Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking Yasuko Iwakiri*†‡, Ayano Satoh§, Suvro Chatterjee¶, Derek K. Toomre§ʈ, Cecile M. Chalouni§, David Fulton**, Roberto J. Groszmann*‡, Vijay H. Shah¶, and William C. Sessa†,†† *Section of Digestive Diseases, Departments of †Pharmacology and §Cell Biology, and ʈInstitute for Cancer Research, Yale University School of Medicine, New Haven, CT 06510; ‡Hepatic Hemodynamic Laboratory, VA Connecticut Healthcare System, West Haven, CT 06516; ¶Gastroenterology Research Unit, Department of Physiology and Tumor Biology Program, Mayo Clinic, Rochester, MN 55905; and **Vascular Biology Center and Department of Pharmacology, Medical College of Georgia, Augusta, GA 30912 Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved October 23, 2006 (received for review July 13, 2006) Nitric oxide (NO) is a highly diffusible and short-lived physiological ating NO, suggesting that the compartmentalization of NO equiv- messenger. Despite its diffusible nature, NO modifies thiol groups alents is physiologically important to regulate diverse cellular of specific cysteine residues in target proteins and alters protein function. (ii) In red blood cells, S-nitrosohemoglobin binds to and function via S-nitrosylation. Although intracellular S-nitrosylation transnitrosylates the plasma membrane anion exchange protein is a specific posttranslational modification, the defined localization AE1, whereas NO itself does not S-nitrosylate AE1 in the absence of an NO source (nitric oxide synthase, NOS) with protein S- of hemoglobin (13, 14). (iii) Inducible NOS (iNOS) can only nitrosylation has never been directly demonstrated. Endothelial S-nitrosylate cyclooxygenase after a direct protein–protein inter- NOS (eNOS) is localized mainly on the Golgi apparatus and in action; if the interaction between iNOS and cyclooxygenase is plasma membrane caveolae. Here, we show by using eNOS tar- prevented (with an inhibitory peptide), iNOS generates NO, but geted to either the Golgi or the nucleus that S-nitrosylation is does not S-nitrosylate cyclooxygenase (15). (iv) NOSs are them- concentrated at the primary site of eNOS localization. Furthermore, selves S-nitrosylated, thus NO would appear to be acting ‘‘very localization of eNOS on the Golgi enhances overall Golgi protein locally’’ (16, 17). (v) Privileged sites of S-nitrosylation have been CELL BIOLOGY S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide- identified in particular, mitochondrial procaspase-3 is constitu- sensitive factor and reduces the speed of protein transport from tively S-nitrosylated whereas the cytosolic form is not (5). (vi)OxyR the endoplasmic reticulum to the plasma membrane in a reversible is S-nitrosylated more readily by S-nitrosoglutathione (GSNO) than manner. These data indicate that local NOS action generates S-nitrosocysteine (CysNO) (18), whereas the opposite is true for organelle-specific protein S-nitrosylation reactions that can regu- hemoglobin (19). GSNO (but not CysNO) binds directly to OxyR, late intracellular transport processes. whereas it is too large to access the buried Cys in hemoglobin. In comparison to the actions of NO-regulating metal-centered endothelial nitric oxide synthase ͉ Golgi ͉ targeting processes (activation of soluble guanylyl cyclase and inhibition of cytochrome oxidase) or its interaction with other radicals, thiol itric oxide (NO), produced by the nitric oxide synthase (NOS) modification via S-nitrosylation is thought to require higher con- Nfamily of proteins, regulates a variety of important physiolog- centrations of NO among the primary biological reactions with NO ical responses, including vasodilation, respiration, cell migration, (20) to sustain protein S-nitrosylation in vitro (21). Therefore, the and apoptosis (1–5). NO has been considered to mediate these flux of NO generated by NOS may regulate specific cellular responses by activating the primary NO effector soluble guanylyl functions despite the diffusible and short-lived properties of NO; cyclase to produce cGMP (6) or by NO-based chemical modifica- however, this has never been directly demonstrated. tions of proteins or perhaps lipids. One clear example of cGMP- eNOS is unique among the NOS family members as it is localized independent actions of NO is protein thiol group modification by mainly to specific intracellular membrane domains, including the NO known as S-nitrosylation (7). This posttranslational control cytoplasmic aspect of the Golgi apparatus and plasma membrane mechanism regulates important physiological activities of proteins caveolae (22–25). Particularly, on the Golgi, it is shown that eNOS in response to endogenously or exogenously generated NO (8). is colocalized with well known Golgi proteins, such as Golgi matrix Thus, S-nitrosylation of proteins is an emerging area of investigation protein 130 (GM130), 53K Golgi protein, and mannosidase II for NO-mediated physiological responses (8). (22–24, 