The Proline-rich Domain of Dynamin-2 Is Responsible for
Dynamin-dependent in Vitro Potentiation of
Endothelial Nitric-oxide Synthase Activity via Selective Effects
on Reductase Domain Function*
Sheng
Cao
,
Janet
Yao
, and
Vijay
Shah
§¶
From the
Gastrointestinal Research Unit and
Department of Medicine and the § Tumor Biology Program,
Mayo Clinic, Rochester, Minnesota 55905
Received for publication, December 9, 2002
 |
ABSTRACT |
The GTPase dynamin-2 (dyn-2)
binds and positively regulates the nitric oxide-generating enzyme,
endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y.,
McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we
demonstrate, using purified proteins, that this occurs through a
selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the
eNOS reductase domain. In vitro studies demonstrate that
dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed
35S-eNOS reductase domain but not the 35S-eNOS
oxygenase domain. Furthermore GST-dyn-2 PRD binds a
35S-labeled eNOS reductase domain fragment (amino acids
645-850) that partially overlaps with the FAD binding domain of eNOS.
A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as
the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a
concentration-dependent manner in an order of magnitude
similar to that observed with dyn-2 full-length and in a manner that
requires calmodulin. Although dyn-2 PRD does not influence eNOS
oxygenase domain function or ferricyanide reduction, it does potentiate
the ability of recombinant eNOS to reduce cytochrome c,
supporting an influence of dyn-2 PRD on electron transfer between FAD
and FMN. (These data indicate that the binding domains of dyn-2 and
eNOS reside within the dyn-2 PRD domain and the FAD binding region of
the eNOS reductase domains, respectively, and that dyn-2 PRD is
sufficient to mediate dyn-2-dependent potentiation of eNOS
activity, at least in part, by potentiating electron transfer.)
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INTRODUCTION |
Endothelial nitric-oxide synthase
(eNOS)1 is a
membrane-associated protein that catalyzes the conversion of
L-arginine to L-citrulline and nitric oxide
(NO) (1). eNOS function is regulated in part by post-translational
mechanisms including acylation, phosphorylation, and protein
interactions (2-7). Indeed, specific proteins have been identified
which interact with eNOS, thereby regulating enzyme function, including
the large GTPase, dynamin-2 (dyn-2) (8-12). With regard to the latter,
it has been demonstrated that dyn-2 specifically binds eNOS in a direct
manner and potentiates the ability of eNOS to convert
L-arginine to L-citrulline in a
concentration-dependent manner (12). However, delineation
of binding domains and the mechanism of activation remain unexplored.
eNOS is a bi-domain enzyme that requires a number of cofactors and
substrates to generate NO optimally via an orchestrated electron
transfer mechanism (1). An oxygenase domain (amino acids 1-491)
contains binding sites for tetrahydrobiopterin, heme, and
L-arginine, and a reductase domain (amino acids 492-1205) contains binding sites for calmodulin, FMN, FAD, and NADPH (1). Electron flux is initiated at the reductase domain where NADPH-derived electrons are transferred sequentially through the bound flavins, FAD
and FMN. Electron transfer from the reductase domain to the oxygenase
domain is facilitated by calmodulin and allows for reduction of heme
iron and the ensuing binding and activation of oxygen. Subsequent
oxidation of the amino group of L-arginine allows for the
formation of L-citrulline, water, and NO (1). Assessment of
the individual reductase and oxygenase components of this biochemical paradigm allows for mechanistic dissection of how eNOS is influenced by
putative regulatory events. Specifically, eNOS reductase domain function can be dissected in isolation from the oxygenase domain by
exploiting the ability of moieties within this domain to transfer electrons to exogenous heme protein acceptors (13-16). Specifically, the rate of electron transfer from NADPH to FAD can be estimated by
utilizing the electron acceptor ferricyanide (FeCN), which accepts
electrons directly from the FAD; the rate of electron transfer from FAD
to FMN can be estimated by utilizing the exogenous electron acceptor
cytochrome c (15, 17). Conversely, oxygenase domain function
can be analyzed, independent of reductase domain-dependent heme reduction, by measuring NO synthesis from the NOS enzyme reaction
intermediate N
-hydroxy-L-arginine
(NOHA) (14, 18, 19).
Members of the dynamin family of proteins, including dyn-2, are
recognized as modulators of membrane scission events (20). However,
these proteins also facilitate specific and well characterized signaling functions by virtue of distinct protein interactions (21). In
this regard, the dyn-2 protein consists of several characterized
subdomains: the GTPase domain (amino acids 1-399), pleckstrin homology
domain (PHD; amino acids 521-623), GTPase effector domain (GED; amino
acids 623-746), and the proline-rich domain (PRD; amino acids
746-870). Each of these domains maintains distinct functions, and they
act in concert to facilitate the cellular functions of dyn-2 (22). The
GTPase domain is responsible for the binding and hydrolysis of GTP,
events that are promoted by GED. PHD and PRD regions are prominently
implicated in regulatory binding interactions. More specifically, PHD
binds phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol
4-phosphate, whereas PRD binds specific proteins, often notable for the
presence of an SH3 domain, including phospholipase C
, Nck,
AP-2, Grb2, and phosphatidylinositol 3-kinase (23-30).
