Acetylation by p300 Regulates Nuclear Localization and Function of the Transcriptional Corepressor CtBP2
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m509051200
ISSN1083-351X
AutoresLing‐Jun Zhao, T. Subramanian, Yun Zhou, G. Chinnadurai,
Tópico(s)Genetic Syndromes and Imprinting
ResumoCtBP family members, CtBP1 and CtBP2, are unique transcriptional regulators that adapt a metabolic enzyme fold, and their activities are regulated by NAD(H)-binding. CtBP1 is both cytoplasmic and nuclear, and its subcellular localization is regulated by sumoylation, phosphorylation, and binding to a PDZ protein. In contrast, we showed that CtBP2 is exclusively nuclear. CtBP1 and CtBP2 are highly similar, but differ at the N-terminal 20 amino acid region. Substitution of the N-terminal domain of CtBP1 with the corresponding CtBP2 domain confers a dominant nuclear localization pattern to CtBP1. The N-terminal domain of CtBP2 contains three Lys residues. Our results show that these Lys residues are acetylated by the nuclear acetylase p300. Although all three Lys residues of CtBP2 (Lys-6, Lys-8, and Lys-10) appear to be acetylated, acetylation of Lys-10 is critical for nuclear localization. CtBP2 with a single amino acid substitution at Lys-10 (K10R) is predominantly localized in the cytoplasm. The cytoplasmic localization of the K10R mutant is correlated with enhanced nuclear export that is inhibited by leptomycin B. Furthermore, lack of acetylation at Lys-10 renders CtBP2 to be more efficient in repression of the E-cadherin promoter. Our studies have revealed the important roles of acetylation in regulating subcellular localization and transcriptional activity of CtBP2. CtBP family members, CtBP1 and CtBP2, are unique transcriptional regulators that adapt a metabolic enzyme fold, and their activities are regulated by NAD(H)-binding. CtBP1 is both cytoplasmic and nuclear, and its subcellular localization is regulated by sumoylation, phosphorylation, and binding to a PDZ protein. In contrast, we showed that CtBP2 is exclusively nuclear. CtBP1 and CtBP2 are highly similar, but differ at the N-terminal 20 amino acid region. Substitution of the N-terminal domain of CtBP1 with the corresponding CtBP2 domain confers a dominant nuclear localization pattern to CtBP1. The N-terminal domain of CtBP2 contains three Lys residues. Our results show that these Lys residues are acetylated by the nuclear acetylase p300. Although all three Lys residues of CtBP2 (Lys-6, Lys-8, and Lys-10) appear to be acetylated, acetylation of Lys-10 is critical for nuclear localization. CtBP2 with a single amino acid substitution at Lys-10 (K10R) is predominantly localized in the cytoplasm. The cytoplasmic localization of the K10R mutant is correlated with enhanced nuclear export that is inhibited by leptomycin B. Furthermore, lack of acetylation at Lys-10 renders CtBP2 to be more efficient in repression of the E-cadherin promoter. Our studies have revealed the important roles of acetylation in regulating subcellular localization and transcriptional activity of CtBP2. The CtBP (E1A C-terminal binding protein) family proteins are highly conserved in higher eukaryotes and are linked to important biological processes (1.Chinnadurai G. Mol. Cell. 2002; 9: 213-224Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 2.Turner J. Crossley M. BioEssays. 2001; 23: 683-690Crossref PubMed Scopus (146) Google Scholar). The vertebrate genomes contain two different genes that code for two highly related proteins, CtBP1 (3.Boyd J.M. Subramanian T. Schaeper U. La Regina M. Bayley S. Chinnadurai G. EMBO J. 1993; 12: 469-478Crossref PubMed Scopus (260) Google Scholar, 4.Schaeper 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 CtBP2 (5.Katsanis N. Fisher E.M. Genomics. 1998; 47: 294-299Crossref PubMed Scopus (54) Google Scholar, 6.Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar), whereas genomes of invertebrates such as Drosophila and Caenorhabditis elegans contain a single Ctbp gene. Studies with mutant mice with targeted inactivation of the Ctbp genes suggest that they play important roles in multiple developmental processes during mouse development (7.Hildebrand J.D. Soriano P. Mol. Cell Biol. 2002; 22: 5296-5307Crossref PubMed Scopus (229) Google Scholar). In addition to CtBP1 and CtBP2, mammals also express an isoform of CtBP1 termed CtBP1-S/BARS (8.Spano S. Silletta M.G. Colanzi A. Alberti S. Fiucci G. Valente C. Fusella A. Salmona M. Mironov A. Luini A. Corda D. Spanfo S. J. Biol. Chem. 1999; 274: 17705-17710Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and an isoform of CtBP2 designated as RIBEYE (9.Schmitz F. Konigstorfer A. Sudhof T.C. Neuron. 2000; 28: 857-872Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) that are generated by differential RNA splicing and promoter utilization. The vertebrate CtBPs (6.Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar, 10.Sollerbrant K. Chinnadurai G. Svensson C. Nucleic Acids Res. 1996; 24: 2578-2584Crossref PubMed Scopus (68) Google Scholar, 11.Criqui-Filipe P. Ducret C. Maira S.M. Wasylyk B. EMBO J. 1999; 18: 3392-3403Crossref PubMed Scopus (136) Google Scholar, 12.Brannon M. Brown J.D. Bates R. Kimelman D. Moon R.T. Development (Camb.). 1999; 126: 3159-3170Crossref PubMed Google Scholar, 13.Sewalt R.G. Gunster M.J. van der Vlag J. Satijn D.P. Otte A.P. Mol. Cell Biol. 1999; 19: 777-787Crossref PubMed Scopus (160) Google Scholar) and the Drosophila homolog, dCtBP (14.Poortinga G. Watanabe M. Parkhurst S.M. EMBO J. 1998; 17: 2067-2078Crossref PubMed Scopus (206) Google Scholar, 15.Nibu Y. Zhang H. Levine M. Science. 1998; 280: 101-104Crossref PubMed Scopus (220) Google Scholar), function as transcriptional corepressors. Consistent with the high sequence similarity between vertebrate CtBP1 and CtBP2, they exhibit overlapping transcriptional regulatory activities during mouse development (7.Hildebrand J.D. Soriano P. Mol. Cell Biol. 2002; 22: 5296-5307Crossref PubMed Scopus (229) Google Scholar). The CtBP corepressors are recruited by various repressors through a conserved CtBP-binding motif (PXDLS) that was first identified in adenovirus E1A proteins (4.Schaeper 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).Unlike other transcription factors, CtBPs share a high degree of sequence homology with a group of metabolic enzymes of the 2-hydroxy acid dehydrogenase family (4.Schaeper 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). The crystal structure of CtBP1 has revealed that it is indeed a 2-hydroxy acid dehydrogenase (16.Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 17.Nardini M. Spano S. Cericola C. Pesce A. Massaro A. Millo E. Luini A. Corda D. Bolognesi M. EMBO J. 2003; 22: 3122-3130Crossref PubMed Scopus (126) Google Scholar). Consistent with the structural analyses, CtBP1 also possesses a slow dehydrogenase activity (16.Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 18.Balasubramanian P. Zhao L.J. Chinnadurai G. FEBS Lett. 2003; 537: 157-160Crossref PubMed Scopus (70) Google Scholar). At present, the relationship between the intrinsic dehydrogenase activity of CtBP and the transcriptional repression activity is not clear and remains controversial (6.Turner J. Crossley M. EMBO J. 1998; 17: 5129-5140Crossref PubMed Scopus (277) Google Scholar, 16.Kumar V. Carlson J.E. Ohgi K.A. Edwards T.A. Rose D.W. Escalante C.R. Rosenfeld M.G. Aggarwal A.K. Mol. Cell. 2002; 10: 857-869Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 19.Grooteclaes M. Deveraux Q. Hildebrand J. Zhang Q. Goodman R.H. Frisch S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4568-4573Crossref PubMed Scopus (155) Google Scholar). However, binding of CtBP1 with NAD(H) promotes its oligomerization and enhances the affinity for PXDLS-containing transcription factors (18.Balasubramanian P. Zhao L.J. Chinnadurai G. FEBS Lett. 2003; 537: 157-160Crossref PubMed Scopus (70) Google Scholar, 20.Zhang Q. Piston D.W. Goodman R.H. Science. 2002; 295: 1895-1897PubMed Google Scholar) and has been postulated to link the cellular metabolic status to transcriptional regulation (21.Fjeld C.C. Birdsong W.T. Goodman R.