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Binding of Neuronal Nitric-oxide Synthase (nNOS) to Carboxyl-terminal-binding Protein (CtBP) Changes the Localization of CtBP from the Nucleus to the Cytosol

2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês

10.1074/jbc.m106503200

1083-351X

Gary M. Riefler, Bonnie L. Firestein,

Nuclear Receptors and Signaling

Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DX L, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization. Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DX L, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization. nitric oxide carboxyl-terminal-binding protein glutathioneS-transferase neuronal nitric-oxide synthase postsynaptic density Madin-Darby canine kidney bovine serum albumin Nitric oxide (NO)1 is a gaseous molecule that plays an important role in the central nervous system (1Christopherson K.S. Bredt D.S. J. Clin. Invest. 1997; 100: 2424-2429Crossref PubMed Scopus (273) Google Scholar, 2Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1532) Google Scholar). It is produced by the enzyme neuronal nitric-oxide synthase (nNOS), which can be stimulated when glutamate receptors of the N-methyl-d-aspartate receptor family are activated, and calcium enters the neuron (2Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1532) Google Scholar). Calcium binds to calmodulin, and the complex then activates nNOS. Furthermore, the NO produced can increase the production of 3′,5′-cyclic guanosine monophosphate levels, which can activate other enzymes including protein kinases (3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1494) Google Scholar, 4Jaffrey S.R. Snyder S.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 417-440Crossref PubMed Scopus (304) Google Scholar). Recent evidence supports a role for NO in synaptic plasticity (5Son H. Hawkins R.D. Martin K. Kiebler M. Huang P.L. Fishman M.C. Kandel E.R. Cell. 1996; 87: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), long-term potentiation (6Hawkins R.D. Neuron. 1996; 16: 465-467Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and aspects of learning and memory (7Kendrick K.M. Guevara-Guzman R. Zorrilla J. Hinton M.R. Broad K.D. Mimmack M. Ohkura S. Nature. 1997; 388: 670-674Crossref PubMed Scopus (226) Google Scholar).nNOS contains a PDZ (P SD-95, d iscs-large and z ona occludens-1) domain, a consensus sequence of approximately 90 amino acids that has been shown to mediate protein-protein interactions (8Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). In neurons, nNOS is targeted to synaptic sites via its interaction with the PDZ domains of PSD-95 and PSD-93 (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). In fact, PSD-95 also interacts withN-methyl-d-aspartate receptors via one of its PDZ domains. Through its concurrent interaction with nNOS, PSD-95 serves as a physical tether to allow nNOS signaling byN-methyl-d-aspartate receptor activity (10Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1619) Google Scholar, 11Kim E. Cho K.O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). By abolishing expression of PSD-95 protein either by knockout technology in mice or by antisense technology in tissue culture, it has been shown that the presence of PSD-95 is essential for NO production by glutamate stimulation (12Sattler R. Xiong Z. Lu W.Y. Hafner M. MacDonald J.F. Tymianski M. Science. 1999; 284: 1845-1848Crossref PubMed Scopus (709) Google Scholar, 13Migaud M. Charlesworth P. Dempster M. Webster L.C. Watabe A.M. Makhinson M. He Y. Ramsay M.F. Morris R.G. Morrison J.H. O'Dell T.J. Grant S.G. Nature. 1998; 396: 433-439Crossref PubMed Scopus (952) Google Scholar). Thus, the PDZ domain of nNOS plays an important role in helping to localize nNOS to appropriate sites in the neuron.Subcellular fractionation shows that roughly half of nNOS is soluble, and half is particulate (14Hecker M. Mulsch A. Busse R. J. Neurochem. 1994; 62: 1524-1529Crossref PubMed Scopus (137) Google Scholar). As such, binding partners of the PDZ domain of nNOS can serve to regulate nNOS localization in the cell. In fact, it has been shown that the soluble CAPON (ca rboxy-terminal P DZ ligand of n NOS) competes with PSD-95 and PSD-93 for binding of nNOS and thus may participate in localization of nNOS to the cytoplasm (15Jaffrey S.R. Snowman A.M. Eliasson M.J. Cohen N.A. Snyder S.H. Neuron. 