(Received for publication, November 2, 1994; and in revised form, November 22, 1994)
From the
The nitric oxide synthases (NOS) comprise a family of enzymes
which differ in primary structure, biological roles, subcellular
distribution, and post-translational modifications. The endothelial
nitric oxide synthase (ecNOS) is unique among the NOS isoforms in being
modified by N-terminal myristoylation, which is necessary for its
targeting to the endothelial cell membrane. The subcellular
localization of the ecNOS, but not enzyme myristoylation, is
dynamically regulated by agonists such as bradykinin, which promote
ecNOS translocation from membrane to cytosol, as well as enhancing
enzyme phosphorylation. Using transiently transfected endothelial
cells, we now show that a myristoylation-deficient mutant ecNOS
undergoes phosphorylation despite restriction to the cytosol,
suggesting that phosphorylation may be a consequence rather than a
cause of ecNOS translocation. We therefore explored whether other
post-translational modifications might regulate ecNOS targeting and now
report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of
endothelial cells with [H]palmitic acid followed
by immunoprecipitation of ecNOS revealed that the enzyme is
palmitoylated; the label is released by hydroxylamine, consistent with
formation of a fatty acyl thioester, and authentic palmitate can be
recovered from labeled ecNOS following acid hydrolysis. Importantly,
pulse-chase experiments in endothelial cells biosynthetically labeled
with [
H]palmitate show that bradykinin treatment
promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation
is dynamically regulated by bradykinin and propose that
depalmitoylation of the enzyme may result in its cytosolic
translocation and subsequent phosphorylation.
Nitric oxide (NO) is now recognized as a ubiquitous signaling
and effector molecule involved in diverse physiological processes,
including neurotransmission, cell-mediated cytotoxicity, and blood
pressure regulation(1, 2) . NO is synthesized by a
family of NO synthases (NOS) ()which share many structural
and biochemical properties despite their divergent tissue distribution,
regulatory mechanisms, and biological
functions(3, 4) . In the vasculature, the endothelial
isoform of NOS (ecNOS) plays a key role in the transduction of signals
from the bloodstream to the underlying smooth muscle to induce vascular
relaxation(1) . ecNOS is unique among the NOS isozymes in being
predominantly membrane-associated, but in response to agonists, such as
bradykinin, the enzyme translocates from membrane to
cytosol(5) . Agonist-stimulated subcellular translocation has
been described for other proteins involved in cell signaling pathways,
including G-proteins and protein kinases, and has been implicated in
their functioning in signal
transduction(6, 7, 8, 9) . The
subcellular redistribution of ecNOS in response to extracellular
signals may also be important to its specific biological functions.
The ecNOS undergoes N-terminal myristoylation, and this modification is necessary for membrane association(10) , primarily via hydrophobic interactions between the ecNOS myristate moiety and membrane phospholipids(11) . However, translocation of the enzyme from membrane to cytosol does not appear to result from loss of the myristate moiety; this modification is co-translational and typically irreversible, precluding its dynamic regulation by agonists(8, 12, 13) . Myristoylated proteins are in fact found both in the soluble and particulate subcellular fractions. Moreover, the stable membrane association of myristoylated proteins may require hydrophobic or electrostatic interactions in addition to those between myristate and membrane lipids(14) , although we have previously found no evidence for a polybasic domain that might stabilize ecNOS association with the membrane(11) . Reversible post-translational modifications, such as phosphorylation or palmitoylation, may determine the subcellular localization of myristoylated proteins and provide a mechanism for their regulation(7, 8) .
We have shown previously that ecNOS undergoes phosphorylation and that its phosphorylation is enhanced by agonists, such as bradykinin, which also stimulate dissociation of the enzyme from cell membranes(5) . Furthermore, phosphorylated ecNOS is found predominantly in the cytosolic fraction even when the majority of the protein is membrane bound. These observations raised the possibility that phosphorylation of ecNOS at the cell membrane triggers its translocation to the cytosol, perhaps by altering electrostatic interactions with membrane lipids, as has been described for the myristoylated alanine rich protein kinase C substrate (MARCKS) protein(15, 16, 17) . However, in this paper we demonstrate that an exclusively cytosolic, myristoylation-deficient mutant of ecNOS is, nevertheless, phosphorylated when expressed in endothelial cells, suggesting that phosphorylation may follow, rather than cause, enzyme translocation to the cytosol. We now provide evidence that the subcellular localization of ecNOS may be determined by palmitoylation: membrane-bound ecNOS is palmitoylated via a reversible thioester linkage, and palmitoylation is regulated by enzyme agonists such as bradykinin.
