©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Agonist-modulated Palmitoylation of Endothelial Nitric Oxide Synthase (*)

(Received for publication, November 2, 1994; and in revised form, November 22, 1994)

Lisa J. Robinson(§)(¶) Liliana Busconi (¶) Thomas Michel (**)

From the Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [^3H]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 [^3H]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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

The epitope-tagged ecNOS was prepared using a plasmid (gift of Dr. Tomas Kirchausen) containing the sequence of the influenza hemagglutinin (HA) epitope followed by a stop codon cloned into the EcoRV site of pBluescript (Stratagene). The ecNOS cDNA (18) was cloned into the EcoRI site of this plasmid, upstream from the HA epitope, and then a fragment of the C terminus was prepared by PCR in which the stop codon was replaced by a BamHI site. After blunting this BamHI site, the PCR fragment was used to replace the C terminus of ecNOS and the vector sequence between the EcoRI site and the EcoRV site at the start of the HA epitope. The sequence of the PCR fragment was verified by dideoxynucleotide sequencing. This construct was subsequently cloned into the pBK-CMV expression vector (Stratagene), between the NheI and XhoI sites, to form ecNOS-HA. An epitope-tagged form of a previously characterized myristoylation-deficient mutant (myr) (10) of ecNOS was subsequently prepared by substituting the 5` 1.5 kilobases of the myristoylation-deficient mutant for that of the epitope-tagged ecNOS at a propitious BglII restriction site to form myr-HA.

Cell Culture and Transfection

Bovine aortic endothelial cells (BAEC) were purchased from Cell Systems and cultured as described previously(18) . Cells were used between passages 4 and 10. BAEC were transfected using Lipofectin (Life Technologies, Inc.) according to the manufacturer's directions. The COS-7 cell line was grown and transfected with previously described (10) wild-type or myr mutant ecNOS cDNAs using the DEAE-dextran method. NOS enzymatic activity in transfected COS-7 cells was assayed as described previously (10) by measuring the conversion of [^3H]arginine (Amersham Corp.) to [^3H]citrulline.

Biosynthetic Labeling

Cultures were labeled with [S]methionine (TranS-label, ICN) or with [P]orthophosphate (DuPont NEN) as described previously (5, 10) . To label cells with [^3H]palmitate, cultures were incubated in RPMI containing 10% dialyzed fetal bovine serum and 1 mCi/ml [^3H]palmitate (DuPont NEN) for 2 h. To examine depalmitoylation, pulse-chase experiments were performed; after labeling with 1 mCi/ml [^3H]palmitate in RPMI plus 10% dialyzed fetal bovine serum for 2 h, cultures were washed with RPMI plus 10% dialyzed fetal bovine serum and 100 µM unlabeled palmitate, then incubated in RPMI plus 10% dialyzed fetal bovine serum and 100 µM unlabeled palmitate in the presence or absence of 10 µM bradykinin for the indicated time. For cell fractionation, cells were lysed by sonication in hypotonic buffer and separated into soluble and particulate fractions by ultracentrifugation (100,000 times g, 30 min) as described previously(10) .

Immunoprecipitation and Polyacrylamide Gel Electrophoresis

Immunoprecipitations with polyclonal antiserum to ecNOS were performed as described previously(10) . Epitope-tagged proteins were immunoprecipitated using the 12CA5 monoclonal antibody to the HA epitope (BabCo.) at a final dilution of 1:100(19) . Immunoprecipitates were eluted from Protein A-Sepharose with SDS-PAGE sample buffer containing either 5 mM dithiothreitol (for [^3H]palmitate-labeled samples) or 5% beta-mercaptoethanol (all others). Immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography (for P) or fluorography (for S and ^3H). To test for hydroxylamine-sensitive [^3H]palmitate labeling, gels containing replicate samples were incubated for 4 h in either 1 M Tris-HCl, pH 7 (control), or 1 M hydroxylamine, pH 7, as described previously(20) , and then processed for fluorography.

Analysis of Bound Fatty Acids

The identity of the tritiated fatty acids attached to immunoprecipitated ecNOS was determined as described previously(21) . Briefly, ecNOS from [^3H]palmitate-labeled cells was immunoprecipitated, separated by SDS-PAGE, and electrophoretically transferred to a polyvinylidene difluoride membrane. The region of the membrane containing ecNOS was excised and treated with 6 N HCl in vacuo at 110 °C for 18 h. The acid hydrolysis products were extracted with toluene, dried under nitrogen, and analyzed by thin layer chromatography on KC-18 reverse phase plates (Whatman) developed with 1:1 acetic acid:acetonitrile, using samples of [^3H]myristate and [^3H]palmitate (both from DuPont NEN) as standards. The plates were sprayed with EN^3HANCE (DuPont NEN), and the tritiated fatty acids were detected by fluorography.


RESULTS AND DISCUSSION

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 [^3H]citrulline from [^3H]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 [^3H]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 ^3H-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 [^3H]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 [^3H]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 [^3H]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 [^3H]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 [^3H]palmitate. Following SDS-PAGE, the gel was treated either with Tris-HCl, pH 7 (Control) or hydroxylamine, pH 7 (NH(2)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: [^3H]palmitic acid standard, [^3H]myristic acid standard, and tritiated fatty acid released by acid hydrolysis from ecNOS immunoprecipitated from BAEC that were biosynthetically labeled for 2 h with [^3H]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 [^3H]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 [^3H]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 [^3H]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 beta-adrenergic receptor(26) , and, more recently, for a variety of G-protein alpha 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 alpha(s) 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).^2 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 [^3H]palmitate for 2 h and then incubated for the indicated times in medium plus either H(2)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 ^3H 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 alpha-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 [^3H]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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL46457. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Bugher-AHA fellow in Cardiovascular Molecular Biology.

These authors contributed equally to this report.

**
Wyeth-Ayerst Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115. Tel.: 617-732-7376; Fax: 617-732-5132.

(^1)
The abbreviations used are: NOS, nitric oxide synthase; ecNOS, endothelial isoform of nitric oxide synthase; HA, influenza hemagglutinin; BAEC, bovine aortic endothelial cells; myr, myristoylation-deficient mutant of ecNOS; ecNOS-HA, HA epitope-tagged ecNOS; myr-HA, HA epitope-tagged myr; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

(^2)
T. Michel, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful for Gordon K. Li for superlative technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.