Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.

form responsible for cardiovascular homeostasis including regulation of blood pressure, vessel remodeling, and angiogenesis. In addition to the profound physiological role of eNOS-derived NO, eNOS is unique among NOS family members since it is a peripheral membrane protein that is modified by co-translational N-myristoylation and post-translational cysteine palmitoylation (1,2). Both N-myristoylation and cysteine palmitoylation are necessary for the subcellular targeting of eNOS onto peripheral aspects of the Golgi complex and to cholesterol-rich microdomains of the plasma membrane including caveolae/ lipid rafts (3,4). Moreover, mislocalization of the enzyme to either domain impairs agonist-stimulated NO release from cells, implying that the proper subcellular localization of eNOS is critical for stimulus-dependent coupling to the enzyme (5,6).
Recently many investigators have shown that protein phosphorylation of eNOS by several serine/threonine kinases is a critical control step for NO production by endothelial cells. Phosphorylation by AMP kinase (7), Akt (or protein kinase B) (8 -11), or protein kinase A (12) on serine 1179 (bovine) or serine 1177 (human) of eNOS leads to enhanced activity of the enzyme and, thus, augmented production of NO. Indeed, mutation of serine 1179 to an alanine residue (eNOS S1179A) prevents Akt-dependent phosphorylation and NO production, proving that this residue is indispensable for activation of the enzyme by this kinase (8,9). Mechanistically, phosphorylation of eNOS by Akt on serine 1179, which can be mimicked by an aspartate substitution at this residue (eNOS S1179D) (8,9), enhances the rate of electron flux from the reductase to the oxygenase domain of the protein and reduces the relative calcium requirement for the enzyme, thus increasing the rate of NO synthesis (13). Thus, the phosphorylation of serine 1179 may be considered synonymous with the activation state of eNOS in response to two important physiological agonists for NO production, vascular endothelial growth factor (VEGF) and fluid shear stress. Indeed, eNOS-derived NO is necessary for VEGF-induced angiogenesis and permeability changes in vivo (14,15).
The mechanism by which localization influences eNOS activation is not completely understood. Previously it has been suggested that the localization of eNOS changes upon stimulation from membrane to cytosol and that the translocation is either important for activation or inactivation of the enzyme. In addition, post-translational phosphorylation (16) or depalmitoylation (17) has been proposed to initiate translocation; however, the phenomenon of membrane to cytosol or membrane to membrane translocation is controversial (12, 18 -20). Previously we have shown that localization of eNOS is critical for its * This work was supported by National Institutes of Health Grants RO1 HL57665, HL61371, and HL64793 (to W. C. S.), T32HL10183 (to D. F.), and HL48038 (to D. R.) and a grant-in-aid from the American Heart Association (National Grant to W. C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the Canadian Institutes of Health Research.
** An Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-737-2291; Fax: 203-737-2290; E-mail: william.sessa@yale.edu. 1 The abbreviations used are: eNOS, endothelial nitric-oxide synthase; P-eNOS, phosphorylated-eNOS; VEGF, vascular endothelial growth factor; Ab, antibody; BAEC, bovine aortic endothelial cells; HU-VEC, human umbilical vein endothelial cells; WT, wild type; MES, 4-morpholineethanesulfonic acid; DAF-2-DA, 4,5-diaminofluorescein diacetate. activation by Akt since co-transfection of Akt with a non-acylated form of eNOS (G2A eNOS) abrogates Akt-dependent NO production (8). This suggested to us that the localization of eNOS phosphorylated on serine 1179 reflected an "activated pool" of the enzyme. Thus, the goal of the present study was to gain insights into where activated eNOS resides and where NO is produced in cells and to examine the relative distribution of total versus phosphorylated eNOS in Golgi versus cholesterolrich microdomains of the plasma membrane.

MATERIALS AND METHODS
Generation of a Phosphorylation-specific Antibody for Serine 1179 -Rabbits were injected with a phosphopeptide (QEVTSRIRTQpSFS-LQERHLRG (where pS is phosphorylated serine) corresponding to amino acids 1169 -1189 on bovine eNOS) coupled to KLH. Antibodies (Ab) were first purified by positive selection on phosphopeptide columns followed by negative selection on nonphospho-peptide columns (Dr. Mark Amano, Zymed Laboratories Inc. Laboratories). The resultant Ab was identical in recognizing only eNOS phosphorylated on serine 1179 similar to the original phospho-eNOS (P-eNOS) Ab described (7).