1998; 20: 115-124Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Furthermore, phosphofructokinase binds to the PDZ domain of nNOS and co-localizes with nNOS in the cytosol of inhibitory interneurons in the hippocampus (16Firestein B.L. Bredt D.S. J. Biol. Chem. 1999; 274: 10545-10550Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The binding of phosphofructokinase to nNOS does not alter the enzymatic activity of either phosphofructokinase or nNOS (16Firestein B.L. Bredt D.S. J. Biol. Chem. 1999; 274: 10545-10550Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); thus, the possibility exists that the binding of these two enzymes is important for subcellular localization.Based on the fact that the PDZ domain of nNOS is important for protein localization, we tested whether other protein complexes may bind to this domain. To address this question, we performed affinity chromatography using a GST fusion protein column containing the PDZ domain of nNOS. Purification of brain lysates on the column yielded a protein of 48 kDa, which was identified as the carboxyl-terminal-binding protein (CtBP), a protein originally identified as a phosphoprotein that interacts with the adenovirus E1A protein (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar) and acts as a transcriptional co-repressor (18Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (166) Google Scholar, 19Postigo A.A. Dean D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6683-6688Crossref PubMed Scopus (223) Google Scholar). Endogenous CtBP associates with nNOS in the brain and binds to the peptide-binding site of the nNOS PDZ domain via a consensus sequence at its carboxyl terminus. At the cellular level, CtBP protein is expressed in the nucleus, whereas when CtBP is co-expressed with nNOS, it relocalizes to the cytosol. Furthermore, this translocation is dependent on nNOS binding. Taken together, these results suggest a novel function for a PDZ domain-containing protein, namely nNOS, in regulation of the localization of a transcriptional co-repressor to the cytosol.DISCUSSIONThe primary finding of this study is that nNOS associates with the transcription factor CtBP. CtBP specifically binds to the peptide-binding site in the PDZ domain of nNOS because its binding can be competed with the peptide VSPDFGDAV. The carboxyl terminus of CtBP contains a consensus sequence shown to bind to the peptide-binding site, and mutation of crucial amino acids at the carboxyl terminus of CtBP abolishes its binding to nNOS. Furthermore, CtBP and nNOS associate in vivo as evidenced by the co-immunoprecipitation of nNOS by incubation of brain extract with an antibody raised against CtBP. Most exciting is the fact that the binding of CtBP to nNOS results in the change in localization of CtBP from the nucleus to the cytosol.CtBP was originally isolated as a cellular phosphoprotein that interacts with the carboxyl terminus of the adenovirus protein E1A that negatively regulates oncogenic transformation (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar). Most work has focused on the role of CtBP as a short range repressor, although little is known about the mechanism by which CtBP acts (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar). CtBP can interact with a subset of transcription factors including theDrosophila Knirps (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar) and Hairy (25Poortinga G. Watanabe M. Parkhurst S.M. EMBO J. 1998; 17: 2067-2078Crossref PubMed Scopus (206) Google Scholar), the vertebrate CtIP (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar, 26Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), and the transcription factor MEF2. It is thought that CtBP functions as a co-repressor protein that can recruit histone deacetylase 1 (27Sundqvist A. Sollerbrant K. Svensson C. FEBS Lett. 1998; 429: 183-188Crossref PubMed Scopus (89) Google Scholar) or DNA-binding proteins, which can inhibit transcription. The localization of CtBP to the cytosol by nNOS may make CtBP unavailable to interact with these nuclear proteins (i.e. histone deacetylase and transcription factors), thereby hindering the function of CtBP as a co-repressor. Perhaps, by binding CtBP, nNOS helps to maintain homeostasis of transcriptional repression and activation.In addition, CtBP has been shown to have a role in Golgi maintenance (28Weigert R. Silletta M.G. Spano S. Turacchio G. Cericola C. Colanzi A. Senatore S. Mancini R. Polishchuk E.V. Salmona M. Facchiano F. Burger K.N. Mironov A. Luini A. Corda D. Nature. 