Here we seek to elucidate further interaction regulation mechanisms of
dyn-2 and eNOS, using recombinant proteins. The present study
demonstrates that dyn-2 binds eNOS by virtue of dyn-2 PRD and
conversely that the binding domain of dyn-2 on eNOS is contained within
its reductase domain, specifically within a sequence overlapping the
FAD binding domain. This interaction is competed by the SH3-containing protein Fyn, thereby implicating the SH3-like region within this eNOS
reductase domain fragment as the dyn-2 PRD binding site. Furthermore,
glutathione S-transferase (GST)-dyn-2 PRD potentiates eNOS
activity in a concentration-dependent manner, with potency similar to that of the full-length protein and in a manner that requires bound calmodulin. Analysis of individual NOS subdomain function demonstrates that although GST-dyn-2 PRD does not influence eNOS oxygenase domain function independently, it does influence eNOS
reductase domain activity by potentiating electron transfer between the
reductase domain flavins, FAD and FMN. Thus these studies
provide mechanistic insights as to how dyn-2 enhances eNOS protein
function in vitro.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant
Proteins--
Recombinant eNOS protein was purified from
Escherichia coli as we and others have described previously
(12, 31). 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 affinity column with high
purity as evidenced by a single major band on SDS-PAGE. Purified eNOS
was stabilized with 0.5 mM L-arginine and 5-fold molar
excess of tetrahydrobiopterin. Dyn-2 and dyn-2 subdomain constructs
(provided courtesy of Mark McNiven) were subcloned into the GST fusion
protein vector, pGEX-1. GST constructs were transformed into BL21 (DE3)
and purified as we described previously (12). Specificity and quality
of GST-dyn-2 subdomain constructs were assessed by Coomassie staining
of SDS-polyacrylamide gels as shown in Fig. 1A. These
constructs have been documented previously to be functional for
in vitro assays of binding and activity, including
assessment of GTPase activity, molecular interaction, microtubule
responsiveness, and polymeric assembly (21, 32). eNOS oxygenase and
reductase subdomain constructs were generated from the full-length
bovine eNOS (bovine eNOS was courtesy of Bill Sessa), using PCR with
primers incorporating restriction endonuclease cutting sites for
HindIII and EcoRI, whereas the reductase domain
fragments encoding amino acids 511-850, 511-645, and 645-850 were
generated by PCR with primers flanked by EcoRI and
XhoI restriction endonuclease cutting sites. Individual
subdomains and fragments were subcloned into the vector
pcDNA3. [35S]Methionine-labeled eNOS
full-length or subdomains were translated in rabbit reticulocyte
lysates using the TNT coupled reticulocye lysate system
(Promega, Madison, WI) as we described previously (12).
[35S]Methionine was obtained from Amersham Biosciences.
In brief, the reaction mix, containing vectors encoding bovine
full-length, subdomains, or reductase domain fragments of eNOS DNA (or
alternatively negative control containing no DNA), T7 RNA polymerase,
and [35S]methionine, was incubated at 30 °C for 90 min. Translation products were examined by SDS-PAGE analysis and
autoradiography of dried gels. Purity and specificity of these reagents
are shown in the autoradiograph in Fig. 2, B and
D. Recombinant Fyn was obtained from PanVera (Madison, WI).
In Vitro Binding Assays and Binding Affinity--
In
vitro binding of GST-dyn-2 subdomain proteins with purified
recombinant eNOS from E. coli was performed by incubating
recombinant eNOS protein (15 pmol) with GST-dyn-2 fusion proteins (15 pmol), or GST beads alone, at 4 °C overnight, in a 300-µl reaction
buffer containing 50 mM Tris-HCl, 0.1 mM EGTA,
0.1 mM EDTA, 2 µM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, pH 7.5. For competition experiments, a
fixed amount of recombinant eNOS (3 pmol) was incubated with varying
amounts of recombinant Fyn (0-15 pmol) and with GST beads that
contained 10 pmol of GST-dyn-2 PRD, or alternatively GST beads alone,
overnight at 4 °C in 300 µl of binding buffer. Bound proteins were
washed three times with a buffer containing 50 mM Tris-HCl,
200 mM NaCl, 1 mM EDTA, then eluted with
Laemmli buffer and used for gel electrophoresis. Bound proteins were
analyzed by SDS-PAGE and Western blot analysis, using an eNOS
monoclonal antibody and a Fyn monoclonal antibody (Transduction
Laboratories, Lexington, KY) (33). In vitro binding of
GST-fused dyn-2 subdomain proteins with in vitro transcribed
35S-eNOS subdomains and reductase domain fragments was
examined by incubating 15 pmol of GST-dyn-2 beads or GST beads alone
with a fixed concentration of in vitro translated eNOS (3 µl of rabbit reticulocyte lysate) in 300 µl, using incubation and
wash conditions identical to those described above. Bound
35S-eNOS was examined by SDS-PAGE and autoradiography of
dried gels. Calculation of the equilibrium dissociation constant
(Kd) was performed by incubating a fixed
concentration of GST-dyn-2 or GST-dyn-2 PRD beads (5 nM)
with purified recombinant eNOS (0-640 nM), premixed with
proportionate volume of 35S-eNOS (0-32 µl) used as a
radiolabel tracer in a 300-µl reaction mix. Previous analyses
performed over a logarithmic range of immobilized GST-dyn-2 protein
concentrations (5-45 nM) incubated with saturating concentrations of eNOS ligand have demonstrated that varying the concentration of immobilized GST-dynamin in this experimental protocol
does not affect the Kd estimation (12).
Quantitation of bound and free 35S-eNOS, assessed directly
by scintillation counting, allowed for determination of bound and free
recombinant eNOS. Radioactive counts detected after incubation of GST
beads with 35S-eNOS, which were of low level, were
attributed to background and were subtracted from all subsequent
values. Kd was calculated by Scatchard plot
analysis of bound and free levels of recombinant eNOS.
