Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m402155200
ISSN1083-351X
AutoresDavid Fulton, Roger W. Babbitt, Stefan Zoellner, Jason Fontana, Lisette M. Acevedo, Timothy J. McCabe, Yasuko Iwakiri, William C. Sessa,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Akt-mediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane- and Golgi complex-targeted constructs, Ser1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane- and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes. The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Akt-mediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane- and Golgi complex-targeted constructs, Ser1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane- and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes. Within the vascular endothelium, nitric oxide (NO) is generated via the enzyme endothelial nitric-oxide synthase (eNOS). 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; WT, wild-type. This highly labile and reactive gas plays a key role in regulating cardiovascular homeostasis, influencing systemic and pulmonary blood pressure, vascular remodeling, and angiogenesis. Given the important and pervasive action of endothelium-derived NO in the cardiovascular system, it is not surprising that the activity of eNOS is highly regulated in a temporal and spatial manner (1Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar). eNOS was originally characterized as a calcium/calmodulin-dependent enzyme (2Singer H.A. Peach M.J. Hypertension. 1982; 4: 19-25PubMed Google Scholar, 3Forstermann U. Pollock J.S. Schmidt H.H. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (550) Google Scholar, 4Pollock J.S. Forstermann U. Mitchell J.A. Warner T.D. Schmidt H.H. Nakane M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10480-10484Crossref PubMed Scopus (899) Google Scholar), and although this is the primary means by which eNOS activity is controlled, discrepancies between the levels of eNOS protein, the amount of NO released, calcium regulation of the enzyme, and the impaired function of mislocalized eNOS have led to the discovery of additional post-translational control mechanisms regulating eNOS activity. It is now generally accepted that eNOS activity is also influenced directly by regulated protein-protein interactions and phosphorylation and indirectly by fatty acylation, which targets the enzyme to specific intracellular domains (1Fulton D. Gratton J.P. Sessa W.C. J. Pharmacol. Exp. Ther. 2001; 299: 818-824PubMed Google Scholar, 5Kone B.C. Kuncewicz T. Zhang W. Yu Z.Y. Am. J. Physiol. 2003; 285: F178-F190Crossref PubMed Scopus (243) Google Scholar, 6Fleming I. Busse R. Am. J. Physiol. 2003; 284: R1-R12PubMed Google Scholar). Activation of endothelial cells in response to vascular endothelial growth factor (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 8Takahashi S. Mendelsohn M.E. J. Biol. Chem. 2003; 278: 30821-30827Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), sphingosine-1-phosphate (9Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 10Morales-Ruiz M. Lee M.J. Zollner S. Gratton J.P. Scotland R. Shiojima I. Walsh K. Hla T. Sessa W.C. J. Biol. Chem. 2001; 276: 19672-19677Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), estrogen (11Simoncini T. Hafezi-Moghadam A. Brazil D.P. Ley K. Chin W.W. Liao J.K. Nature. 2000; 407: 538-541Crossref PubMed Scopus (1231) Google Scholar, 12Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (487) Google Scholar), insulin (13Zeng G. Nystrom F.H. Ravichandran L.V. Cong L.N. Kirby M. Mostowski H. Quon M.J. Circulation. 