Western Blotting-Cells were washed twice with phosphate-buffered saline and lysed on ice in 50 mM Tris HCl, pH 7.5, 1% Nonidet P-40 (v/v), 10 mM NaF, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin), and lysates were transferred to an Eppendorf tube and rotated for 45 min at 4°C. Lysates were Douncehomogenized (50 strokes), and insoluble material was removed by centrifugation at 12,000 ϫ g for 10 min at 4°C and subjected to Western analysis as described previously (8).
Cellular Fractionation-Confluent BAEC (150-mm dish) were serum-starved for 12 h before stimulation with VEGF (50 ng/ml) for 3 min. Cells were washed twice with phosphate-buffered saline and scrapped in ice-cold hypotonic buffer (50 mM Tris, pH 7.4, 20 mM NaF, 5 mM MgCl 2 , 1 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin) and lysed by homogenization (Dounce, 25 strokes). Nuclei were removed by centrifugation at 1000 ϫ g. Membrane and cytosolic fractions were obtained by additional centrifugation at 100,000 ϫ g for 45 min. Low density caveolin-enriched membrane fractions were isolated as described previously (19). BAEC or transfected COS-7 cells were washed twice with Dulbecco's phosphate-buffered saline, scraped into 2 ml of 500 mM sodium carbonate, pH 11, Dounce-homogenized, and sonicated (three 20-s bursts at 30% of maximal power). The homogenate was then adjusted to 42.5% sucrose by the addition of 2 ml of 85% sucrose prepared in MBS (25 mM MES, pH 6.5, 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A 5-30% discontinuous sucrose gradient was formed above (3 ml of 5% sucrose, 5 ml of 30% sucrose; both in MBS containing 250 mM sodium carbonate) and centrifuged at 35,000 rpm for 18 h in an SW40 rotor Beckman Instruments). A light-scattered band confined to the 5-30% sucrose interface was observed that contained caveolin but excluded most other proteins. Gradient fractions (1 ml) were collected from the top of the tube to yield a total of 12 fractions, and 50 l of each fraction (2-10) was used for Western analysis. After transfer, nitrocellulose membranes were stained with Ponceau S to visualize protein bands and subjected to immunoblotting as described previously (19). Blots were probed with the P-eNOS antibody (as described above), eNOS monoclonal antibody (9D10, Zymed Laboratories Inc.), rabbit anti-caveolin-1 polyclonal antibody (1:1000; Transduction Laboratories), anti-␤-COP polyclonal anti-body (1:1000; Affinity BioReagents), anti-␤-actin, and anti-Akt and phospho-Akt (P-Akt, Cell Signaling) and followed by horseradish peroxidase-conjugated goat antirabbit secondary antibody and ECL to detect the immunoreactive proteins. The percentage of total membrane protein in different gradient fractions was determined by laser densitometry and plotted as a percentage of total protein.
Immunofluorescence-COS cells were transfected as described and plated onto sterile coverslips. Cells were fixed in acetone/methanol 1:1 for 3 min at Ϫ20°C and rinsed twice with Dulbecco's phosphate-buffered saline plus 0.1% bovine serum albumin (w/v, Dulbecco's phosphate-buffered saline/bovine serum albumin) for 5 min at room temperature. The cells were then incubated with 5% goat serum in Dulbecco's phosphate-buffered saline/bovine serum albumin for 30 min at room temperature followed by a 2-h incubation with polyclonal P-eNOS antibody and monoclonal eNOS antibody (H32, Transduction Labs) for 2 h at room temperature. The P-eNOS antibody was preabsorbed against a 10:1 molar ratio of either P-eNOS peptide (as described above) or unlabeled peptide. Anti-rabbit Texas Red-labeled (diluted 1:100) and anti-mouse fluorescein isothiocyanate-labeled (1:100) secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were incubated for 1 h at room temperature. Slides were mounted with Slowfade (Molecular Probes, Inc., Eugene, OR), and cells were observed with an inverted Zeiss microscope fitted with a Bio-Rad MRC 600 confocal imaging system.
Primary HUVEC were plated onto gelatin-coated coverslips and serum-starved for 24 h. Endothelial cells were stimulated with VEGF (50 ng/ml) for 3 min and fixed in 1:1 methanol/acetone solution for 3 min at Ϫ20°C. Primary and secondary antibodies were incubated as described above, and cells were then mounted using gel mount (Biomedia) and visualized using confocal microscopy.