1999; 402: 429-433Crossref PubMed Scopus (277) Google Scholar). CtBP is ribosylated by brefeldin A, and this ribosylation leads to Golgi disassembly. It is believed that this function of CtBP is independent of its function as a transcriptional co-repressor. By localizing CtBP to the cytosol, nNOS may make CtBP available to perform a cellular function independent of co-repression.Until now, reports have suggested that the PDZ domain of nNOS serves to regulate the localization of nNOS protein. Interactions with other proteins containing PDZ domains function to target nNOS to noncytosolic sites. For example, in brain, nNOS binding to PSD-95 localizes nNOS to the postsynaptic density (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). Similarly, in skeletal muscle, association of nNOS with α1-syntrophin localizes nNOS to the sarcolemma (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar, 29Brenman J.E. Chao D.S. Xia H. Aldape K. Bredt D.S. Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (840) Google Scholar). In contrast, proteins that contain carboxyl-terminal motifs that bind to the PDZ domain of nNOS often target nNOS to the cytosol or inhibit nNOS activity. For example, when CAPON binds to nNOS, nNOS is not targeted to postsynaptic sites in the brain; instead, it is localized to the cytosol (15Jaffrey S.R. Snowman A.M. Eliasson M.J. Cohen N.A. Snyder S.H. Neuron. 1998; 20: 115-124Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). PIN (p rotein i nhibitor of n NOS) associates with nNOS in the cytosol and inhibits nNOS activity (30Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (423) Google Scholar, 31Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Interestingly, we found no effect on nNOS activity when we added purified GST-CtBP to brain extract. 2B. L. Firestein, unpublished observations. Thus, we believe that the binding of CtBP to nNOS plays a role in protein localization rather than regulation of enzymatic activity, suggesting a novel role for nNOS as a targeting molecule.Recently, there have been a number of reports describing the role of PDZ domain-containing proteins in localization to the nucleus. For example, the PDZ-containing protein zona occludens-1 shuttles between tight junctions and the nucleus in epithelial cells and is thought to regulate gene expression (32Gonzalez-Mariscal L. Islas S. Contreras R.G. Garcia-Villegas M.R. Betanzos A. Vega J. Diaz-Quinonez A. Martin-Orozco N. Ortiz-Navarrete V. Cereijido M. Valdes J. Exp. Cell Res. 1999; 248: 97-109Crossref PubMed Scopus (42) Google Scholar). When it is localized to the nucleus, the SH3 domain of zona occludens-1 binds to a nucleic acid-binding protein known as ZONAB, and together zona occludens-1 and ZONAB bind to the Erb-2 promoter and regulate Erb-2 transcription (33Balda M.S. Matter K. EMBO J. 2000; 19: 2024-2033Crossref PubMed Scopus (352) Google Scholar). Similarly, CASK/LIN-2, a multi-PDZ-containing protein, can translocate from the cytosol to the nucleus and bind to Tbr-1, a T-box transcription factor involved in brain development (34Hsueh Y.P. Wang T.F. Yang F.C. Sheng M. Nature. 2000; 404: 298-302Crossref PubMed Scopus (285) Google Scholar). The binding of the guanylate kinase domain of CASK to Tbr-1 induces gene transcription. Thus, evidence is beginning to emerge to support a role for PDZ-containing proteins in transcription factor activity. Our data support the idea that nNOS protein is present in the nucleus; however, unlike the other PDZ proteins mentioned above, nNOS may traffic nuclear proteins to the cytosol, thereby making them inaccessible to regulate transcription. Furthermore, targeting of proteins by nNOS occurs via its PDZ domain, unlike the proteins described above, which contain PDZ domains but regulate nuclear events via other domains.There are two possible mechanisms by which nNOS may target proteins from the nucleus. Because a majority of nNOS protein is found in the cytosol, nNOS may act to "trap" nuclear proteins in the cytosol once they diffuse or are transported from the nucleus. Alternatively, nNOS may bind nuclear proteins and actively transport them from the nucleus to the cytosol. In line with this mechanism, we find some nNOS protein, although only a small amount, in the nucleus (Fig.6 A). In addition, this localized nNOS associates with CtBP in brain nuclei (Fig. 6 B). nNOS contains putative nuclear export sequences; however, treatment of cells with leptomycin B, a drug that inhibits crm-1-mediated nuclear transport, has no effect on nNOS protein localization. 