2000; 101: 1539-1545Crossref PubMed Scopus (656) Google Scholar), and shear stress (14Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar) results in the Akt-dependent phosphorylation of Ser1179 on eNOS. Although there is increasing evidence that other phosphorylation sites (Ser116, Thr497, Ser617, and Ser635) (15Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 16Fleming I. Fisslthaler B. Dimmeler S. Kemp B.E. Busse R. Circ. Res. 2001; 88: E68-E75Crossref PubMed Scopus (604) Google Scholar, 17Bauer P.M. Fulton D. Boo Y.C. Sorescu G.P. Kemp B.E. Jo H. Sessa W.C. J. Biol. Chem. 2003; 278: 14841-14849Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 18Lin M.I. Fulton D. Babbitt R. Flemming I. Busse R. Pritchard Jr., K.A. Sessa W.C. J. Biol. Chem. 2003; 278: 44719-44726Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) contribute to temporal regulation of eNOS function, mutation of Ser1179 prevents both Akt-mediated phosphorylation and NO release, and dominant-negative Akt reduces endothelium-dependent vascular responses in vitro and in vivo (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 14Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 19Luo Z. Fujio Y. Kureishi Y. Rudic R.D. Daumerie G. Fulton D. Sessa W.C. Walsh K. J. Clin. Investig. 2000; 106: 493-499Crossref PubMed Scopus (178) Google Scholar), thus highlighting the importance of this regulatory site. The ability of both Akt-phosphorylated and S1179D eNOS (a constitutively active allele in which Ser1179 is mutated to an aspartate reside) to release more NO in the absence of a stimulus has led to the proposal that phosphorylation of Ser1179 mediates the calcium-independent activation of eNOS (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 14Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar, 20McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). eNOS is modified by post-translational myristoylation of Gly2 and palmitoylation of Cys15 and Cys26 (21Pollock J.S. Klinghofer V. Forstermann U. Murad F. FEBS Lett. 1992; 309: 402-404Crossref PubMed Scopus (81) Google Scholar, 22Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar, 23Robinson L.J. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11776-11780Crossref PubMed Scopus (131) Google Scholar). These N-terminal fatty acid modifications target eNOS to the Golgi complex and to cholesterol-enriched domains of the plasma membrane, including caveolae and “lipid rafts” (24Liu J. Hughes T.E. Sessa W.C. J. Cell Biol. 1997; 137: 1525-1535Crossref PubMed Scopus (158) Google Scholar, 25Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 26García-Cardeña G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (578) Google Scholar). Whether in situ within blood vessels or in cultured endothelial cells, the relative proportion of eNOS in the Golgi complex versus the plasma membrane is variable and can depend on the vascular bed, species, and state of confluency (27Andries L.J. Brutsaert D.L. Sys S.U. Circ. Res. 1998; 82: 195-203Crossref PubMed Scopus (120) Google Scholar, 28O'Brien A.J. Young H.M. Povey J.M. Furness J.B. Histochem. Cell Biol. 1995; 103: 221-225Crossref PubMed Scopus (54) Google Scholar, 29Govers R. Rabelink T.J. Am. J. Physiol. 2001; 280: F193-F206Crossref PubMed Google Scholar). The N-myristoylation site mutant of eNOS is catalytically competent, remains cytosolic, and displays reduced NO release. Biochemically, this is due to a reduction in Ser1179 phosphorylation (30Fulton D. Fontana J. Sowa G. Gratton J.P. Lin M. Li K.X. Michell B. Kemp B.E. Rodman D. Sessa W.C. J. Biol. Chem. 2002; 277: 4277-4284Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 31Gonzalez E. Kou R. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2002; 277: 39554-39560Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 32Lin S. Fagan K.A. Li K.X. Shaul P.W. Cooper D.M. Rodman D.M. J. Biol. Chem. 2000; 275: 17979-17985Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Phosphorylation of eNOS at Ser1179 has been observed in the Golgi complex and in the plasma membrane of endothelial cells; however, due to the presence of eNOS in both locations, the individual functions of Golgi versus plasmalemmal eNOS, if any, are not yet known and are matter of mere speculation (30Fulton D. Fontana J. Sowa G. Gratton J.P. Lin M. Li K.X. Michell B. Kemp B.E. Rodman D. Sessa W.C. J. Biol. Chem. 2002; 277: 4277-4284Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Therefore, the goal of this study was to determine the individual function of eNOS in the cytoplasmic aspect of the Golgi complex and plasma membrane using molecular targeting with organelle-restricted “zip codes” to deliver functional eNOS to specific intracellular sites and to examine whether eNOS targeted to endomembranes