Mammalian Two-hybrid Screen--
A GAL4-based mammalian
two-hybrid screen was used to detect subdomain interactions in HEK
cells (34). All tissue culture reagents for HEK cells were obtained
from Invitrogen. pM and pVP16 vectors were used to create fusion
proteins with the GAL4 DNA binding domain and DNA activation domain,
respectively (35) (mammalian two-hybrid matchmaker assay,
Clontech). cDNA encoding eNOS full-length and
subdomains and dyn-2 full-length and subdomains were subcloned into
vectors pM and pVP16, respectively, using PCR with primers
incorporating restriction endonuclease cutting sites for
BamHI and XbaI, and HindIII and
XbaI, respectively. cDNA encoding reductase domain
fragments with amino acids 511-850, 511-645, and 645-850 were
generated by PCR with primers incorporating restriction sites for
EcoRI and BamHI and inserted into pM. HEK cells,
grown in 12-well plates, were transfected with pM and pVP16 vectors
(0.2 µg) containing appropriate inserts, as well as vectors encoding
CAT and Renilla luciferase reporter vectors (0.1 µg and 0.01 µg, respectively). Cell lysates were prepared 24 h after transfection, and CAT reporter gene expression was quantified from cell
lysates from triplicate wells using a spectrophotometric assay (CAT
enzyme-linked immunosorbent assay, Roche Molecular Biochemicals).
Variation in transfection efficiency and protein concentration was
corrected by normalizing CAT readings with Renilla luciferase values. Negative controls in each experiment included transfection with empty pM and pVP16 vectors. Positive controls in each
experiment included transfection with pM-53, which encodes mouse p53
protein, and pVP16-T, which encodes SV40 large T-antigen, which is
known to interact with p53.
NOS Activity Assays--
NOS activity from E. coli-derived recombinant eNOS protein was assessed by measuring
the L-nitroarginine methylester
(L-NAME)-inhibited conversion of 3H-labeled
L-arginine to 3H-labeled
L-citrulline, as we described previously (12). GST protein,
GST-dyn-2, and subdomains were eluted from glutathione affinity beads
with reduced glutathione and dialyzed. 6.25 pmol of GST-fused protein
was incubated with 6.25 pmol of purified recombinant eNOS protein for
60 min at 4 °C in 300 µl of binding buffer identical to that
described for in vitro binding assays. In some experiments
the concentration of dyn-2 PRD protein was varied between 1.5 and 8 pmol to generate a molar range of 0.25:1, to 1.25:1, of dyn-2 PRD:eNOS.
To determine NOS activity, duplicate samples of the preincubated
recombinant proteins were added to a 50-µl reaction mix 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 some experiments the calmodulin concentration was varied from 0 to 100 nM. All samples were analyzed in the presence and
absence of the NOS inhibitor L-NAME (1.0 mM)
for specificity of effect. 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 50W-X8 resin column. Radiolabeled counts/min of generated
L-citrulline were measured and used to determine
L-NAME-inhibitable NOS activity.
Cytochrome c Reduction Assay and FeCN Reduction
Assay--
Components of reductase domain function were determined by
measuring the ability of E. coli-derived recombinant eNOS to
reduce cytochrome c and alternatively FeCN, in an
NADPH-dependent manner using spectrophotometric assays
(13-17, 36). GST, GST-dyn-2, and GST-dyn-2 subdomains were eluted with
reduced glutathione and dialyzed. 4 nM recombinant eNOS
protein was incubated for 1 h with 4 nM GST-fused
dyn-2 subdomain protein in 300 µl of reaction buffer buffer
containing 50 mM HEPES, pH 7.4, 250 mM NaCl at
4 °C. In some experiments, assays were performed using varying
concentrations of GST-dyn-2 PRD (2-7 nM) or alternatively,
preboiled GST-dyn-2 PRD protein. For cytochrome c reduction
assay, 100-µl aliquots from the preincubation mix were added to a
1-ml reaction containing 50 mM HEPES, pH 7.4, 250 mM NaCl, 40 µM cytochrome c, 0.1 mM NADPH, 0.12 µM calmodulin, and 0.2 mM CaCl2. Cytochrome c reduction was measured at 550 nm at 25 °C, using a Beckman spectrophotometer with
temperature control (model DU 650), with slight modification from the
protocol described by McCabe et al. (13). Briefly, the
reaction was monitored for 60 s, immediately upon addition of the
preincubated recombinant protein mix to the reaction buffer, a period
during which the reduction rate was linear. The change in absorbance
was calculated between the 30-s period of 10-40 s. Turnover number was
calculated using the absorbance change during this 30-s interval and an
extinction coefficient of 0.021 µM
1. For
FeCN reduction assay, 100-µl aliquots from the preincubation protein
mix were added to a 1-ml reaction buffer containing 50 mM
HEPES, pH 7.6, 250 mM NaCl, 0.2 mM NADPH, 0.12 µM calmodulin, 0.2 mM
CaCl2. Reaction was initiated with
the addition of 0.5 mM potassium ferricyanide, after which
the reaction was monitored for 10 min at 420 nm at 25 °C. The
turnover number was then calculated using an extinction coefficient of
1.02 mM
1.