3G. M. Riefler and B. L. Firestein, unpublished observations. This suggests that nNOS does not shuttle from the nucleus to cytosol by binding crm-1. Future studies will serve to identify which mechanism plays a role in the targeting of nuclear proteins by nNOS.Thus, we believe that we have found a novel function for nNOS as a regulator of nuclear protein localization. Although other forms of nitric-oxide synthase, such as endothelial and inducible nitric-oxide synthase, also produce NO, nNOS is the only isoform that contains a PDZ domain. Furthermore, nNOS is expressed at the highest levels in brain and skeletal muscle, with very little expression in other tissues (35Park C.S. Krishna G. Ahn M.S. Kang J.H. Chung W.G. Kim D.J. Hwang H.K. Lee J.N. Paik S.G. Cha Y.N. Nitric Oxide. 2000; 4: 459-471Crossref PubMed Scopus (46) Google Scholar). As such, the PDZ of nNOS may play a novel role in nNOS function in these two tissues. Nitric oxide (NO)1 is a gaseous molecule that plays an important role in the central nervous system (1Christopherson K.S. Bredt D.S. J. Clin. Invest. 1997; 100: 2424-2429Crossref PubMed Scopus (273) Google Scholar, 2Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1532) Google Scholar). It is produced by the enzyme neuronal nitric-oxide synthase (nNOS), which can be stimulated when glutamate receptors of the N-methyl-d-aspartate receptor family are activated, and calcium enters the neuron (2Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1532) Google Scholar). Calcium binds to calmodulin, and the complex then activates nNOS. Furthermore, the NO produced can increase the production of 3′,5′-cyclic guanosine monophosphate levels, which can activate other enzymes including protein kinases (3Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1494) Google Scholar, 4Jaffrey S.R. Snyder S.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 417-440Crossref PubMed Scopus (304) Google Scholar). Recent evidence supports a role for NO in synaptic plasticity (5Son H. Hawkins R.D. Martin K. Kiebler M. Huang P.L. Fishman M.C. Kandel E.R. Cell. 1996; 87: 1015-1023Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), long-term potentiation (6Hawkins R.D. Neuron. 1996; 16: 465-467Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and aspects of learning and memory (7Kendrick K.M. Guevara-Guzman R. Zorrilla J. Hinton M.R. Broad K.D. Mimmack M. Ohkura S. Nature. 1997; 388: 670-674Crossref PubMed Scopus (226) Google Scholar). nNOS contains a PDZ (P SD-95, d iscs-large and z ona occludens-1) domain, a consensus sequence of approximately 90 amino acids that has been shown to mediate protein-protein interactions (8Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). In neurons, nNOS is targeted to synaptic sites via its interaction with the PDZ domains of PSD-95 and PSD-93 (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). In fact, PSD-95 also interacts withN-methyl-d-aspartate receptors via one of its PDZ domains. Through its concurrent interaction with nNOS, PSD-95 serves as a physical tether to allow nNOS signaling byN-methyl-d-aspartate receptor activity (10Kornau H.C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1619) Google Scholar, 11Kim E. Cho K.O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). By abolishing expression of PSD-95 protein either by knockout technology in mice or by antisense technology in tissue culture, it has been shown that the presence of PSD-95 is essential for NO production by glutamate stimulation (12Sattler R. Xiong Z. Lu W.Y. Hafner M. MacDonald J.F. Tymianski M. Science. 1999; 284: 1845-1848Crossref PubMed Scopus (709) Google Scholar, 13Migaud M. Charlesworth P. Dempster M. Webster L.C. Watabe A.M. Makhinson M. He Y. Ramsay M.F. Morris R.G. Morrison J.H. O'Dell T.J. Grant S.G. Nature. 1998; 396: 433-439Crossref PubMed Scopus (952) Google Scholar). Thus, the PDZ domain of nNOS plays an important role in helping to localize nNOS to appropriate sites in the neuron. Subcellular fractionation shows that roughly half of nNOS is soluble, and half is particulate (14Hecker M. Mulsch A. Busse R. J. Neurochem. 1994; 62: 1524-1529Crossref PubMed Scopus (137) Google Scholar). As such, binding partners of the PDZ domain of nNOS can serve to regulate nNOS localization in the cell. In fact, it has been shown that the soluble CAPON (ca rboxy-terminal P DZ ligand of n NOS) competes with PSD-95 and PSD-93 for binding of nNOS and thus may participate in localization of nNOS to the cytoplasm (15Jaffrey S.R. Snowman A.M. Eliasson M.J. Cohen N.A. Snyder S.H. Neuron. 