Localization of NO Production with DAF-2 DA-To localize NO production in cultured bovine pulmonary artery endothelial cells, a membrane-permeable fluorescent indicator DAF-2 DA (Calbiochem) (21-23) was used. We have previously reported that over the physiological range, DAF-2 fluorescence is linearly related to NO production and sensitive to changes in NOS activation due to capacitative calcium entry (21)(22)(23). Briefly, endothelial cells attached on a 25-mm glass coverslip were washed with physiological saline solution and loaded with 10 M DAF-2 DA at 37°C for 45 min in the dark and allowed to de-esterify for an additional 20 min. The glass coverslip with cells attached was mounted on an Olympus IX70 inverted microscope equipped with Photometrics PXL camera and a Silicon Graphics computer with Delta Vision deconvolution software (Applied Precision, Seattle, WA). Fluorescence section images from individual DAF-2 DAloaded cells were obtained at 0.2 M increments with 490-nm wavelength excitation and 528-nm wavelength emission. The deconvolved images before and after (30 min) treatment with VEGF (50 ng/ml) were used for localization of DAF-2 fluorescence.

RESULTS
To examine the phosphorylation of eNOS on serine 1179 in cells, we characterized the specificity of a P-eNOS antibody that recognizes this residue. As seen in Fig. 1A, incubation of recombinant eNOS with active Akt in the presence of ATP resulted in detection of P-eNOS (upper panel), whereas omission of ATP from the kinase reaction did not yield a phosphorylated product. Reprobing the blot with a total eNOS antibody showed the presence of the substrate (bottom panel). Next, COS cells were transfected with wild-type or eNOS S1179A in the absence or presence of Akt. As seen in 1B, the P-eNOS Ab only recognized phosphorylated wild-type but not the phosphorylation site mutant eNOS (upper panel). Finally, the relative level of P-eNOS was examined in endothelial cells infected with adenoviruses expressing ␤-galactosidase (control) or constitutively activated Akt. As seen in 1C (left panel), the P-eNOS Ab detected enhanced phosphorylation in cells infected with activated Akt, with low level basal phosphorylation in cells infected with ␤-galactosidase virus, whereas total eNOS levels were unchanged (right panel).
Next, we examined the time course of eNOS and Akt phosphorylation in BAEC in response to VEGF (50 ng/ml). As seen in Fig. 2A, VEGF stimulated time-dependent increases in eNOS and Akt phosphorylation with maximal phosphorylation of both occurring at 3 min post-stimulation. Moreover, the total levels of eNOS and Akt were unchanged throughout. eNOS phosphorylation lasted longer than did Akt, suggesting that the kinetics of dephosphorylation were different for these proteins. Recently several papers have shown that the G-proteincoupled receptor agonist bradykinin and in some but not all papers, VEGF, promotes the dephosphorylation of threonine 495 (or 497 in bovine eNOS) concomitantly with an increase in serine 1179 phosphorylation (24 -26). The authors suggest that both dephosphorylation and phosphorylation of these residues are requisite for activation of eNOS and NO release. To test this hypothesis in our cells, BAEC were treated with VEGF, and phosphorylation of eNOS on Ser-1179 and T497 was examined. As seen in Fig. 2B, VEGF stimulated P-eNOS on serine 1179 and did not reduce the phosphorylation on Thr-497 as seen with bradykinin. Identical results were seen using early passage porcine and human endothelial cells, and no changes were observed at later time points up to 60 min (not shown). We also wanted to see if Akt-stimulated NO release was associated with changes in threonine 497 phosphorylation. Previously, we and others have shown that infection of endothelial cells with an adenovirus expressing activated Akt promotes a large increase in NO release (8,9). As seen in Fig. 2C, Akt-induced NO release occurs simultaneously with enhanced Ser-1179 phosphorylation but no change in threonine 497 phosphorylation. Thus, VEGF and activated Akt stimulate serine 1179 phosphorylation but not threonine 497 dephosphorylation in our cells. In A, bovine aortic endothelial cells were stimulated with VEGF (50 ng/ ml) for the times indicated and Western blotted for P-eNOS, total eNOS, P-Akt, and total Akt. In B, endothelial cells were stimulated with VEGF as in A for different time points, and P-eNOS was examined with phospho-specific antibodies. Based on published work, threonine dephosphorylation is maximal from 15 s to 3 min (24 -26). In C, endothelial cells were infected with adenoviral myristoylated (myr)-Akt and NO 2 Ϫ accumulation, and phospho-eNOS levels were examined. The results are representative of 3-5 separate experiments. P-, phosphorylated.
