamin with eNOS as shown in the representative Western blot
(WB). Note that equal amounts of eNOS are immunoprecipitated
from cell lysates in each group. Right panel, transfection
of 6 µg of dynamin-2 DNA results in a significant increase in NOS
activity (*, p < 0.05) as assessed by the
L-NAME-inhibited conversion of L-arginine to
L-citrulline in cell lysates. Transfection of 3 µg of
dynamin DNA also increases NOS activity but not in a statistically
significant manner (p > 0.05; not significant
(NS)). Data shown are the mean and standard error from five
separate experiments, each performed in duplicate.
|
|
 |
DISCUSSION |
Several NOS-associated proteins have been identified that bind in
a direct and specific manner with eNOS. Both calmodulin and Hsp 90 bind
and directly activate eNOS, whereas the binding of caveolin and the
bradykinin 2 receptor inhibit NOS activity (4, 6-9). The findings in
this study indicate that dynamin-2 is a NOS-associated protein. We
demonstrate a direct, specific, and high affinity interaction in
vitro, which is regulated by ionic stringency and intracellular
signals in vivo. Additionally, these studies provide
evidence for a functional interaction as dynamin-2 augmenting eNOS
catalysis in in vitro assays.
eNOS contains a unique amino-terminal consensus sequence, and acylation
of this site targets the protein to membrane compartments including
Golgi membranes and caveolae (1-3, 28). In prior studies, the
distribution of dynamin protein within cells has been detected most
prominently within Golgi membranes and associated clathrin-coated
vesicles, as well as within the neck of endothelial cell caveolae as
suggested by colocalization with the nascent Golgi protein, TGN 38 and
with caveolin, respectively (13, 14). In our studies eNOS and dynamin-2
colocalize predominantly in a perinuclear distribution in Clone 9 cells
heterologously expressing both proteins as well as in BAEC, expressing
both proteins endogenously. Additionally, the perinuclear distribution
of both proteins colocalizes with the Golgi marker, Golgi 58-kDa
protein, suggesting that within endothelial cells, endogenous eNOS and
dynamin likely colocalize and interact within Golgi membranes. Thus
functional eNOS protein interactions may not be exclusive to
plasmalemmal caveolae and may occur in other membrane compartments
within which eNOS resides.
eNOS and dynamin-2 coimmunoprecipitates within cells, and
additionally, dynamin in the form of a GST fusion protein
coprecipitates both recombinant eNOS from solution as well as
35S-eNOS transcribed in vitro. Further
specificity of binding is supported by a variety of experimental
approaches including the demonstration of stoichiometric, high affinity
binding kinetics in vitro, modulation of the interaction by
ionic strength, and intracellular signals in vivo. The
binding kinetics of eNOS and dynamin demonstrate interesting contrasts
in vitro as compared with cell lysates. Our in
vitro studies indicate that the recombinant proteins bind avidly
in solution with an estimated Kd of 27.6 nM. Of
note, a previously published affinity analysis of dynamin with
-adaptin has demonstrated an estimated half-maximal binding of
~200 nM (20). In our studies, at a 1:1 molar ratio, purified recombinant eNOS protein in solution binds stoichiometrically with GST-dynamin. However, in vivo, tetrameric dynamin and
dimeric eNOS may bind in an alternative kinetic manner. In our studies, significantly smaller pools of bound protein are detected from cell
lysates by immunoprecipitation. Although the precise size of the bound
pools of protein is confounded by detergents in the immunoprecipitation
buffer which may disrupt protein interactions and thereby underestimate
true binding, based on the signal intensity of dynamin bound to eNOS
(IP) compared with the intensity of dynamin in the
supernatant (Post IP) in Fig. 2, it would appear that ~5% of the pool of cellular dynamin is coprecipitated by eNOS in BAEC. Additionally, as demonstrated in the representative blot in Fig. 3,
binding in response to ionomycin is increased suggesting that agonist
stimulation is associated with enhanced binding of dynamin and eNOS.