is functionally responsive to extracellular stimuli. The cytosolic myristoylation site mutant (G2A eNOS) is catalytically identical to the wild-type (WT) enzyme (33Sessa W.C. García-Cardeña G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and was used as an enzymatic scaffold for attaching the different targeting sequences as outlined below. GRIP-eNOS—The GRIP domain (∼42 amino acids) targets cargo to a distinct population of trans-Golgi tubulovesicular membranes. This domain is shared by a family of coiled-coil peripheral membrane proteins, including p230, golgin-97, and golgin-245 (34Kjer-Nielsen L. Teasdale R.D. van Vliet C. Gleeson P.A. Curr. Biol. 1999; 9: 385-388Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). A region of golgin-97 containing the GRIP domain (87 amino acids) was isolated from human umbilical vein endothelial cell mRNA; fused in-frame to G2A eNOS using PCR overlap extension with primers 5′-TGT GCC TCG AGC GGG GCC ACA TGT T-3′ (sense), 5′-CCA GAC ACC CCC GGC CCC GTC ACG AAT AAC ACT GAC-3′ (middle sense), 5′-GTC AGT GTT ATT CGT GAC GGG GCC GGG GGT GTC TGG-3′ (middle sense), and 5′-ACT AGT TGA GTC TAG ACT AGG ACC ATG GTA TCC GAG GG TT-3′ (antisense); and encodes the GRIP domain (VTNNTDLTDAREINFEYLKHVVLKFMSCRESEAFHLIKAVSVLLNFSQEEENMLKETLEYKMSWFGSKPAPKGSIRPSISNPRIPWS). Syn17-eNOS—The length of the transmembrane domain of syntaxin-3 determines membrane association and subcellular targeting (35Watson R.T. Pessin J.E. Am. J. Physiol. 2001; 281: C215-C223Crossref PubMed Google Scholar). The truncated transmembrane domain of syntaxin-3 (referred to as Syn17; 18 amino acids, KLIIIIVLVVVLLGILAL) that targets the cis/medial-Golgi complex was fused in-frame to the C terminus of G2A eNOS using primers 5′-TGT GCC TCG AGC GGG GCC ACA TG TT-3′ (sense) and 5′-AAA TCT AGA TCT AGA TCA AAT CAA TGC TAA AAT GCC CAG CAA CAC AAC TAC TAG CAC AAT GAT AAT TAT CAA TTT GGG GCC GGG GGT GTC TGG GC-3′ (antisense). CAAX-eNOS—The K-Ras polybasic (polylysine) domain containing the CAAX (where A is aliphatic amino acid) motif, which undergoes farnesylation, proteolysis of AAX, and methyl esterification (GKKKKKKSKTKCVIM), was fused in-frame to the C terminus of G2A eNOS using primers 5′-TGT GCC TCG AGC GGG GCC ACA TGT T-3′ (sense) and 5′-TAT CTA GAT CTA GAT TAC ATA ATT ACA CAC TTT GTC TTT GAC TTC TTT TTC TTC TTT TTA CCG GGG CCG GGG GTG TCT GGG CCG GG-3′ (antisense) (36Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (846) Google Scholar). Syn25-eNOS—A region of syntaxin-3 containing the full-length plasma membrane-targeting domain (27 amino acids, KLIIIIVLVVVLLGILALIIGLSVGLN) was fused in-frame to the C terminus of G2A eNOS using primer 5′-ATC TAG ATC TAG ATC AAT TCA GCC CAA CGG AAA GTC CAA TAA TCA ATG CTA AAA TGC CCA GCA ACA CAA CTA CTA GCA CAA TGA TAA TTA TCA ATT TGG GGC CGG GGG TGT CTG GGC-3′ (antisense). COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% (v/v) fetal calf serum. For transfection, COS-7 cells were seeded at a density of 1.3 × 106 cells/100-mm dish and transfected the next day with the cDNAs for WT eNOS, G2A eNOS, GRIP-eNOS, CAAX-eNOS, Syn17-eNOS, Syn25-eNOS, Flk, and Akt using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Cells were washed twice with phosphate-buffered saline and lysed on ice in 50 mm Tris-HCl (pH 7.5), 1% (v/v) Nonidet P-40, 10 mm NaF, 1 mm vanadate, 1 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin, and lysates were transferred to an Eppendorf tube and rotated for 45 min at 4 °C. Lysates were homogenized in a Dounce homogenizer (50 strokes), and insoluble material was removed by centrifugation at 12,000 × g for 10 min at 4 °C, size-fractionated by SDS-PAGE, and Western-blotted as described previously (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar). COS-7 cells were transfected as described above and plated onto sterile coverslips. The cells were then fixed in 1:1 acetone/methanol for 3 min at –20 °C and rinsed twice with phosphate-buffered saline plus 0.1% bovine serum albumin for 5 min at room temperature. The cells were incubated with 5% goat serum in phosphate-buffered saline plus 0.1% bovine serum albumin for 30 min at room temperature, followed by