26). Previously, we hypothesized that a NO pool is formed NO is a lipophillic, highly diffusible, and short-lived physiological around and within the Golgi and may create a favorable environ- messenger (9). On the one hand, NO is thought to diffuse over a ␮ wide area (100 m), moving freely through membranes of neigh- Author contributions: Y.I., C.M.C., and R.J.G. designed research; Y.I., A.S., S.C., and C.M.C. boring cells (9). On the other hand, given the apparent promiscuity performed research; D.K.T., D.F., and V.H.S. contributed new reagents/analytic tools; Y.I., of NO, the question arises as to how S-nitrosylation might occur in S.C., D.K.T., C.M.C., V.H.S., and W.C.S. analyzed data; and Y.I., R.J.G., V.H.S., and W.C.S. a precisely regulated manner, i.e., protein S-nitrosylation occurs on wrote the paper. specific thiol residues in proteins that are targeted to specific The authors declare no conflict of interest. organelles in cells (10), and low concentrations of NO activate the This article is a PNAS direct submission. ryanodine receptor via thiol modification, whereas higher concen- Abbreviations: NOS, nitric oxide synthase; eNOS, endothelial NOS; GSNO, S-nitrosogluta- trations inhibit the receptor (11). There are several compelling thione; GSNOR, GSNO reductase; NLS, nuclear localization signal; RFP, red fluorescent arguments in favor of the concept that all sources of NO are not protein; DAF, diaminofluorescein; DAF-2T, DAF-2 triazole; DAF-2DA, 4-amino-5-methyl- amino-2Ј,7Јdifluorescein; SNAP, S-nitroso-N-acetyl-D-L-penicillamine; L-NAME, L-nitroargi- bioequivalent. (i) In cardiac myocytes, which express two forms of nine methylester; VSVG, vesicular stomatitis virus glycoprotein; ER, endoplasmic reticulum; NOS, gene deletion of either neuronal NOS or endothelial NOS NSF, N-ethylmaleimide-sensitive factor. (eNOS) exerts isoform-specific phenotypes, arguing that the source ††To whom correspondence should be addressed. E-mail: [email protected]. of NO favors local control of different cellular functions (12). This article contains supporting information online at www.pnas.org/cgi/content/full/ Moreover, in many circumstances, the actions of endogenously 0605907103/DC1. generated NO from NOS cannot be recapitulated by drugs liber- © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605907103 PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19777–19782 Downloaded by guest on September 30, 2021 Fig. 2. Kinetics of DAF-2DA (an intracellular NO indicator) in response to Fig. 1. The functional analysis of eNOS constructs. (A) Subcellular localiza- exogenous and endogenous NO in living cells. The y axis of each graph is relative tion of eNOS in COS cells. eNOS constructs fused with RFP include: WT-eNOS- fluorescent intensity (RFI) of DAF-2 fluorescence. (A) NO donor, NONOate, in- RFP, which is WT eNOS that primarily localizes at the Golgi apparatus, and creased DAF-2 fluorescence in living cells. Images of DAF-2 taken every minute ␮ RFP-eNOS-NLS to target the nucleus. (Scale bar: 10 m.) (B) eNOS expression after COS-7 cells were loaded with DAF-2DA. The fluorescent intensity of DAF-2T of eNOS constructs. (C) NO production in COS-7 cells transfected with eNOS was measured after the dye was loaded. The average Ϯ SEM in each time point constructs. Forty-eight hours after the transfection of eNOS constructs or was obtained from three independent experiments. (B) An inhibitor of NOS control plasmid (RFP only), the media were processed for the measurement of (L-NAME) blocked DAF-2 fluorescence in cells that expresses WT-eNOS-RFP. Be- nitrite, a stable breakdown product of NO in aqueous solution by chemilu- fore being stimulated with ATP (100 ␮M) for NO production, cells were treated minescence. (Left) NO release from samples collected from basal (4 h) accu- with or without L-NAME (100 ␮M). A stock solution of L-NAME (100 mM) was mulation of nitrite in serum-containing media is shown. The same cells were prepared in water and diluted in culture medium (1:1,000) to have a final then incubated with serum-free DMEM for 6 h and the fresh media containing concentration of 100 ␮M. The treatment of cells with ATP resulted in DAF-2T agonist ATP (100 ␮M) for 30 min. (Right) NO release as a result of ATP fluorescence (Upper), which was blocked by L-NAME (Lower). Control RFI values stimulation is shown. The cells were lysed to determine the total protein were obtained from COS cells expressing WT-eNOS-RFP treated without a pres- concentration and evaluate equal eNOS expression by Western blot. Data are ence of L-NAME. * and **, P Ͻ 0.05. (Scale bars: 100 ␮m.) presented as means Ϯ SEM; n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.05. ing eNOS to nucleus reduces NOS activation presumably because ment for local S-nitrosylation of proteins in living cells (1–5). Thus, of insufficient access to calcium/calmodulin and impaired phos- the purpose of this study is to examine local NO production and phorylation (28).