NADPH-independent Oxygenase Domain Assay--
Selective,
NADPH-independent function of the oxygenase domain was assessed by
determining the ability of E. coli-derived recombinant eNOS
to convert NOHA into nitrite (14, 18, 19). GST, GST-dyn-2, or GST-dyn-2
PRD was eluted from beads with reduced glutathione. After dialysis, 62 pmol of purified recombinant eNOS was preincubated with an equimolar
concentration of the GST-fused protein in 60 mM EPPS for 90 min at 4 °C. Aliquots of the preincubation mix were then added to a
100-µl reaction mix containing 60 mM EPPS, pH 7.5, 1 mM NOHA, 0.5 mM dithiothreitol, 25 units/ml
superoxide dismutase, 50 µg/ml bovine serum albumin, and 4 µM tetrahydrobiopterin. The reaction was initiated by
adding 30 mM H2O2 at 37 °C and
stopped after 10 min by adding 1,300 units of catalase. To measure
nitrite generation, 100 µl of Griess reagent was added to triplicate
samples and incubated at room temperature for 15 min to allow for color development. Absorbance was measured at 570 nm using a microplate reader (Bio-Rad). Nitrite production was quantified based on
NaNO2 standards.
Statistical Analysis--
All data are given as the means ± S.E. Data were analyzed using an unpaired Student's t test.
 |
RESULTS |
Dyn-2 Binds with eNOS Reductase Domain by Virtue of Its
PRD--
We have demonstrated previously that eNOS binds with dyn-2 in
a direct and specific manner (12). To examine the mechanism of
interaction, binding domains of eNOS and dyn-2 were delineated through
the use of complementary approaches, including GST binding assays,
competition study, estimation of binding affinities, and mammalian
two-hybrid screen. To delineate the domain of dyn-2 required for eNOS
interaction, we performed binding assays using recombinant eNOS,
purified from E. coli, incubated at a 1:1 molar ratio with
the characterized subdomains of dyn-2, including GTPase, PHD, GED, and
PRD, each in the form of a GST fusion. The Coomassie stained
SDS-polyacrylamide gel, in Fig.
1A, depicts the purity of the
GST-dyn-2 subdomain proteins as shown by a single dominant protein band
of the corresponding molecular size. As seen in the representative
Western blot in Fig. 1B, the specific binding of purified
recombinant eNOS is detected with GST-dyn-2 PRD, when both proteins are
incubated at an equimolar ratio. Conversely, no binding is detected
between recombinant eNOS and other GST-dyn-2 subdomains.

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Fig. 1.
GST-dyn-2 binds eNOS by virtue of the dyn-2
PRD. Binding assays were performed by incubating E. coli-derived purified recombinant eNOS with GST-dyn-2 subdomain
fusion proteins. Bound proteins were assessed by gel electrophoresis
and Western blot analysis. A, Coomassie-stained SDS-PAGE
demonstrates the purity of GST-dyn subdomains (GTPase, PHD, GED, and
PRD), as evidenced by a single dominant band of the correct molecular
size for each subdomain. B, the representative Western blot
demonstrates that when incubated at an equimolar ratio, GST-dyn-2 PRD
binds recombinant eNOS protein. Conversely, other GST-dyn-2 subdomains
do not bind eNOS. The blot is representative of three independent
experiments that yielded similar results. C, a series of
concentrations (0-640 nM) of E. coli-derived
purified recombinant eNOS protein, premixed with proportionate amounts
of 35S-eNOS tracer (0-32 µl), was incubated with 5.0 nM GST-dyn-2 or alternatively GST-dyn-2 PRD. Bound
35S-eNOS and an 35S-eNOS standard curve were
analyzed directly by scintillation counting. Scatchard plot analysis of
bound and free recombinant eNOS is shown with individual data points
from a representative experiment using GST-dyn-2 (black
triangles; Kd = 62.4 ± 16.7 nM, Bmax = 0.97 ± 0.12 nM) and GST-dyn-2 PRD (white squares;
Kd = 129.8 ± 12.6, Bmax = 1.13 ± 0.16).
Kd and Bmax, the maximal
binding of eNOS ligand at the indicated concentrations of recombinant
proteins, are mean values derived from three independent
experiments, each with duplicate readings.
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We next sought to compare the binding affinity between eNOS and dyn-2
PRD in vitro using radiolabel tracer experiments and Scatchard analysis of binding data. 5.0 nM GST-dyn-2 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 and free
radioactive counts were measured directly by scintillation counting in
duplicate. In Fig. 1C, a Scatchard plot analysis of binding
data is shown. Scatchard analysis demonstrates a
Kd of 62.4 nM between eNOS and dyn-2
(black triangles), similar to that reported previously (12).
Kd analysis of eNOS and dyn-2 PRD (white
squares) was detected to be 129.8, which is comparable although
somewhat higher than that observed with the dyn-2 full-length.
Bmax, the maximal binding of eNOS ligand at the
indicated concentrations of recombinant GST proteins, was similar,
0.97 ± 0.12 nM and 1.13 ± 0.16 nM
for GST-dyn-2 and GST-dyn-2 PRD, respectively. The somewhat greater
affinity of dyn-2 full-length for eNOS may reflect beneficial effects
of tertiary structure of dyn-2 on binding kinetics which cannot be
achieved by the PRD subdomain alone.
Upon determining the relevant domain of dyn-2 which is responsible for
binding with eNOS, we next sought to determine the region of eNOS which
is required for binding to dyn-2. For this purpose, we produced a
series of in vitro 35S-labeled subdomain and
deletion constructs of eNOS for use in the GST binding assay (Fig.