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9202-9207Crossref PubMed Scopus (221) Google Scholar). The dinucleotide binding activity of dCtBP has been shown to be critically important for the transcriptional repression activity in a native chromatin environment (22.Sutrias-Grau M. Arnosti D.N. Mol. Cell Biol. 2004; 24: 5953-5966Crossref PubMed Scopus (27) Google Scholar).Although the transcriptional regulatory mechanisms of CtBPs remain to be fully elucidated, an analysis of a nuclear CtBP1 protein complex has revealed that CtBP may effect its transcriptional repression activity at least in part by recruitment of other enzymatic components, such as histone deacetylases and a histone methyl transferase (23.Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (630) Google Scholar) and a novel lysine-specific demethylase (24.Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3093) Google Scholar). CtBP1 has also been reported to inhibit the general transcriptional machinery through direct interaction with nuclear acetylases such as p300 via a PXDLS motif located within the bromodomain of these enzymes (25.Kim J.H. Cho E.J. Kim S.T. Youn H.D. Nat. Struct. Mol. Biol. 2005; 12: 423-428Crossref PubMed Scopus (94) Google Scholar).In addition to the nuclear transcriptional regulatory activities, CtBPs also have certain diverse cytoplasmic activities (26.Chinnadurai G. BioEssays. 2003; 25: 9-12Crossref PubMed Scopus (82) Google Scholar). For example, CtBP1-S, which is identical to CtBP1 except for the absence of the N-terminal 12 amino acid region, has been implicated in Golgi membrane fission in in vitro studies (8.Spano S. Silletta M.G. Colanzi A. Alberti S. Fiucci G. Valente C. Fusella A. Salmona M. Mironov A. Luini A. Corda D. Spanfo S. J. Biol. Chem. 1999; 274: 17705-17710Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 27.Bonazzi M. Spano S. Turacchio G. Cericola C. Valente C. Colanzi A. Kweon H.S. Hsu V.W. Polishchuck E.V. Polishchuck R.S. Sallese M. Pulvirenti T. Corda D. Luini A. Nat. Cell Biol. 2005; 7: 570-580Crossref PubMed Scopus (145) Google Scholar, 28.Weigert 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, 29.Hidalgo Carcedo C. Bonazzi M. Spano S. Turacchio G. Colanzi A. Luini A. Corda D. Science. 2004; 305: 93-96Crossref PubMed Scopus (107) Google Scholar). It appears that the membrane fission activity of CtBP1-S might be related to a slow acyltransferase activity associated with CtBP1/CtBP1-S (17.Nardini M. Spano S. Cericola C. Pesce A. Massaro A. Millo E. Luini A. Corda D. Bolognesi M. EMBO J. 2003; 22: 3122-3130Crossref PubMed Scopus (126) Google Scholar, 28.Weigert 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). However, the existence of an intrinsic acyltransferase activity of CtBP1-S remains controversial (30.Gallop J.L. Butler P.J. McMahon H.T. Nature. 2005; 438: 675-678Crossref PubMed Scopus (70) Google Scholar). In the retina, the photoreceptor ribbon synaptic complex contains RIBEYE (9.Schmitz F. Konigstorfer A. Sudhof T.C. Neuron. 2000; 28: 857-872Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) and CtBP1 (31.tom Dieck S. Altrock W.D. Kessels M.M. Qualmann B. Regus H. Brauner D. Fejtova A. Bracko O. Gundelfinger E.D. Brandstatter J.H. J. Cell Biol. 2005; 168: 825-836Crossref PubMed Scopus (326) Google Scholar). RIBEYE and CtBP1 are important for the assembly and function of central nervous system synapses. Thus, CtBP family proteins in addition to their dominant transcriptional regulatory activity also perform diverse activities in the cytoplasmic compartment.The regulation of subcellular localization of CtBP1 has recently been examined in some detail. CtBP1 have been reported to be a target for the p21-activated protein kinase (Pak) 1 (32.Barnes C.J. Vadlamudi R.K. Mishra S.K. Jacobson R.H. Li F. Kumar R. Nat. Struct. Biol. 2003; 10: 622-628Crossref PubMed Scopus (78) Google Scholar). Pak1 specifically phosphorylates CtBP1 at Ser-158 resulting in CtBP1 redistribution from the nucleus to the cytoplasm, thereby blocking the corepressor function of CtBP1 under specific growth conditions. Although sequences of CtBP2 are conserved at the Pak1 phosphorylation site, it is not known whether CtBP2 is a target for Pak1. The polycomb protein Pc2 forms a specific complex with CtBP1 (13.Sewalt R.G. Gunster M.J. van der Vlag J. Satijn D.P. Otte A.P. Mol. Cell Biol. 1999; 19: 777-787Crossref PubMed Scopus (160) Google Scholar, 23.Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (630) Google Scholar, 33.Dahiya A. Wong S. Gonzalo S. Gavin M. Dean D.C. Mol. Cell. 2001; 8: 557-569Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The human Pc2 recruits CtBP1 and Ubc9 to the PcG bodies (34.Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) resulting in sumoylation of CtBP1 at a single Lys (Lys-428) residue (34.Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 35.Lin X. Sun B. Liang M. Liang Y.Y. Gast A. Hildebrand J. Brunicardi F.C. Melchior F. Feng X.H. Mol. Cell. 2003; 11: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The SUMO modification of CtBP1 appears to be critical for its nuclear accumulation. CtBP1 is also subject to an additional regulatory process that governs its nucleocytoplasmic distribution. The C-terminal region of CtBP1 has a PDZ-binding motif (1.Chinnadurai G. Mol. Cell. 2002; 9: 213-224Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar), which mediates interaction with a PDZ domain containing protein, neuronal nitric-oxide synthase, changing the localization pattern of CtBP1 from nuclear to cytoplasmic (36.Riefler G.M. Firestein B.L. J. Biol. Chem. 2001; 276: 48262-48268Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, sumoylation and PDZ binding exert opposing effects on subcellular localization of CtBP1 (35.Lin X. Sun B. Liang M. Liang Y.Y. Gast A. Hildebrand J. Brunicardi F.C. Melchior F. Feng X.H. Mol. Cell. 2003; 11: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In contrast to CtBP1, CtBP2 is not sumoylated (34.Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 35.Lin X. Sun B. Liang M. Liang Y.Y. Gast A. Hildebrand J. Brunicardi F.C. Melchior F. Feng X.H. Mol. Cell. 2003; 11: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and does not contain the PDZ-binding motif, suggesting that the regulation of subcellular localization of CtBP might be different from that CtBP1. In this report, we show that CtBP2 has a dominant nuclear localization pattern, which is dictated by the N-terminal 20 amino acid domain. Further, we demonstrate that CtBP2 is acetylated by p300 at Lys-6, Lys-8, and Lys-10 and that acetylation of Lys-10 is critically important for nuclear retention of CtBP2.EXPERIMENTAL PROCEDURESExpression Constructs—The mammalian expression vector pIRESneo3 (Clontech) was modified to express Flag-HA 2The abbreviations used are: HA, hemagglutinin; siRNA, small interfering RNA; wt, wild type; 3KR, three mutated Lys residues. tag at the N terminus of a protein of interest. A double-stranded oligonucleotide encoding the Flag-HA epitopes was ligated to the StuI/EcoRI sites of pIRESnes3 to generate pFH-IRESneo vector. All CtBP1 and CtBP2 expression constructs were based on pFH-IRESneo, using PCR-amplified coding sequences. All PCR-amplified regions in the clones were sequence-confirmed by automatic DNA sequence analysis.Cell Culture and Transfection—HeLa and A549 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Transfections were performed with the Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's recommendations. Typically, 2.5 × 105 cells were plated in each well of a 6-well plate 1 day prior to transfection, and DNA precipitates were prepared using 3 μg of a plasmid DNA and 7 μlofthe Lipofectamine 2000 reagent unless indicated otherwise. For siRNA experiments, HeLa cells were first transfected with SmartPool siRNAs (Dharmacon) with Dharmafect 1 (Dharmacon) and 24 h later were transfected with the CtBP2 expression plasmid using Lipofectamine 2000 reagent. Protein analyses were carried out 24 h after the second transfection. For E-cadherin promoter assays, the reporter plasmid pE-Cad-Luc and promoterless