1998; 20: 115-124Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Furthermore, phosphofructokinase binds to the PDZ domain of nNOS and co-localizes with nNOS in the cytosol of inhibitory interneurons in the hippocampus (16Firestein B.L. Bredt D.S. J. Biol. Chem. 1999; 274: 10545-10550Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The binding of phosphofructokinase to nNOS does not alter the enzymatic activity of either phosphofructokinase or nNOS (16Firestein B.L. Bredt D.S. J. Biol. Chem. 1999; 274: 10545-10550Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); thus, the possibility exists that the binding of these two enzymes is important for subcellular localization. Based on the fact that the PDZ domain of nNOS is important for protein localization, we tested whether other protein complexes may bind to this domain. To address this question, we performed affinity chromatography using a GST fusion protein column containing the PDZ domain of nNOS. Purification of brain lysates on the column yielded a protein of 48 kDa, which was identified as the carboxyl-terminal-binding protein (CtBP), a protein originally identified as a phosphoprotein that interacts with the adenovirus E1A protein (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar) and acts as a transcriptional co-repressor (18Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (166) Google Scholar, 19Postigo A.A. Dean D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6683-6688Crossref PubMed Scopus (223) Google Scholar). Endogenous CtBP associates with nNOS in the brain and binds to the peptide-binding site of the nNOS PDZ domain via a consensus sequence at its carboxyl terminus. At the cellular level, CtBP protein is expressed in the nucleus, whereas when CtBP is co-expressed with nNOS, it relocalizes to the cytosol. Furthermore, this translocation is dependent on nNOS binding. Taken together, these results suggest a novel function for a PDZ domain-containing protein, namely nNOS, in regulation of the localization of a transcriptional co-repressor to the cytosol. DISCUSSIONThe primary finding of this study is that nNOS associates with the transcription factor CtBP. CtBP specifically binds to the peptide-binding site in the PDZ domain of nNOS because its binding can be competed with the peptide VSPDFGDAV. The carboxyl terminus of CtBP contains a consensus sequence shown to bind to the peptide-binding site, and mutation of crucial amino acids at the carboxyl terminus of CtBP abolishes its binding to nNOS. Furthermore, CtBP and nNOS associate in vivo as evidenced by the co-immunoprecipitation of nNOS by incubation of brain extract with an antibody raised against CtBP. Most exciting is the fact that the binding of CtBP to nNOS results in the change in localization of CtBP from the nucleus to the cytosol.CtBP was originally isolated as a cellular phosphoprotein that interacts with the carboxyl terminus of the adenovirus protein E1A that negatively regulates oncogenic transformation (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar). Most work has focused on the role of CtBP as a short range repressor, although little is known about the mechanism by which CtBP acts (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar). CtBP can interact with a subset of transcription factors including theDrosophila Knirps (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar) and Hairy (25Poortinga G. Watanabe M. Parkhurst S.M. EMBO J. 1998; 17: 2067-2078Crossref PubMed Scopus (206) Google Scholar), the vertebrate CtIP (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar, 26Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), and the transcription factor MEF2. It is thought that CtBP functions as a co-repressor protein that can recruit histone deacetylase 1 (27Sundqvist A. Sollerbrant K. Svensson C. FEBS Lett. 1998; 429: 183-188Crossref PubMed Scopus (89) Google Scholar) or DNA-binding proteins, which can inhibit transcription. The localization of CtBP to the cytosol by nNOS may make CtBP unavailable to interact with these nuclear proteins (i.e. histone deacetylase and transcription factors), thereby hindering the function of CtBP as a co-repressor. Perhaps, by binding CtBP, nNOS helps to maintain homeostasis of transcriptional repression and activation.In addition, CtBP has been shown to have a role in Golgi maintenance (28Weigert R. Silletta M.G. Spano S. Turacchio G. Cericola C. Colanzi A. Senatore S. Mancini R. Polishchuk E.V. Salmona M. Facchiano F. Burger K.N. Mironov A. Luini A. Corda D. Nature. 