To explore the relationship between ecNOS phosphorylation and
subcellular translocation in endothelial cells, we constructed
epitope-tagged wild-type and myristoylation-deficient mutant
(myr) ecNOS cDNAs that would express protein that
could be selectively immunoprecipitated from transfected endothelial
cells by an antibody to the epitope tag. To check that addition of this
epitope did not alter the subcellular distribution of ecNOS, we
analyzed the localization of the epitope-tagged wild-type ecNOS and the
epitope-tagged myr
mutant in transfected BAEC. The
epitope-tagged, wild-type ecNOS is predominantly membrane-associated (Fig. 1A, upper panel), as is the endogenous
enzyme, immunoprecipitated alone from sham transfected cells with the
ecNOS antiserum (Fig. 1A, lowerpanel). Epitope-tagged, myristoylation-deficient ecNOS is
found exclusively in the cytosol (Fig. 1A, upperpanel), as was previously observed for the untagged
myristoylation mutant in heterologous expression
systems(10, 11) . Thus, addition of the epitope tag
does not in itself appear to influence the subcellular localization of
ecNOS. In separate experiments we also found no difference in the
enzymatic activity of wild-type and epitope-tagged ecNOS transiently
expressed in COS-7 cells (as measured by formation of
[
H]citrulline from
[
H]arginine in cell lysates: 6.7 ± 1.9 versus 6.9 ± 1.2 pmol of citrulline/min/mg of protein
for the wild-type and epitope-tagged ecNOS, respectively). To determine
whether membrane association was necessary for phosphorylation, BAEC
transfected with epitope-tagged wild-type (ecNOS-HA) or
myristoylation-deficient (myr
-HA) ecNOS cDNAs were
biosynthetically labeled with [
P]orthophosphate
and then immunoprecipitated using either ecNOS antiserum or the
antibody to the epitope tag (Fig. 1B). Both the
wild-type and myristoylation-deficient mutant ecNOS were
phosphorylated, despite restriction of the latter to the cytoplasm (Fig. 1A). Furthermore, both constructs yielded the
same single phosphopeptide on two-dimensional tryptic phosphopeptide
analysis that was previously reported for the endogenous ecNOS (data
not shown; see (5) ). These data suggest that phosphorylation
of ecNOS in BAEC may occur in the cytosol. As we have previously
reported, the majority of phosphorylated ecNOS is found in the cytosol,
even with a preponderance of total ecNOS protein in the membrane
fraction(5) .
Figure 1:
Subcellular distribution and
phosphorylation of epitope-tagged wild-type and
myristoylation-deficient ecNOS. A, BAEC were transfected with
the pBK-CMV vector alone (sham), or with cDNA for the HA
epitope-tagged wild-type ecNOS (ecNOS), or the HA
epitope-tagged myr mutant ecNOS (myr
) and biosynthetically labeled with
[
S]methionine for 3 h, then lysed and
fractionated by ultracentrifugation. Proteins were immunoprecipitated
from the cytosolic fraction (C) and the membrane fraction (M) and analyzed by SDS-PAGE and fluorography. The upper
panel shows the subcellular distribution of the transfected
enzyme, immunoprecipitated with the antibody to the HA epitope tag. The lowerpanel shows the subcellular distribution of
endogenous and transfected enzyme combined, which are both
immunoprecipitated using antiserum to ecNOS. The arrow indicates a 135-kDa protein specifically immunoprecipitated by the
HA antibody from cells transfected with the epitope-tagged wild-type
and myr
ecNOS cDNA but not from the sham-transfected
cells. Immunoprecipitation using the HA antibody also yielded several
nonspecific lower molecular weight bands of variable intensity from
culture to culture. B, shown is an autoradiogram of SDS-PAGE
analysis of phosphoproteins immunoprecipitated with either the HA
antibody or the ecNOS antiserum from BAEC transfected with vector alone (sham), epitope-tagged wild-type (ecNOS-HA),
or epitope-tagged myr
ecNOS (myr
-HA) cDNAs, and
biosynthetically labeled with [
P]orthophosphate
for 3 h. The arrow indicates the 135-kDa phosphoprotein
corresponding to endogenous plus transfected ecNOS (immunoprecipitated
with the ecNOS antiserum) in the leftpanel or
epitope-tagged transfected ecNOS (immunoprecipitated with the HA
antibody) in the rightpanel.