1A). In Fig. 2B, an
autoradiograph is shown which depicts the radiolabeled eNOS
full-length, reductase, and oxygenase domains, indicated by a single
dominant protein band of the corresponding molecular size. GST,
GST-dyn-2, or characterized GST dyn-2 subdomains, including GTPase,
PHD, GED, and PRD, were incubated with 35S-eNOS
full-length, reductase, or oxygenase domain proteins. Binding was
assessed by SDS-PAGE and autoradiography. As seen in the representative autoradiograph in Fig. 2C, dyn-2 PRD binds the
35S-eNOS reductase domain, as well as 35S-eNOS
full-length, but not the 35S-eNOS oxygenase domain. Note
that binding of 35S-eNOS full-length or its subdomains is
not detected with other GST-dyn-2 subdomains. To dissect further the
relevant PRD binding region within the eNOS reductase domain, deletion
constructs spanning the eNOS reductase domain were constructed for
in vitro translation and then analyzed for their ability to
bind dyn-2 PRD in the GST binding assay (Fig. 2A). As seen
in the autoradiograph in Fig. 2D, dyn-2 PRD binds eNOS
511-850 and eNOS 645-850 but not eNOS 511-645. These data suggests
that dyn-2 PRD interacts within amino acids 645-850 within the eNOS
reductase domain.

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Fig. 2.
GST-dyn-2 PRD binds with an SH3-like sequence
within the reductase domain of eNOS. Purified recombinant eNOS
derived from E. coli or alternatively, 35S-eNOS
full-length, oxygenase, and reductase domain fragments were transcribed
in vitro and incubated with GST-dyn-2 full-length or one of
its subdomains (GTPase, PHD, GED, PRD). In some experiments, increasing
amounts of recombinant Fyn were also added to the reaction mix. Binding
was assessed by SDS-PAGE and autoradiography of dried gels, Western
blot analysis, and Coomassie staining of gels, as indicated.
A, physical maps of the eNOS constructs utilized for mapping
of the domain contributing to the interaction between eNOS and dyn-2.
The numbers shown on the top of each
box correspond to amino acid residues. B,
autoradiograph of SDS-PAGE demonstrates the purity and specificity of
35S-eNOS full-length, reductase, and oxygenase subdomains
as assessed by a single dominant band of the correct molecular size for
each protein. C, a representative SDS-PAGE autoradiograph
shows that GST-dyn-2 PRD binds the 35S-eNOS full-length
(top panel) and 35S-eNOS reductase domain
(middle panel) but not the 35S-eNOS oxygenase
domain (bottom panel). Note that other dyn-2 subdomains do
not bind 35S-eNOS full-length or eNOS subdomains. The
radiograph is representative of three independent experiments that
yielded comparable results. D, a representative SDS-PAGE
autoradiograph shows that GST-dyn-2 PRD binds 35S-eNOS
511-850 (lane 2) and 35S-eNOS 645-850
(lane 4) but not eNOS 511-645 (lane 6).
Lanes 1, 3, and 5 depict the input of
the respective 35S-eNOS fragments in the binding assay.
E, the sequence of the putative SH3 domain of eNOS contained
within the region that binds with dyn-2 PRD is shown in comparison with
SH3 domains from phospholipase C , Nck, and Fyn (white
boxes denote identical amino acids residues between eNOS and the
SH3 domains of phospholipase C , Nck, and Fyn). Comparison was made
using SeqWeb Version 1.2. F, a representative Western blot
using eNOS monoclonal antibody and Fyn monoclonal antibody is shown and
demonstrates that when added at increasing concentrations, recombinant
Fyn competes with purified recombinant eNOS protein for binding with
GST dyn-2 PRD (lanes 2-8). Lane 1 indicates the
eNOS input in the binding reactions; lane 9 indicates the
maximum Fyn input in the binding reactions. The bottom panel
is a Coomassie-stained gel demonstrating similar levels of GST-dyn-2
PRD in each binding reaction. The experiment was repeated twice and
yielded similar results.
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Because many dynamin-associated proteins bind by virtue of an SH3
domain-PRD interaction (24, 25, 28) and the eNOS sequence that binds
dyn-2 PRD contains an SH3-like region at amino acids 767-823 (Fig.
2E), we next sought to determine whether dyn-2 PRD binding
with eNOS could be competed by other SH3 domain-containing proteins.
For this purpose, purified recombinant Fyn was added at increasing
concentrations to the eNOS:GST-dyn-2 PRD binding assay, and bound
proteins were assessed by Western blot analysis. Fig. 2F
demonstrates that adding increasing amounts of Fyn protein competes off
the binding of eNOS with dyn-2 PRD in a
concentration-dependent manner (lanes 2-8),
thereby inferring that the aforementioned SH3-like region within eNOS
is likely responsible for dyn-2 PRD binding and also indicating that
the binding of both Fyn and eNOS may reside on a similar polyproline
sequence within the dyn-2 PRD region.
Next, to examine binding in the cellular context, we performed a
mammalian two-hybrid screen using vectors encoding eNOS full-length, oxygenase, and reductase domains and reductase domain deletion fragments, fused with the GAL4 DNA binding domain, and vectors encoding
dyn-2 or characterized dyn-2 subdomains, fused with an activation
domain, derived from herpes simplex virus VP16 protein. Cells were
transfected, and after 36 h lysates were prepared for CAT
enzyme-linked immunosorbent assay and Renilla luciferase
measurement. These analyses demonstrate a positive interaction between
heterologously expressed eNOS full-length or eNOS reductase domain, and
either dyn-2 or dyn-2 PRD, as assessed by a prominent increase in CAT expression (Table I). Furthermore,
dyn-2 and dyn-2 PRD interact positively with eNOS full-length, eNOS
511-850, and eNOS 645-850, but not eNOS 511-645 (Table I). No
substantive increase in CAT expression is detected upon expression of
other dyn-2 subdomains with either full-length eNOS or the oxygenase
domain of eNOS, providing specificity of effect. Additionally,
transfection with empty pM and pVP16 vectors results in no significant
CAT expression, whereas transfection with pM-53, which encodes mouse
p53 protein, and pVP16-T, which encodes SV40 large T-antigen, a high
affinity p53 binding partner, results in a level of CAT expression
~2-fold that observed with the positive screens depicted on Table I
(data not shown). Thus, these studies corroborate the results obtained in the GST binding assays and delineate the relevant subdomain interactions in the cellular context as well.