1999; 402: 429-433Crossref PubMed Scopus (277) Google Scholar). CtBP is ribosylated by brefeldin A, and this ribosylation leads to Golgi disassembly. It is believed that this function of CtBP is independent of its function as a transcriptional co-repressor. By localizing CtBP to the cytosol, nNOS may make CtBP available to perform a cellular function independent of co-repression.Until now, reports have suggested that the PDZ domain of nNOS serves to regulate the localization of nNOS protein. Interactions with other proteins containing PDZ domains function to target nNOS to noncytosolic sites. For example, in brain, nNOS binding to PSD-95 localizes nNOS to the postsynaptic density (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). Similarly, in skeletal muscle, association of nNOS with α1-syntrophin localizes nNOS to the sarcolemma (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar, 29Brenman J.E. Chao D.S. Xia H. Aldape K. Bredt D.S. Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (840) Google Scholar). In contrast, proteins that contain carboxyl-terminal motifs that bind to the PDZ domain of nNOS often target nNOS to the cytosol or inhibit nNOS activity. For example, when CAPON binds to nNOS, nNOS is not targeted to postsynaptic sites in the brain; instead, it is localized to the cytosol (15Jaffrey S.R. Snowman A.M. Eliasson M.J. Cohen N.A. Snyder S.H. Neuron. 1998; 20: 115-124Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). PIN (p rotein i nhibitor of n NOS) associates with nNOS in the cytosol and inhibits nNOS activity (30Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (423) Google Scholar, 31Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Interestingly, we found no effect on nNOS activity when we added purified GST-CtBP to brain extract. 2B. L. Firestein, unpublished observations. Thus, we believe that the binding of CtBP to nNOS plays a role in protein localization rather than regulation of enzymatic activity, suggesting a novel role for nNOS as a targeting molecule.Recently, there have been a number of reports describing the role of PDZ domain-containing proteins in localization to the nucleus. For example, the PDZ-containing protein zona occludens-1 shuttles between tight junctions and the nucleus in epithelial cells and is thought to regulate gene expression (32Gonzalez-Mariscal L. Islas S. Contreras R.G. Garcia-Villegas M.R. Betanzos A. Vega J. Diaz-Quinonez A. Martin-Orozco N. Ortiz-Navarrete V. Cereijido M. Valdes J. Exp. Cell Res. 1999; 248: 97-109Crossref PubMed Scopus (42) Google Scholar). When it is localized to the nucleus, the SH3 domain of zona occludens-1 binds to a nucleic acid-binding protein known as ZONAB, and together zona occludens-1 and ZONAB bind to the Erb-2 promoter and regulate Erb-2 transcription (33Balda M.S. Matter K. EMBO J. 2000; 19: 2024-2033Crossref PubMed Scopus (352) Google Scholar). Similarly, CASK/LIN-2, a multi-PDZ-containing protein, can translocate from the cytosol to the nucleus and bind to Tbr-1, a T-box transcription factor involved in brain development (34Hsueh Y.P. Wang T.F. Yang F.C. Sheng M. Nature. 2000; 404: 298-302Crossref PubMed Scopus (285) Google Scholar). The binding of the guanylate kinase domain of CASK to Tbr-1 induces gene transcription. Thus, evidence is beginning to emerge to support a role for PDZ-containing proteins in transcription factor activity. Our data support the idea that nNOS protein is present in the nucleus; however, unlike the other PDZ proteins mentioned above, nNOS may traffic nuclear proteins to the cytosol, thereby making them inaccessible to regulate transcription. Furthermore, targeting of proteins by nNOS occurs via its PDZ domain, unlike the proteins described above, which contain PDZ domains but regulate nuclear events via other domains.There are two possible mechanisms by which nNOS may target proteins from the nucleus. Because a majority of nNOS protein is found in the cytosol, nNOS may act to "trap" nuclear proteins in the cytosol once they diffuse or are transported from the nucleus. Alternatively, nNOS may bind nuclear proteins and actively transport them from the nucleus to the cytosol. In line with this mechanism, we find some nNOS protein, although only a small amount, in the nucleus (Fig.6 A). In addition, this localized nNOS associates with CtBP in brain nuclei (Fig. 6 B). nNOS contains putative nuclear export sequences; however, treatment of cells with leptomycin B, a drug that inhibits crm-1-mediated nuclear transport, has no effect on nNOS protein localization. 