Myristoylation and, more generally, membrane
association of ecNOS thus do not appear to be prerequisites for
phosphorylation, arguing strongly against the hypothesis that
phosphorylation at the cell membrane triggers the dissociation and
translocation of ecNOS from membrane to cytosol. We speculated that
other post-translational modifications might be responsible for the
agonist-regulated association of ecNOS with the cell membrane.
Palmitoylation, like phosphorylation, is a reversible,
post-translational modification that can determine the subcellular
distribution of proteins, including other myristoylated signaling
proteins(8, 17) . To determine whether ecNOS might be
palmitoylated as well as myristoylated, we biosynthetically labeled
BAEC with [H]palmitate and then
immunoprecipitated ecNOS. As shown in Fig. 2A, ecNOS is
indeed labeled under these conditions. However, because palmitate may
be converted to myristate in cells, it was possible that the labeling
we observed reflected the known N-terminal myristoylation of ecNOS. To
address this question, we investigated the nature of the chemical
linkage between the tritiated moiety and ecNOS by treating samples of
the
H-labeled enzyme in SDS-PAGE gels with hydroxylamine,
which will cleave the fatty acyl thioester bonds, characteristic of
protein palmitoylation via a cysteine sulfhydryl, but will not
hydrolyze the N-terminal acyl amide linkage to
myristate(12, 13) , as shown previously for
ecNOS(22) . As shown in Fig. 2A, hydroxylamine
treatment releases all of the label from ecNOS immunoprecipitated from
BAEC biosynthetically labeled with [
H]palmitate,
indicating that this acylation is distinct from the known N-terminal
myristoylation, and probably represents palmitoyl thioester formation
at a cysteine residue(s) in ecNOS. To confirm the chemical identity of
the tritiated group attached to ecNOS, we subjected ecNOS
immunoprecipitated from [
H]palmitate-labeled
cells to acid hydrolysis, which releases fatty acids linked by either
amide or ester bonds(12, 13) . Reverse-phase thin
layer chromatography revealed that the tritiated fatty acid released
from ecNOS co-migrated with a [
H]palmitate
standard (Fig. 2B). Thus, the labeling of ecNOS did not
result from metabolism of palmitate to myristate and subsequent
N-terminal myristoylation, but clearly represents a second, distinct
acylation of this protein.
Figure 2:
ecNOS labeling by
[H]palmitate via an hydroxylamine sensitive bond. A, shown are the results of SDS-PAGE and
autofluorography of ecNOS immunoprecipitated with ecNOS antiserum from
BAEC biosynthetically labeled for 2 h with
[
H]palmitate. Following SDS-PAGE, the gel was
treated either with Tris-HCl, pH 7 (Control) or hydroxylamine,
pH 7 (NH
OH) for 4 h, as described in the text. The
fluorogram was exposed for 30 days on Kodak XAR film at -70
°C using an intensifying screen. B, shown is a fluorogram
of a reverse phase thin layer chromatographic separation of tritiated
fatty acids: [
H]palmitic acid standard,
[
H]myristic acid standard, and tritiated fatty
acid released by acid hydrolysis from ecNOS immunoprecipitated from
BAEC that were biosynthetically labeled for 2 h with
[
H]palmitate. The fluorogram was exposed for 8
days.
We next explored the subcellular
distribution of the palmitoylated protein. If palmitoylation of ecNOS
anchors ecNOS to the membrane by providing additional hydrophobic
interactions with membrane lipids, then palmitoylated ecNOS should be
restricted to the particulate fraction. As shown in Fig. 3, in
transiently transfected COS-7 cells biosynthetically labeled with
[H]palmitate, we found that the palmitoylated
wild-type ecNOS is located exclusively in the particulate subcellular
fraction. To confirm that the absence of signal from cytosolic ecNOS
was not due simply to the smaller amounts of ecNOS in this subcellular
fraction, equal quantities of ecNOS (as determined by Western blotting)
from cytosolic and membrane fractions of
[
H]palmitate-labeled BAEC were analyzed by
SDS-PAGE and fluorography; again, no signal was observed for the
cytosolic protein, although membrane-associated ecNOS was clearly
labeled (data not shown). Fig. 3also shows that the
myr
mutant ecNOS does not undergo palmitoylation.
This is consistent with previous observations for
myristoylation-deficient mutants of other dually acylated
proteins(23, 24, 25) . Myristoylation may be
required for initial targeting to the cell membrane, where subsequent
palmitoylation (perhaps by a membrane-bound palmitoyl transferase) may
stabilize ecNOS membrane association.