To examine if phosphorylation on serine 1179 influenced the bulk subcellular distribution of eNOS, we examined the proportional subcellular distribution of P-eNOS in high speed membranes (Fig. 3A, pellet (P)) and supernatants (S) prepared from control or VEGF treated (50 ng/ml for 3 min) BAEC. As seen in fractions prepared from serum-starved BAEC, greater than 90% of the total eNOS is in the pellet, with very little in the supernatant, and the basal levels of phosphorylation were low in both pellet and supernatant. In contrast, all of the detectable total and P-Akt was in the supernatant fraction. Upon challenge with VEGF, the proportion of eNOS distributed in the pellet versus supernatant did not change, and both pools were phosphorylated on serine 1179. Under these conditions, the distribution of total and P-Akt did not change. Densitometry of the proportion of P-eNOS in the supernatants and pellet shows that the VEGF-stimulated phosphorylation occurred in both pools. Thus, in response to VEGF, eNOS in both cytosol and membranes is phosphorylated, and this does not result in the bulk translocation of total eNOS or Akt. To directly determine if the localization of eNOS was necessary for VEGFinduced phosphorylation on serine 1179, COS cells were transfected with the cDNA for the VEGF 2 receptor (Flk-1) and either wild-type eNOS or a non-acylated form of the enzyme, G2A eNOS (Fig. 3B). COS cells do not endogenously express VEGF receptors or eNOS. Previous work has demonstrated that G2A eNOS is not myristoylated or palmitoylated and is catalytically competent but does not localize to Golgi or plasma membrane and is distributed throughout the cytosol. Furthermore, upon agonist challenge, cells expressing G2A eNOS produce less NO (5). Expression of Flk-1 in COS cells results in VEGF-induced phosphorylation of WT eNOS on serine 1179. In contrast, VEGF receptor engagement does not lead to significant phosphorylation in cells transfected with G2A eNOS, thus demonstrating that localization is necessary for agonist-dependent phosphorylation on serine 1179.
To more accurately characterize the distribution of activated eNOS in transfected COS cells and in endothelial cells, we utilized sodium carbonate extraction of cells followed by a discontinuous sucrose gradient. In this procedure, cholesterolrich microdomains including lipid rafts and caveolae float as buoyant membranes at the 5-30% sucrose interface (fractions 2-3), whereas soluble proteins and heavy membranes remain at the bottom of the gradient (fractions 7-10). Initially, we examined the proportional distribution of P-eNOS and total eNOS and Akt in COS cells transfected with cDNAs for eNOS alone or eNOS in the presence of co-transfected Akt (Fig. 4A). In all gradients, the distribution of caveolin-1 (CAV-1), the coat protein of caveolae, ␤-COP, a marker for Golgi and post-Golgi vesicles, and ␤-actin, a cytoskeletal marker, were also examined to confirm the adequate separation of the membrane fractions. In the absence or presence of Akt, eNOS distributed primarily into two distinct pools, light membranes highly enriched in caveolin-1 and heavy membranes enriched in ␤-COP and ␤-actin. P-eNOS was also distributed in both fractions, with the relative ratio of P-eNOS/total eNOS enhanced in cells co-transfected with Akt (see right panel, bottom). No obvious redistribution of eNOS into or out of each domain was observed (see right panels, top and center). Endogenous Akt was primarily in heavy membranes and was also basally phosphorylated. Overexpression of Akt increased the levels of both P-Akt and total Akt. Next, we examined the relative distribution of a form of eNOS that cannot be phosphorylated, eNOS S1179A, and a constitutively active form that produces eNOS basally, eNOS S1179D. As seen in Fig. 4B, the distribution of both eNOS mutants were virtually identical (see relative quantitation in right panel). Both eNOS S1179A and eNOS S1179D were distributed in light and heavy membranes similar to data found in Fig. 4A.