3G. M. Riefler and B. L. Firestein, unpublished observations. This suggests that nNOS does not shuttle from the nucleus to cytosol by binding crm-1. Future studies will serve to identify which mechanism plays a role in the targeting of nuclear proteins by nNOS.Thus, we believe that we have found a novel function for nNOS as a regulator of nuclear protein localization. Although other forms of nitric-oxide synthase, such as endothelial and inducible nitric-oxide synthase, also produce NO, nNOS is the only isoform that contains a PDZ domain. Furthermore, nNOS is expressed at the highest levels in brain and skeletal muscle, with very little expression in other tissues (35Park C.S. Krishna G. Ahn M.S. Kang J.H. Chung W.G. Kim D.J. Hwang H.K. Lee J.N. Paik S.G. Cha Y.N. Nitric Oxide. 2000; 4: 459-471Crossref PubMed Scopus (46) Google Scholar). As such, the PDZ of nNOS may play a novel role in nNOS function in these two tissues. The primary finding of this study is that nNOS associates with the transcription factor CtBP. CtBP specifically binds to the peptide-binding site in the PDZ domain of nNOS because its binding can be competed with the peptide VSPDFGDAV. The carboxyl terminus of CtBP contains a consensus sequence shown to bind to the peptide-binding site, and mutation of crucial amino acids at the carboxyl terminus of CtBP abolishes its binding to nNOS. Furthermore, CtBP and nNOS associate in vivo as evidenced by the co-immunoprecipitation of nNOS by incubation of brain extract with an antibody raised against CtBP. Most exciting is the fact that the binding of CtBP to nNOS results in the change in localization of CtBP from the nucleus to the cytosol. CtBP was originally isolated as a cellular phosphoprotein that interacts with the carboxyl terminus of the adenovirus protein E1A that negatively regulates oncogenic transformation (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar). Most work has focused on the role of CtBP as a short range repressor, although little is known about the mechanism by which CtBP acts (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar). CtBP can interact with a subset of transcription factors including theDrosophila Knirps (24Nibu Y. Zhang H. Bajor E. Barolo S. Small S. Levine M. EMBO J. 1998; 17: 7009-7020Crossref PubMed Scopus (170) Google Scholar) and Hairy (25Poortinga G. Watanabe M. Parkhurst S.M. EMBO J. 1998; 17: 2067-2078Crossref PubMed Scopus (206) Google Scholar), the vertebrate CtIP (17Schaeper U. Boyd J.M. Verma S. Uhlmann E. Subramanian T. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10467-10471Crossref PubMed Scopus (308) Google Scholar, 26Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), and the transcription factor MEF2. It is thought that CtBP functions as a co-repressor protein that can recruit histone deacetylase 1 (27Sundqvist A. Sollerbrant K. Svensson C. FEBS Lett. 1998; 429: 183-188Crossref PubMed Scopus (89) Google Scholar) or DNA-binding proteins, which can inhibit transcription. The localization of CtBP to the cytosol by nNOS may make CtBP unavailable to interact with these nuclear proteins (i.e. histone deacetylase and transcription factors), thereby hindering the function of CtBP as a co-repressor. Perhaps, by binding CtBP, nNOS helps to maintain homeostasis of transcriptional repression and activation. In addition, CtBP has been shown to have a role in Golgi maintenance (28Weigert R. Silletta M.G. Spano S. Turacchio G. Cericola C. Colanzi A. Senatore S. Mancini R. Polishchuk E.V. Salmona M. Facchiano F. Burger K.N. Mironov A. Luini A. Corda D. Nature. 1999; 402: 429-433Crossref PubMed Scopus (277) Google Scholar). CtBP is ribosylated by brefeldin A, and this ribosylation leads to Golgi disassembly. It is believed that this function of CtBP is independent of its function as a transcriptional co-repressor. By localizing CtBP to the cytosol, nNOS may make CtBP available to perform a cellular function independent of co-repression. Until now, reports have suggested that the PDZ domain of nNOS serves to regulate the localization of nNOS protein. Interactions with other proteins containing PDZ domains function to target nNOS to noncytosolic sites. For example, in brain, nNOS binding to PSD-95 localizes nNOS to the postsynaptic density (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar). Similarly, in skeletal muscle, association of nNOS with α1-syntrophin localizes nNOS to the sarcolemma (9Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 757-767Abstract Full Text Full Text PDF PubMed Scopus (1433) Google Scholar, 29Brenman J.E. Chao D.S. Xia H. Aldape K. Bredt D.S. Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (840) Google Scholar). In contrast, proteins that contain carboxyl-terminal motifs that bind to the PDZ domain of nNOS often target nNOS to the cytosol or inhibit nNOS activity. For example, when CAPON binds to nNOS, nNOS is not targeted to postsynaptic sites in the brain; instead, it is localized to the cytosol (15Jaffrey S.R. Snowman A.M. Eliasson M.J. Cohen N.A. Snyder S.H. Neuron. 