Figure 3:
Palmitoylation is restricted to
membrane-associated, myristoylated ecNOS. Shown is a fluorogram of the
SDS-PAGE analysis of wild-type and myr mutant ecNOS
immunoprecipitated with ecNOS antiserum from transiently transfected
COS-7 cells biosynthetically labeled with
[
H]palmitate, lysed to form homogenates (H), then fractionated by ultracentrifugation into cytosolic (C) and membrane (M) fractions as described in the
text. This fluorogram was exposed for 1
month.
Because palmitoylation, unlike
myristoylation, is a reversible post-translational modification, it
provides a potential mechanism for agonist regulation of ecNOS
subcellular localization. Agonist regulation of protein palmitoylation
has been described previously for membrane receptors, such as the
-adrenergic receptor(26) , and, more recently, for a
variety of G-protein
subunits(7, 25, 27) . Moreover, for the
latter, peripheral membrane proteins, the loss of palmitate may
correlate with protein redistribution to the cytosolic subcellular
fraction(7) . Agonists for receptors linked to G-protein
appear to stimulate palmitate turnover, specifically
accelerating depalmitoylation(7, 27) . To test whether
agonists of ecNOS might also stimulate its depalmitoylation, we
performed pulse-chase experiments in the presence or absence of
bradykinin. As shown in Fig. 4, bradykinin clearly stimulates
depalmitoylation of ecNOS. The half-life of the palmitoyl-ecNOS
declines from
40 min to less than 10 min after addition of
bradykinin (Fig. 4B). Bradykinin does not alter the
half-life of the ecNOS protein (t
=
20 h).
Loss of palmitate, and its hydrophobic
interactions with cell membranes, could be the mechanism for release of
ecNOS from the cell membrane and translocation to the cytosol in
response to bradykinin.
Figure 4:
Agonist-stimulated depalmitoylation of
ecNOS. BAEC were biosynthetically labeled with
[H]palmitate for 2 h and then incubated for the
indicated times in medium plus either H
O (Control)
or 10 µM bradykinin (+Bradykinin), and ecNOS
was immunoprecipitated and analyzed by SDS-PAGE and autofluorography as
described in the text. PanelA, a fluorogram (exposed
for 10 days) of the SDS-PAGE analysis of proteins immunoprecipitated
with the ecNOS antiserum; B, a graph of the relative intensity
of
H labeling of ecNOS, analyzed by densitometry; upper
curve, control; lower curve, plus
bradykinin.
The palmitoylation of several signaling
proteins has been shown to influence their activity, protein
interactions, and subcellular
localization(7, 8, 9) . However, the
biochemical processes that regulate reversible palmitoylation of these
proteins remain less well understood, and few enzymes involved in the
formation or hydrolysis of palmitoyl-protein thioesters have been
characterized extensively. No general consensus sequence for protein
palmitoylation has been identified, although some dually acylated
G-protein -subunits and members of the Src family of tyrosine
kinases are palmitoylated at a cysteine residue within a conserved
N-terminal sequence: MGCXXS. However, this cysteine-containing
sequence is not found in ecNOS. Very recently, a protein
palmitoylthioesterase was isolated and
cloned(28, 29) , but its regulatory characteristics
are not fully defined, and its relationship to ecNOS palmitoylation is
completely unknown. The mechanisms by which bradykinin promotes ecNOS
depalmitoylation are thus unclear. It is possible that stimulation of
the bradykinin receptor leads directly to activation of a protein
palmitoyl thioesterase. Alternatively, ecNOS activation and nitric
oxide production may influence depalmitoylation, as it has recently
been shown that nitric oxide reduces [
H]palmitate
labeling of two nerve growth cone-associated proteins(20) . NO
might regulate palmitoyl thioesterase activity or directly influence
ecNOS palmitoylation via nitrosothiol formation at the site(s) of
palmitoylation; these possibilities represent novel mechanisms for
product regulation of an enzyme.
The regulation of ecNOS palmitoylation is likely to have important implications for NO-mediated signal transduction. For example, depalmitoylation and translocation of ecNOS could influence NO signaling in the vasculature by removing the enzyme from proximity to membrane receptors and/or intracellular effectors, thereby modulating the response to extracellular signals. Reversible palmitoylation of ecNOS may thus represent an important control point for the regulation of NO biological activities in the vascular wall.