incubation for 2 h with primary antibody (polyclonal or monoclonal) at room temperature. Texas Red-labeled anti-rabbit (diluted 1:100) or fluorescein isothiocyanate-labeled anti-mouse (diluted 1:100) secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was 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. Antibodies for eNOS were obtained from BD Biosciences (anti-eNOS monoclonal antibody), and those for GM130 were from G. Warren (Yale University). Medium (100 μl) containing NO (primarily NO–2) was ethanol-precipitated to remove proteins and refluxed in sodium iodide/glacial acetic acid to convert NO–2 to NO. NO was quantitated via specific chemiluminescence following reaction with ozone (Sievers). Net NO–2 was calculated after subtracting unstimulated basal release (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar). The activity of WT and targeted eNOS was determined in detergent-solubilized lysates of transfected COS-7 cells by measuring the conversion of [14C]arginine to [14C]citrulline as described previously (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar). NO release data are expressed as means ± S.E. Comparisons were made using two-tailed Student's t test or analysis of variance with a post-hoc test where appropriate. Differences were considered as significant at p < 0.05. Generation and Characterization of eNOS Targeting Constructs—A cytosolic mutant of eNOS (G2A) that cannot be N-myristoylated was used to generate eNOS fusion proteins that specifically target the Golgi complex or plasma membrane (Fig. 1). Previous work has shown that G2A eNOS retains full catalytic ability in vitro; however, the stimulated release of NO from intact cells expressing this mutant is impaired because the enzyme fails to target the Golgi complex and plasmalemmal rafts/caveolae and is not efficiently phosphorylated by Akt at Ser1179 (30Fulton D. Fontana J. Sowa G. Gratton J.P. Lin M. Li K.X. Michell B. Kemp B.E. Rodman D. Sessa W.C. J. Biol. Chem. 2002; 277: 4277-4284Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 33Sessa W.C. García-Cardeña G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 37Sakoda T. Hirata K. Kuroda R. Miki N. Suematsu M. Kawashima S. Yokoyama M. Mol. Cell. Biochem. 1995; 152: 143-148Crossref PubMed Scopus (48) Google Scholar). By exploiting these properties of G2A eNOS, we generated Golgi complex- and plasma membrane-targeted eNOS to assess the functional importance of subcellular targeting (Fig. 1). To determine whether the eNOS fusion proteins target their intended destinations, indirect immunofluorescence for eNOS and GM130, a resident peripheral Golgi protein, was performed in transfected COS cells. As shown in Fig. 2, WT eNOS was present in both the plasma membrane (WT-eNOS, left panel, arrows) and the Golgi complex (arrowhead), co-localizing with the Golgi marker GM130 (right panel, arrowhead), whereas G2A eNOS was diffusely distributed throughout the cell (G2A-eNOS, left panel) and did not specifically co-localize with GM130 (right panel). Targeting of eNOS to the Golgi complex with the GRIP domain was confirmed by the presence of eNOS concentrated in a perinuclear pattern (GRIP-eNOS, left panel), but it was also found in a pattern reminiscent of post-Golgi vesicles with the absence of plasma membrane staining. GRIP-eNOS partially co-localized with the Golgi marker GM130 (right panel). Similarly, fusion of eNOS with the syntaxin-3 cis/medial-Golgi complex-targeting motif (Syn17-eNOS) demonstrated Golgi targeting (S17-eNOS, left panel) and partial co-localization with GM130 and no plasma membrane staining (right panel). Fusion of eNOS to the full-length transmembrane domain of syntaxin-3 (Syn25-eNOS) resulted in dramatic plasma membrane staining, with minor amounts present in the Golgi complex (S25-eNOS, left panel). Membrane targeting of eNOS with the CAAX motif derived from K-Ras (CAAX-eNOS) also showed the majority of eNOS in the plasma membrane (CAAX-eNOS, left panel). Thus, using these targeting motifs, eNOS was targeted to the peripheral aspect of the Golgi network (GRIP-eNOS and Syn17-eNOS) or the plasma membrane (Syn25-eNOS and CAAX-eNOS). Previous studies have shown that WT and G2A eNOS have similar catalytic abilities and cofactor requirements (38Sessa W.C. Barber C.M. Lynch K.R. Circ. Res. 1993; 72: 921-924Crossref PubMed Scopus (132) Google Scholar, 39Venema R.C. Sayegh H.S. Arnal J.F. Harrison D.G. J. Biol. Chem. 1995; 270: 14705-14711Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) despite the inability of mislocalized G2A eNOS to produce NO after stimulation of cells with calcium-mobilizing agonists (33Sessa W.C. García-Cardeña G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 37Sakoda T. Hirata K. Kuroda R. Miki N. Suematsu M. Kawashima S. Yokoyama M. Mol. Cell. Biochem. 