To examine if a similar distribution exists in endothelial cells, the distribution of phospho-proteins was examined in BAEC challenged with VEGF for 3 min before preparation of cellular extracts (Fig. 5). Consistent with the data in Fig. 4, P-eNOS and total eNOS were enriched in two major pools, as was P-Akt and total-Akt. Upon activation of BAEC with VEGF, the relative levels of P-eNOS and P-Akt increased, but the distribution did not change (see right panels). Collectively, these data suggest that overexpression of Akt or activation of Akt by VEGF will phosphorylate eNOS on serine 1179 in both light and heavy membrane fractions without bulk redistribution of eNOS or Akt. Moreover, the constitutively active form of eNOS distributes similarly to the non-phosphorylated mutant.
Next we examined the intracellular localization of P-eNOS by confocal microscopy in COS cells transfected with eNOS in the absence and presence of ␤-galactosidase (control) or Akt. As seen in Fig. 6, eNOS was enriched in the perinuclear region of cells in a punctate, vesicular pattern (first row, left panel, white arrows) previously shown to co-localize with markers for the Golgi complex (19). In addition, eNOS was found at the periphery of cells (open arrows) previously shown to colocalize with caveolin-1. Transfection of COS cells with ␤-galactosidase did not result in significant detection of P-eNOS (right panel). However, transfection of COS cells with Akt resulted in a similar pattern of total eNOS (second row, left panel) and ample detection of P-eNOS in the perinuclear region (white arrows) and in the plasma membrane (open arrows). Moreover, preabsorption of the P-eNOS Ab with the immunogen peptide (third row) or transfection of eNOS S1179A (data not shown) completely prevented the detection of P-eNOS (third row, right panels) without influencing the detection of total eNOS (third row, left panel).
Next we examined the localization of endogenous eNOS in FIG. 3. VEGF-stimulated phosphorylation and eNOS subcellular distribution. In A, bovine aortic endothelial cells were stimulated with VEGF for 3 min, and high speed membrane pellets (P) and cytosolic supernatant (S) fractions were separated via centrifugation (100,000 ϫ g) and analyzed as above. In B, COS cells were transfected with cDNAs encoding Flk-1 with either WT-eNOS or G2A-eNOS and left to recover for 24 h. Cell were then stimulated with VEGF (50 ng/nl) and Western-blotted for P-eNOS and total eNOS. Results are representative of 3-5 separate experiments. human endothelial cells. Treatment of HUVECs with VEGF resulted in P-eNOS staining in both perinuclear and plasmalemmal pools and P-eNOS was colocalized with total eNOS in the same regions of the cell (Fig. 7A). To document that phosphorylation coincided with NO production, endothelial cells were loaded with DAF-2, a dye that upon binding an oxidized species of NO results in irreversible fluorescence, permitting identification of the source of local NO production in living cells (Fig. 7B). Confocal images of DAF-2 fluorescence demonstrated "hot spots" of activity in two primary sub-cellular compartments. The predominant locus of activity was in the perinuclear region with focal areas of additional activity near the plasma membrane. After treatment with VEGF, increased fluorescence was detected in both compartments along with a generalized increase in cytosolic fluorescence (n ϭ 3). No significant increase in fluorescence was seen in time control experiments or cells pretreated with a NOS inhibitor (n ϭ 2, not shown). DISCUSSION The central findings of this paper are that Akt-dependent phosphorylation of eNOS on serine 1179 occurs primarily while eNOS is localized to discrete domains of cells and that activation of eNOS does not cause bulk translocation of the enzyme using cell fractionation techniques. Moreover, confocal imaging studies reveal that Akt-dependent P-eNOS could be detected in plasma membrane extensions and in the perinuclear region of cells and that the relative distribution of P-eNOS to total eNOS in these pools did not change with overexpression of Akt or by stimulation of cells with VEGF. Basal and VEGF-stimulated NO synthesis, imaged by DAF-2 fluorescence, occurred in both the perinuclear area and plasmalemma. Most importantly, the proper membrane localization of eNOS is necessary for VEGFstimulated phosphorylation of eNOS on serine 1179, thus explaining the original observation that eNOS compartmentalization is necessary for optimal NO production from cells (5,6). These data support the model that eNOS in the Golgi and plasma membrane are dynamically regulated and that localization into both domains are necessary for phosphorylation on serine 1179 and VEGF-dependent NO production.