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Table I
eNOS reductase domain interacts with dyn-2 PRD in mammalian two-hybrid
screen
Interactions between eNOS and dyn-2 subdomains were examined within
cells using a mammalian two-hybrid screen. Dyn-2 and the characterized
subdomains, GTPase, PHD, GED, and PRD, were cloned into pVP16;
full-length eNOS, the reductase, oxygenase subdomains, and reductase
subdomain deletion constructs were subcloned into pM. These vectors
encode a DNA activation domain and a GAL4 DNA binding domain,
respectively. pM and pVP16 were cotransfected into HEK cells in
conjunction with CAT reporter vector and Renilla luciferase
vector. Positive controls in each experiment included transfection with
pM-53, which encodes mouse p53 protein, and pVP16-T, which encodes SV40
large T-antigen, which is known to interact with p53. Positive
interactions were assessed by increase in quantitative CAT activity
(shown in relative units) and normalized for Renilla.
Positive interactions are indicated by an asterisk (*, p < 0.05 compared with negative controls using empty vectors). Data are
compiled from eight separate screens (n = 8; means ± S.E.).
|
|
Dyn-2 PRD is sufficient for dyn-2-dependent potentiation of
eNOS activity. We next explored the functional significance of the
detected binding interaction between dyn-2 PRD and the eNOS reductase
domain as it mechanistically relates to eNOS function. First we sought
to determine whether dyn-2 PRD is responsible for
dyn-2-dependent potentiation of eNOS activity. NOS activity assays were performed by assessing the ability of purified recombinant eNOS to convert L-arginine to L-citrulline
after incubation with dyn-2 full-length or alternatively, equimolar
concentrations of dyn-2 subdomains, including GTPase, PHD, GED, and
PRD. As seen in Fig. 3A, dyn-2
PRD potentiates eNOS catalytic activity with a potency similar to dyn-2
full-length, whereas other dyn-2 subdomains do not influence NOS
activity. Furthermore, as shown in Fig. 3B, dyn-2 PRD
activation of eNOS occurs in a concentration-dependent manner and is dependent on an intact structure, as boiling of the GST
fusion protein, prior to incubation with eNOS, renders dyn-2 PRD
incapable of potentiating NOS activity. To gain further insight into
the mechanism of dyn-2 PRD activation of eNOS, studies were performed
with varying concentrations of calmodulin, a NOS-binding protein, which
enhances eNOS reductase domain and interdomain electron transfer. As
seen in Fig. 3C, dyn-2 PRD potentiates calmodulin-replete (100 nM) NOS activity, whereas dyn-2 PRD activation is
abrogated in the presence of rate-limiting amounts of calmodulin (10 nM) and entirely abolished in the absence of exogenous
calmodulin. These studies indicate that dyn-2 PRD is responsible for
dyn-2-dependent potentiation of NOS activity and
furthermore, that calmodulin is requisite for dyn-2 PRD potentiation of
NOS activity.

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Fig. 3.
Dyn-2 PRD is the requisite
subdomain for dyn-2-dependent potentiation of eNOS
activity. The activity of E. coli-derived purified
recombinant eNOS was examined after incubating recombinant eNOS protein
with GST-dyn-2 full-length or alternatively, dyn-2 subdomains (GTPase,
PHD, GED, and PRD). NOS activity was assessed by measuring the
L-NAME-inhibited conversion of L-arginine to
L-citrulline. A, dyn-2 PRD potentiates eNOS
catalytic activity in a magnitude similar to dyn-2 full-length, whereas
other dyn-2 subdomains do not influence NOS activity (*,
p < 0.05 compared with GST alone; n = 6 experiments each performed in duplicate). B, activation of
eNOS by dyn-2 PRD occurs in a concentration-dependent
manner as assessed by measuring NOS activity at varying molar
concentrations of GST dyn-2 PRD (*, p < 0.05 compared
with 0:1 (GST alone); n = 6 separate experiments, each
performed in duplicate). Note that after boiling the GST-dyn-2 PRD
protein (labeled denatured), dyn-2 PRD is no longer
effective in potentiating NOS activity. C, dyn-2 PRD
activation of eNOS was examined at varying concentrations of calmodulin
(0-100 nM; labeled Cam). Although dyn-2 PRD
potentiates calmodulin-replete (100 nM) NOS activity,
activation is abrogated in the presence of rate-limiting amounts of
calmodulin (10 nM). In the absence of calmodulin, NOS
activity is not detected regardless of the presence of dyn-2 PRD (*,
p < 0.05 compared with other groups; n = 3 separate experiments, each performed in duplicate).
|
|
Dyn-2 PRD selectively potentiates eNOS reductase domain function. eNOS
is a bi-domain enzyme with the carboxyl-terminal reductase domain and
amino-terminal oxygenase domain cooperating to allow electron transfer
and ensuing NO generation (1). Reductase domain function is comprised
in part by a series of electron transfer steps, including electron
transfer from NADPH into the flavin groups via FAD, with subsequent
intraflavin electron transfer to FMN and ensuing transfer into the heme
group within the oxygenase domain (1). The activity of the eNOS
oxygenase domain can also be assessed independently of reductase domain
function by measuring the ability of recombinant eNOS to generate
nitrite from the reaction intermediate, NOHA (14, 18, 19). To determine
how dyn-2 potentiates eNOS activity, we examined the effects of
GST-dyn-2 PRD on some of these individual NOS reductase and oxygenase
subdomain functions. We first examined the influence of dyn-2
full-length and dyn-2 PRD on eNOS oxygenase domain function. In these
experiments, depicted in Fig.