This degree of binding in cell lysates (5-10%) is similar to that
observed with most NOS-associated proteins (7, 29-31), and therefore,
it has been suggested that the interactions of eNOS with its associated
proteins are more akin to transient signaling complexes rather than
stoichiometric receptor-ligand type interactions (29). Additionally,
with the varied cellular functions of dynamin, it would be unexpected
for a large portion of the cellular dynamin pool to be bound to eNOS.
Recent studies suggest that eNOS may reside in a complex in cells with
other proteins including Hsp 90, caveolin, and calmodulin (5, 29). In
this regard, our findings do not exclude the possibility that within
cells binding of dynamin and eNOS is mediated in part through one or
more of these proteins. In fact, as caveolin-1 binds directly with eNOS and additionally colocalizes with dynamin within cells (8, 13, 32), the
inclusion of dynamin in such a protein complex is plausible. However in
our studies, GST-dynamin binds recombinant eNOS and
35S-eNOS in vitro and potentiates eNOS catalysis
in solution, indicating that the proteins can bind directly and in a
functional manner in the absence of an adapter protein.
One of the primary cellular functions of dynamin relates to membrane
fission events, in particular, vesicle budding and internalization of
caveolae (12, 14). However, the dynamin family of proteins also
participates in a variety of signaling cascades such as
mitogen-activated protein kinase signaling, which are independent of
their motor functions (15-17). Based on these divergent cellular
actions of dynamins, one might postulate possible mechanisms by which
dynamin-2 might impact on eNOS biology within the cell. One possibility is that the interaction of these proteins facilitates trafficking or
translocation of eNOS and/or its associated signaling partners (33).
eNOS resides within Golgi membranes and plasmalemmal caveolae, both
compartments from which vesicles may bud in part through the scission
actions of dynamin (1, 2, 12, 14). As eNOS undergoes dynamic movement
between and within each of the two compartments (21, 34), the binding
of the two proteins might facilitate the trafficking of eNOS within
pools of vesicles liberated by dynamin. The present studies provide
evidence for an additional mechanism that dynamin binds with eNOS and
modulates NOS signaling and activity. This scenario is supported by the
following: 1) the enhanced binding of dynamin with eNOS in cells
treated with ionophore, a well characterized agonist of NO production;
2) enhanced activity of purified eNOS protein in the presence of
comparable molar concentrations of recombinant dynamin-2; and 3)
enhanced binding of dynamin with eNOS and potentiation of eNOS
catalytic activity in cells overexpressing dynamin-2. These findings
suggest a novel role for dynamin-2 as a signaling protein for eNOS
activation and are consistent with prior studies demonstrating the
participation of the dynamins in a variety of signaling pathways
(15-17, 35).
In summary, this study demonstrates that eNOS and dynamin bind in a
direct, specific, and regulated manner and that binding results in an
increase in eNOS catalysis. We anticipate that these events may have
relevance for eNOS regulation and trafficking within the vascular endothelium.
 |
ACKNOWLEDGEMENTS |
We thank Raul Urrutia and Guillermo
Garcia-Cardena for their critical review of the manuscript; David Toft
for helpful discussions; Pri Pradhan for assistance with statistical
analyses; and Mark McNiven for enthusiastic support as well as generous
provisions of dynamin-2 pAb, Clone 9 cells, GST-dynamin-2, and
GFP-dynamin constructs.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK-02529 (to V. S.), HL-57665, HL-61371 (to W. C. S.), and HL-53524 (to Z. S. K.), Northland American Heart Association (to V. S.), and the Mayo Clinic Foundation. A portion of this work was presented at the 2000 Nitric Oxide Society Meeting in San Franciso,
CA, June 4-7, 2000.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: GI Research Unit,
Alfred 2-435, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.:
507-255-5040; Fax: 507-255-6318; E-mail: shah.vijay@mayo.edu.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M006258200
 |
ABBREVIATIONS |
The abbreviations used are:
eNOS, endothelial
nitric-oxide synthase;
GFP, green fluorescent protein;
BAEC, bovine
aortic endothelial cells;
GST, glutathione S-transferase;
NO, nitric oxide;
L-NAME, L-nitroarginine
methyl ester;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
IP, immunoprecipitation.
 |
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