1998; 20: 115-124Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). PIN (p rotein i nhibitor of n NOS) associates with nNOS in the cytosol and inhibits nNOS activity (30Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (423) Google Scholar, 31Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Interestingly, we found no effect on nNOS activity when we added purified GST-CtBP to brain extract. 2B. L. Firestein, unpublished observations. Thus, we believe that the binding of CtBP to nNOS plays a role in protein localization rather than regulation of enzymatic activity, suggesting a novel role for nNOS as a targeting molecule. Recently, there have been a number of reports describing the role of PDZ domain-containing proteins in localization to the nucleus. For example, the PDZ-containing protein zona occludens-1 shuttles between tight junctions and the nucleus in epithelial cells and is thought to regulate gene expression (32Gonzalez-Mariscal L. Islas S. Contreras R.G. Garcia-Villegas M.R. Betanzos A. Vega J. Diaz-Quinonez A. Martin-Orozco N. Ortiz-Navarrete V. Cereijido M. Valdes J. Exp. Cell Res. 1999; 248: 97-109Crossref PubMed Scopus (42) Google Scholar). When it is localized to the nucleus, the SH3 domain of zona occludens-1 binds to a nucleic acid-binding protein known as ZONAB, and together zona occludens-1 and ZONAB bind to the Erb-2 promoter and regulate Erb-2 transcription (33Balda M.S. Matter K. EMBO J. 2000; 19: 2024-2033Crossref PubMed Scopus (352) Google Scholar). Similarly, CASK/LIN-2, a multi-PDZ-containing protein, can translocate from the cytosol to the nucleus and bind to Tbr-1, a T-box transcription factor involved in brain development (34Hsueh Y.P. Wang T.F. Yang F.C. Sheng M. Nature. 2000; 404: 298-302Crossref PubMed Scopus (285) Google Scholar). The binding of the guanylate kinase domain of CASK to Tbr-1 induces gene transcription. Thus, evidence is beginning to emerge to support a role for PDZ-containing proteins in transcription factor activity. Our data support the idea that nNOS protein is present in the nucleus; however, unlike the other PDZ proteins mentioned above, nNOS may traffic nuclear proteins to the cytosol, thereby making them inaccessible to regulate transcription. Furthermore, targeting of proteins by nNOS occurs via its PDZ domain, unlike the proteins described above, which contain PDZ domains but regulate nuclear events via other domains. There are two possible mechanisms by which nNOS may target proteins from the nucleus. Because a majority of nNOS protein is found in the cytosol, nNOS may act to "trap" nuclear proteins in the cytosol once they diffuse or are transported from the nucleus. Alternatively, nNOS may bind nuclear proteins and actively transport them from the nucleus to the cytosol. In line with this mechanism, we find some nNOS protein, although only a small amount, in the nucleus (Fig.6 A). In addition, this localized nNOS associates with CtBP in brain nuclei (Fig. 6 B). nNOS contains putative nuclear export sequences; however, treatment of cells with leptomycin B, a drug that inhibits crm-1-mediated nuclear transport, has no effect on nNOS protein localization. 3G. M. Riefler and B. L. Firestein, unpublished observations. This suggests that nNOS does not shuttle from the nucleus to cytosol by binding crm-1. Future studies will serve to identify which mechanism plays a role in the targeting of nuclear proteins by nNOS. Thus, we believe that we have found a novel function for nNOS as a regulator of nuclear protein localization. Although other forms of nitric-oxide synthase, such as endothelial and inducible nitric-oxide synthase, also produce NO, nNOS is the only isoform that contains a PDZ domain. Furthermore, nNOS is expressed at the highest levels in brain and skeletal muscle, with very little expression in other tissues (35Park C.S. Krishna G. Ahn M.S. Kang J.H. Chung W.G. Kim D.J. Hwang H.K. Lee J.N. Paik S.G. Cha Y.N. Nitric Oxide. 2000; 4: 459-471Crossref PubMed Scopus (46) Google Scholar). As such, the PDZ of nNOS may play a novel role in nNOS function in these two tissues. We thank Drs. G. Chinnadurai and T. Subramanian for the generous gift of antibodies raised against CtBP and a cDNA encoding T7-tagged human CtBP, Dr. David Bredt for insightful discussion, Dr. Crista Adamson for technical assistance with microscopy, and Drs. Shu-Chan Hsu, Chris Rongo, and Joe Ramos for comments on the manuscript.