4A, neither GST-dyn-2
full-length nor GST-dyn-2 PRD influences the ability of full-length
recombinant eNOS to generate nitrite from NOHA compared with GST alone.
We next examined the influence of dyn-2 PRD on reductase domain
function using complementary artificial electron acceptors, cytochrome
c and FeCN, which preferentially accept NADPH-derived
electrons from FAD and from FMN, respectively (13-15, 17). First, to
examine the influence of dyn-2 PRD on electron transfer from FAD to
FMN, cytochrome c reduction was examined after incubation of
full-length purified recombinant eNOS with the GST dyn-2 full-length,
or equimolar concentrations of the individual GST dyn-2 subdomains, or
GST alone. This assay is effective in assessing the influence of
protein interactions on eNOS reductase domain function as evidenced by the prominent stimulatory effect of calmodulin on the ability of eNOS
to reduce cytochrome c (data not shown). In these
experiments, eNOS reductase domain activity is potentiated by dyn-2
full-length as well as dyn-2 PRD but not significantly influenced by
other dyn-2 subdomains (Fig. 4B). Furthermore, the effect of
dyn-2 PRD on eNOS-mediated cytochrome c reduction occurs in
a concentration-dependent manner as assessed by varying the
concentration of dyn-2 PRD added to the assay (Fig. 4C).
Next, to examine the influence of dyn-2 PRD on the more proximal
transfer of electrons from NADPH to FAD, the influence of dyn-2 PRD was
examined on electron transfer to the artificial electron acceptor,
FeCN. In these experiments, in contradistinction to its stimulatory
effect on cytochrome c reduction, dyn-2 PRD, at varying
molar concentrations does not influence FeCN reduction in a significant
manner (Fig. 4D). These studies indicate that dyn-2
potentiates eNOS function, at least in part, through selective effects
on the reductase domain of eNOS, with most prominent influence on
intraflavin electron transfer.

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|
Fig. 4.
Dyn-2 PRD potentiates eNOS reductase domain
function in a selective manner. The influence of GST dyn-2
full-length and characterized dyn-2 subdomains on eNOS subdomain
function was examined using complementary spectrophotometric assays.
A, the influence of GST-dyn-2, GST-dyn-2 PRD, or GST alone
on eNOS oxygenase domain was examined by measuring the ability of
recombinant eNOS protein to convert DOHA to nitrite. Nitrite was
measured using the Griess reaction. Neither dyn-2 full-length
(FL) nor dyn-2 PRD influences the oxygenase domain function
of eNOS (n = 3 separate experiments, each performed in
triplicate). B, the cytochrome c reducing
capacity of recombinant eNOS protein was examined after incubation of
eNOS with GST-dyn-2 subdomains or GST alone. Cytochrome c
reduction by eNOS is potentiated by dyn-2 full-length and dyn-2 PRD,
but not significantly influenced by other dyn-2 subdomains (*,
p < 0.05 compared with GST alone; n = 3 separate experiments, each performed in duplicate). C,
dyn-2 PRD activates eNOS reductase domain function in a
concentration-dependent manner as evidenced by incremental
increases in eNOS-dependent cytochrome c
reduction in response to increasing concentrations of dyn-2 PRD (*,
p < 0.05 compared with 0:1 (GST alone);
n = 3 separate experiments, each performed in
duplicate). D, FeCN reduction by eNOS was examined after
incubation of eNOS with varying concentrations of GST dyn-2 PRD.
eNOS-dependent FeCN reduction is not influenced in a
prominent manner by preincubation with GST dyn-2 PRD compared with eNOS
preincubated with equimolar concentrations of GST alone
(n = 5-7 separate experiments, each performed in
duplicate).