The recent identification of serine 1179 as a regulated phosphorylation site in eNOS and the fundamental importance of this site for the activity of the enzyme and NO release in vitro FIG. 4. Subcellular fractionation of P-eNOS, S1179A-eNOS, and S1179D eNOS. In A, COS cells were transfected with cDNAs encoding WT-eNOS with ␤-galactosidase (left panel) or Akt (right panel). In B, COS cells were transfected with cDNAs for S1179A-and S1179D-eNOS. In both experiments cells were processed as described, and equal volumes of each fraction were subjected to SDS-PAGE and Western-blotted for P-eNOS, total eNOS, P-Akt, total Akt, caveolin-1 (CAV-1), ␤-COP, and ␤-actin. The relative distribution of each protein across the sucrose gradient was determined by laser densitometry and plotted as a percentage of total protein (far right). The results are representative of two separate experiments. and in vivo prompted us to examine the subcellular distribution of total versus P-eNOS. Previous studies show that compartmentalization of eNOS to intracellular membrane domains is necessary for agonist-induced NO release from cells (5,18). Both confocal and electron microscopic evaluation of eNOS in cultured cells and blood vessels have localized eNOS exclusively on the cytoplasmic face of the Golgi or in plasma membrane caveolae or both (3,19,20,27). Subcellular fractionation using sodium carbonate to separate light, cholesterol-rich microdomains (rafts and caveolae) from other membrane components has demonstrated distribution of eNOS between caveolin-enriched fractions and Golgi membrane-enriched fractions. Experiments isolating caveolin-enriched membranes thought to only contain caveolae have shown exclusive enrichment of eNOS in caveolae, whereas microscopically, a majority of eNOS is either colocalized with caveolin-1 or in a perinuclear pattern co-localizing with Golgi markers. Upon isolation of caveolinenriched domains versus non-caveolin-enriched domains, the specific activity of eNOS (i.e. NOS activity/mg of protein) is greater in caveolin-enriched domains compared with noncaveolin-enriched domains, leading to the conclusion that active eNOS is exclusively in caveolae (28). However this argument is based on the fact that during caveolae isolation, the paucity of protein recovered in caveolin-1-enriched domains results in a higher specific activity of eNOS in caveolae and dilution of the eNOS activity in non-caveolae membranes due to the presence of a majority of cellular proteins in non-caveolae domains. Because comparisons of eNOS activity normalized to the amounts of eNOS recovered in each fraction have not been performed to date, it is difficult to determine the relative level of activity in each pool. Using the sodium carbonate method for caveolin-1 enrichment in this study removes the inherent bias of protein concentration and dilution from the analysis since the proportional distribution of multiple proteins can be assessed simultaneously with those enriched in caveolin-enriched microdomains. Consistent with a majority of studies using microscopy, here we show that eNOS and more importantly P-eNOS is found in both light (caveolin-1-enriched) and heavy membranes (Golgi) after co-transfection of eNOS with Akt or after VEGF treatment in endothelial cells, suggesting that activated eNOS resides in at least two domains using different cell systems. Moreover, using DAF-2 as a way to detect low level NO production in situ (22) demonstrates that the sites of highest fluorescent intensity completely overlap with the pattern of eNOS trafficking.
Previous work by some (16) but not all labs (12,20,29) shows that agonist-stimulated phosphorylation or depalmitoylation of eNOS may cause translocation of eNOS from membrane to cytosol. Recent data using eNOS-green fluorescent protein expression in cells has documented rapid intra-compartment movement of the enzyme in living cells that is dependent on cell density (19). The idea of intra-or intermembrane movement is supported by experiments showing that bradykinin, ceramide, and estrogen cause movement of eNOS from the plasma membrane to a juxtanuclear membrane location, arguing against bulk movement from membrane to cytosol after agonist stimulation (16,30,31). However, in these experiments, the movement of eNOS was difficult to discern from movement due to cell shape change since all endothelial cell agonists cause actin reorganization and remodeling (32,33). Herein we show that VEGF stimulation of eNOS phosphorylation does not influence bulk eNOS distribution. Moreover, co-transfection of eNOS with Akt to give high levels of eNOS phosphorylation under conditions resulting in tremendous NO release from cells (8) FIG. 5. Subcellular fractionation of phosphorylated eNOS in endothelial cells. Bovine aortic endothelial cells were challenged with vehicle (left) or VEGF (right, 50 ng/ml) for 3 min, and cells were processed as described above. Equal volumes of each fraction were electrophoresed and Western-blotted for P-eNOS, total eNOS, P-Akt, total Akt, caveolin-1 (CAV-1), ␤-COP, and ␤-actin. Results are representative of two separate experiments. and increases in blood flow in vivo or transfection with constitutively active eNOS (8,34) does not result in eNOS redistribution using sodium carbonate gradients or confocal imaging. Interestingly, eNOS S1179A that cannot be phosphorylated on serine 1179 has an identical fractionation pattern to wild-type and constitutively active eNOS. Taken together, although phosphorylation of eNOS on serine 1179 is necessary for NO release induced by VEGF or after Akt transfection, this does not result in bulk eNOS translocation in our cell systems. Our data are consistent with the idea that eNOS within a caveolae microdomain may be liberated from the caveolin-1 inhibitory clamp (20) without bulk movement out of caveolae and that eNOS in other microdomains (cytoplasmic face of Golgi) is a biologically active pool of enzyme.