1995; 152: 143-148Crossref PubMed Scopus (48) Google Scholar). Therefore, we measured the catalytic competency of the newly generated eNOS fusion proteins in vitro using the conversion of l-[3H]arginine to l-[3H]citrulline as an index of NOS activity in detergent-solubilized COS cell extracts. COS cells do not contain any endogenous NOS isoform, and all activity measurements were eliminated by the NOS inhibitor Nω-nitro-l-arginine methyl ester hydrochloride. The activity of GRIP-eNOS was slightly greater than that of WT eNOS (89.7 ± 2.7 and 72.8 ± 5.5 pmol of l-citrulline/mg/min, respectively; n = 3). In a separate series of transfectants, Syn17-eNOS, Syn25-eNOS, and CAAX-eNOS exhibited slightly lower catalytic activities compared with WT eNOS (25.4 ± 0.6, 20.8 ± 1.5, 14.3 ± 0.5, and 32.9 ± 1 pmol of l-citrulline/mg/min, respectively; n = 3). These differences in activity were not due to expression differences as assessed by semiquantitative Western blotting of the same extracts. Plasma Membrane Targeting of eNOS Results in Marked NO Release due to Constitutive Phosphorylation of Ser1179—To determine the relevance of intracellular location to eNOS function in intact cells, COS cells were transfected with the various eNOS constructs, and basal NO release was determined by NO-specific chemiluminescence. Cells were subsequently lysed and immunoblotted for total eNOS or phosphorylated eNOS species using phosphorylation state-specific antibodies (Ser(P)1179, Thr(P)497, Ser(P)617, Ser(P)635). As shown in Fig. 3, non-acylated cytosolic G2A eNOS displayed both significantly reduced NO release (A, graph) and hypophosphorylation of Ser1179 (A) Thr497 (B), and Ser617 and Ser635 (C) compared with WT eNOS (upper gels). On the other hand, the plasma membrane-targeted forms of eNOS fusion proteins (Syn25-eNOS and CAAX-eNOS) produced substantially more basal NO than did WT eNOS, with equivalent levels of eNOS protein expression. As shown in Fig. 3A (upper and lower gels), the level of Ser1179 phosphorylation was also much greater in the plasma membrane-targeted forms of eNOS relative to the WT enzyme. Localization of eNOS to the peripheral aspects of the Golgi network produced divergent results. Restricted localization of eNOS to the trans- and post-Golgi vesicles via the GRIP targeting motif (GRIP-eNOS) resulted in reduced basal NO release compared with the WT enzyme. In contrast, fusion of eNOS to the syntaxin-3 cis-Golgi complex-targeting motif (Syn17-eNOS) produced equivalent amounts of basal NO compared with the WT enzyme. These changes in NO release did not strictly correlate with basal phosphorylation of Ser1179 since GRIP-eNOS and Syn17-eNOS phosphorylation was comparable with WT eNOS phosphorylation, but was again greater than G2A eNOS phosphorylation. We next investigated the relative degree of eNOS phosphorylation of the other regulatory phosphorylation sites. Thr497 phosphorylation is important for the fidelity of eNOS to produce NO versus superoxide since phosphorylation promotes NO release, whereas dephosphorylation promotes superoxide anion production (18Lin M.I. Fulton D. Babbitt R. Flemming I. Busse R. Pritchard Jr., K.A. Sessa W.C. J. Biol. Chem. 2003; 278: 44719-44726Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Phosphorylation of Ser617 and Ser635 regulates temporal aspects of calcium/calmodulin regulation of the enzyme (17Bauer P.M. Fulton D. Boo Y.C. Sorescu G.P. Kemp B.E. Jo H. Sessa W.C. J. Biol. Chem. 2003; 278: 14841-14849Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 40Boo Y.C. Sorescu