|
|
 |
DISCUSSION |
Post-translational mechanisms of eNOS activation are an area of
active investigation. In this regard, eNOS catalytic function is
influenced by specific events, including phosphorylation, acylation, and protein interactions (2, 4-6, 37-39). In support of the latter
concept, specific proteins have been identified which bind eNOS and
regulate enzyme function (9-11, 40, 41). The current study delineates
the eNOS binding and activation mechanisms of one of these proteins,
dyn-2, using purified proteins (12). We have utilized complementary
approaches to establish that dyn-2 PRD and eNOS reductase domain
contain the cognate binding sequences that are responsible for
mediating the binding interaction between dyn-2 and eNOS. These include
in vitro binding assays, utilizing appropriate GST-fused
dyn-2 subdomains incubated with both recombinant eNOS protein derived
from E. coli and radiolabeled in vitro
transcribed subdomain peptides, as well as competition studies using
the SH3-containing protein, Fyn. Corroborative evidence for interaction
between these specific subdomains is demonstrated by a mammalian
two-hybrid screen, thereby providing assurance that these subdomains
can interact in cells as well. Further correlative evidence is provided by the binding affinity detected between eNOS and dyn-2 PRD, which is
relatively similar to that observed between eNOS and the dyn-2 full-length protein. Detection of PRD as the eNOS binding region of
dyn-2 is not entirely unexpected because dyn-2 PRD is responsible for
many of the protein interactions with which dynamins are associated, including AP-2, Grb2, phospholipase C
, and phosphatidylinositol 3-kinase (24, 25, 28). Proline-rich sequences are a common ligand preference for a variety of protein interaction domains (42),
and many dynamin-associated proteins bind by virtue of an SH3
domain-PRD interaction (24, 25, 28). We had detected three regions
within the eNOS reductase domain (amino acids 560-645, 767-823, and
1010-1200) which fit best to these and other SH3-containing proteins
and therefore anticipated that one of these regions in the eNOS
reductase domain contained the proline binding sequence responsible for
the interaction between eNOS and dyn-2. Our deletion analysis indicates
that, of these, the SH3-like region at amino acids 767-823 is likely
responsible for dyn-2 PRD binding because this region lies within the
reductase domain fragment that binds dyn-2 PRD by GST binding assay and
mammalian two-hybrid screen. Further evidence for this inference is
provided by our data demonstrating the ability of the SH3
domain-containing protein, Fyn, to compete quantitatively with eNOS for
binding with dyn-2 PRD. These competitive binding data also suggest
that Fyn and eNOS may bind a common polyproline sequence within the
dyn-2 PRD region. There is precedence for proteins to bind the NOS
reductase domain because calmodulin binds to amino acids 493-512 near
the amino-terminal portion of the reductase domain. A reductase domain
binding site has also been implicated for the inhibitory NOS-associated
protein caveolin-1, in addition to the well characterized binding site
that has been mapped to amino acids 350-358 on the oxygenase domain
(14, 41, 43).
How does dyn-2 PRD influence eNOS function? First, influences on the
eNOS reductase domain are likely to confer effects on overall NOS
enzyme activity as the rate of electron transfer across the reductase
domain has been postulated to be the rate-limiting step in NO formation
from eNOS (13, 44, 45). Consistent with this concept, we find a direct
correspondence among the site of dyn-2 binding on eNOS within the
reductase domain, its ability to potentiate reductase domain function,
and ensuing augmentation of NOS activity. Our studies also indicate
that dyn-2 PRD potentiates calmodulin-replete NOS activity only. Thus,
dyn-2 does not influence the integral calmodulin requirements in the
eNOS pathway. This is evidenced by abolition of the NOS stimulatory
effects of dyn-2 PRD upon depletion of calmodulin. These data also
suggest that calmodulin and dyn-2 are unlikely to compete for a similar
binding site on eNOS. The reductase domain of eNOS catalyzes several
distinct electron transfer steps between NADPH, FAD, FMN, and
subsequently to the heme group. The selective influence of dyn-2 PRD on
cytochrome c reduction in the absence of a prominent effect
on FeCN reduction suggests that dyn-2 PRD likely influences intraflavin
electron transfer rather than electron transfer into the flavins from
NADPH. This is because electron transfer to FeCN proceeds from FAD,
whereas electron transfer to cytochrome c is preferential
from FMN (15, 17). These functional observations are consistent with
the mapping of the dyn-2 PRD binding sequence to a region overlapping
with the eNOS FAD binding domain.
In the present studies, dyn-2 PRD is sufficient for
dyn-2-dependent NOS function. These studies add to a series
of recent observations, which strongly implicate dyn-2, and in some
instances, the dyn-2 PRD domain, as a mediator of cell signaling events
(46). Indeed, dyn-2 signaling has been implicated in the regulation of
diverse cell signaling pathways including mitogen-activated protein
kinase and more recently in the transcriptional regulation of p53 and
downstream apoptosis (27, 46). Furthermore, dyn-2 PRD-protein
interactions are also important in serving the more established
function of dyn-2 as it relates to vesicle scission events, as
evidenced by the demonstration of dyn-2 PRD interaction with the
actin-binding protein cortactin (32). Thus dyn-2 PRD appears
responsible for mediating a variety of dynamin-dependent functions by virtue of direct protein binding events.
In summary, these studies indicate that dyn-2 PRD and the eNOS
reductase domain contain the requisite binding motifs to mediate the
protein interaction between dyn-2 and eNOS. Additionally, dyn-2 PRD is
accountable for the stimulatory effects of dyn-2 on eNOS catalysis
through stimulatory effects on the reductase domain of
calmodulin-replete eNOS at least in part by influencing electron
transfer between the flavins. Together these studies expand our current
understanding of the molecular mechanisms underlying the regulation of
eNOS.
 |
ACKNOWLEDGEMENT |
We thank Raul Urrutia for a critical review of
the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK-02529, DK-59998, and DK-59615 (to V. S.), a grant from the
Northland Affiliate of the American Heart Association (to V. S.), a
fellowship grant from the Northland Affiliate of the American Heart
Association (to S. C.), and by the Mayo Clinic Foundation.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 or 6318; Fax: 507-255-5040 or 6318;
E-mail: shah.vijay@mayo.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M212546200
 |
ABBREVIATIONS |
The abbreviations used are:
eNOS, endothelial
nitric-oxide synthase;
CAT, chloramphenicol acetyltransferase;
dyn-2, dynamin-2;
EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid;
FeCN, ferricyanide;
GED, GTPase effector domain;
GST, glutathione
S-transferase;
HEK, human embryonic kidney;
L-NAME, L-nitroarginine methylester;
NO, nitric
oxide;
NOHA, N
-hydroxy-L-arginine;
PHD, pleckstrin homology domain;
PRD, proline-rich domain;
SH3, Src homology
domain.
 |
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