One interesting finding in this paper is the lack of enrichment of Akt or P-Akt in high speed membranes or caveolin-1enriched domains. This is likely a technical issue based on recent findings. Upon receptor engagement (insulin-like growth factor-1, platelet-derived growth factor, and epidermal growth factor receptors and B cell receptor), Akt is transiently localized to the plasma membrane where it is activated and then moves to the cytosol and nucleus to phosphorylate its respective target proteins (35,36). Our inability to detect Akt co-fractionating with eNOS, although eNOS is phosphorylated by Akt, is most likely due to rapid, reversible kinetics of Akt trafficking, weak interactions of the pleckstrin homology domain of Akt with lipid components important for binding to plasma membrane, and the high pH of the sodium carbonate used for concentrating proteins into light membranes. Any of these scenarios would preclude a stable interaction required for localization into high speed membranes or caveolin-enriched microdomains using the methodologies in this paper. Alternatively, it is possible that eNOS is phosphorylated by Akt while in the cytosol or cytoplasmic face of the Golgi, thus providing the phospho pool of the enzyme for targeting to the plasma membrane.
The rationale for tracking Ser-1179 phosphorylation as a surrogate marker for eNOS activation stemmed from previous observations that phosphorylation of this residue is critical for NO release from cultured endothelial cells, transfected cells, and endothelium-dependent relaxations of isolated blood vessels (8 -11, 34, 37). However, recent data show that stimulusdependent dephosphorylation of threonine 495 (or 497 in bovine eNOS) is necessary for bradykinin-and histamine-stimulated eNOS activation (24 -26). Using VEGF as an eNOS agonist, one report shows marginal dephosphorylation on threonine 495 (26), and the other report shows no dephosphorylation at this site (25). The phosphatase that dephosphorylates this residue is controversial, with pharmacological evidence supporting either calcineurin (25) or PP1 (24,26) as the can-FIG. 6. Cellular location of phosphorylated eNOS in transfected COS cells. COS cells were transfected with plasmids encoding WT-eNOS and S1179A with or without Akt and immunolabeled with total eNOS (left panel) and P-eNOS (right panel) antibodies. Confocal analysis shows that the presence of phosphorylated eNOS was dependent on Akt transfection (upper and middle panels) and was located to the perinuclear region (solid arrows) and also at the plasma membrane (open arrows). The lower panel shows that the P-eNOS peptide (10-fold molar excess) did not influence total eNOS labeling (left panel) but eliminated P-eNOS labeling. The results are representative of 3-5 separate experiments.

FIG. 7. Cellular location of P-eNOS and local NO production in endothelial cells.
In A, HUVEC were stimulated with VEGF (50 ng/ml), and cells were immunolabeled with antibodies to total eNOS or P-eNOS. Both total eNOS (left panel) and P-eNOS (center panel) were localized in the perinuclear region (solid arrows) and also at the plasma membrane (open arrows). The right panel shows the merge of the two images. In B, endothelial cells were loaded with the NO sensitive dye, DAF-2, and fluorescence was monitored in the absence and presence of VEGF (50 ng/ml). Base-line fluorescence demonstrating NO production predominantly in the peri-nuclear region, with additional activity adjacent to the plasma membrane (left panel). Intense DAF-2 fluorescence is seen in both the plasma membrane (arrow) and perinuclear region (arrowhead) after VEGF administration (right panel). The images are representative of three replicates. There was no increase in fluorescence seen in time control experiments (n ϭ 2 experiments, data not shown). Scale bar, 10 M.