G.P. Bauer P.M. Fulton D. Kemp B.E. Harrison D.G. Sessa W.C. Jo H. Free Radic. Biol. Med. 2003; 35: 729-741Crossref PubMed Scopus (78) Google Scholar). As shown in Fig. 3B, WT eNOS and plasma membrane-targeted eNOS (Syn25-eNOS and CAAX-eNOS) exhibited enhanced Thr497 phosphorylation relative to G2A eNOS and the Golgi complex-targeted eNOS constructs. The phosphorylation of Ser617 was similarly distributed, with the greatest degree of phosphorylation observed with the WT and plasma membrane-targeted eNOS constructs. Low levels of Ser617 phosphorylation were observed with cytosolic G2A eNOS and eNOS fusion proteins that target the Golgi network (GRIP-eNOS and Syn17-eNOS). GRIP-eNOS displayed the highest level of Ser635 phosphorylation, followed by WT eNOS and plasma membrane-targeted eNOS (CAAX-eNOS and Syn25-eNOS). Syn17-eNOS (cis-Golgi complex-targeted) was found to have significantly lower phosphorylation of Thr497, Ser635, and Ser617 (Fig. 3, B and C). Plasma Membrane-targeted eNOS Is Highly Sensitive to Transmembrane Calcium Fluxes—Thapsigargin, an inhibitor of SERCA, elevates intracellular calcium through store depletion, which in turn triggers calcium influx via capacitive calcium entry (41Treiman M. Caspersen C. Christensen S.B. Trends Pharmacol. Sci. 1998; 19: 131-135Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). The elevation of intracellular calcium is a key determinant of eNOS activation via the increased association of calcium-activated calmodulin. As shown in Fig. 4A, thapsigargin evoked release of substantially more NO from COS-7 cells transfected with plasma membrane-targeted forms of eNOS (Syn25-eNOS and CAAX-eNOS) compared with cells expressing WT eNOS or Golgi complex-restricted eNOS (GRIP-eNOS and Syn17-eNOS). Compared with the WT enzyme, calcium-dependent activation of cytosolic G2A eNOS resulted in the production of significantly less NO, whereas both trans-Golgi-localized GRIP-eNOS and cis/medial-Golgi targeting with Syn17-eNOS were not significantly different from that with WT eNOS. Golgi Targeting with Syn17-eNOS Is Required for Akt-dependent Activation of eNOS—As stated previously, eNOS can be activated via Akt-dependent phosphorylation of Ser1179 (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 14Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar). As shown in Fig. 4B, cotransfection of WT eNOS with cDNA for Akt-1 resulted in the increased phosphorylation of Ser1179 (upper gels) and enhanced production of NO (graph). Previous work has shown that this effect is abolished if Ser1179 is mutated to alanine (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2232) Google Scholar, 14Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3047) Google Scholar). Fatty acylation and subcellular targeting are required for full activation, as Akt stimulated significantly less NO production (graph) and Ser1179 phosphorylation (upper gels) with cytosolic G2A eNOS versus the WT enzyme. Although a significant increase in NO release was observed in the presence of Akt, this was not greater than the unstimulated levels for the WT enzyme (Fig. 4B, graph). Transfection with Akt did not stimulate statistically significant NO production from GRIP-eNOS; however, restricted cis/medial-Golgi localization with Syn17-eNOS enabled full Akt-dependent activation of eNOS as determined by NO release and phosphorylation of Ser1179 to levels seen with WT eNOS. Plasma membrane-targeted eNOS constructs (CAAX-eNOS and Syn25-eNOS) exhibited constitutive NO release and phosphorylation of Ser1179. In contrast to WT eNOS and Syn17-eNOS, cotransfection with Akt did not stimulate further production of NO or influence the phosphorylation state of Ser1179. Phosphorylation of Ser1179 Is Required for the Activation of Golgi Complex- and Plasma Membrane-restricted eNOS—The ability of Akt to increase eNOS activity is dependent on the specific phosphorylation of Ser1179, and mutation of this site to the phospho-mimetic aspartic acid is sufficient to increase NO release from eNOS in the absence of additional stimuli (7Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossre
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