phRL-0 were cotransfected with different CtBP-expression plasmids into CtBP1–/–/CtBP2–/– mouse embryonic fibroblasts (immortalized with retrovirally introduced SV40 T-antigen), using the jetPEI transfection reagent (ISC BioExpress). Dual luciferase assays were performed with the Dual Luciferase assay kit (Promega) 2 days after transfection.Immunoprecipitation and Western Blot Analyses—Cells were lysed for 30 min at 4 °C in immunoprecipitation (IP) buffer containing 0.2% Nonidet P-40, 20 mm Hepes (pH 7.5), 150 mm NaCl, 10 mm KCl, 0.5 mm EDTA, 10 mm Na Butyrate, 10% glycerol, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride and the protease inhibitor mixture (Roche Applied Science). The cell lysates were clarified by centrifugation and bound to 30 μl of FLAG (Sigma) antibody beads at 4 °C for 1.5 h in IP buffer. After washing three times, immunoprecipitated proteins were examined by Western blot using the ECL-plus (Amersham Biosciences) detection method.Immunofluorescence Analysis—For staining ectopically expressed proteins, transfected (transiently or stably) cells in 6-well plates were fixed with 3.7% formaldehyde/phosphate-buffered saline for 10 min at room temperature and then permeablized with methanol for 6 min at –20 °C. After a brief rinse with phosphate-buffered saline, cells were incubated with Cy3-conjugated FLAG M2 antibody (Sigma) in a humidified chamber for 1 h at 37 °C. Cells were washed with phosphate-buffered saline and photographed under a Nikon fluorescent microscope with a digital camera. The endogenous CtBP proteins were stained with CtBP1 or CtBP2 mAbs (Pharmingen), with a secondary goat anti-mouse antibody conjugated to rhodamine (Pierce).RESULTSCtBP2, But Not CtBP1, Is Localized Exclusively in the Nucleus—Because CtBP2 is not sumoylated and lacks the PDZ-binding motif, we hypothesized that subcellular localization of CtBP2 might be regulated differently from CtBP1. To investigate this aspect, we first examined the subcellular localization of CtBP2 in comparison with CtBP1 by indirect immunofluorescence analysis. The specificities of the antibodies used in these studies were confirmed by their differential reactivity toward CtBP1 or CtBP2 by Western blot analysis of N-terminally tagged versions (Flag-HA) of CtBP1 or CtBP2 immunoprecipitated from stably transfected cell lines (Fig. 1A). The CtBP1 antibody recognized only CtBP1, and the CtBP2 antibody recognized only CtBP2. A minor band revealed in the opposing lane might be because of heterodimerization of endogenous CtBP proteins with the exogenously expressed CtBP proteins. For the immunofluorescence analysis of endogenous CtBP2, two human cell lines, HeLa and A549, were stained with antibodies specific to CtBP1 or CtBP2. These studies revealed striking exclusive nuclear localization of CtBP2 in both HeLa and A549 cells (Fig. 1B), whereas CtBP1 displayed predominant nuclear staining with partial cytoplasmic staining (Fig. 1C). To substantiate the localization patterns further, we examined exogenously introduced CtBP1 and CtBP2 (tagged with FLAG and HA epitopes) in stable HeLa cell lines (Fig. 1D). Immunofluorescence analysis with the FLAG antibody revealed that the patterns of subcellular localization of the ectopically expressed CtBP1 and CtBP2 are similar to that of the endogenous CtBP1 and CtBP2 (compare Fig. 1, B and C with D). We also examined the localization patterns of transiently expressed CtBPs (Fig. 1E), which were found to be highly similar to patterns of endogenous CtBPs and the patterns of CtBPs expressed in stably transfected cell lines (Fig. 1D). Thus, our analyses using three different approaches have shown exclusive nuclear localization of CtBP2 and nuclear and cytoplasmic localization of CtBP1. A similar localization pattern of CtBP1 has been reported by several previous studies (34.Kagey M.H. Melhuish T.A. Wotton D. Cell. 2003; 113: 127-137Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 35.Lin X. Sun B. Liang M. Liang Y.Y. Gast A. Hildebrand J. Brunicardi F.C. Melchior F. Feng X.H. Mol. Cell. 2003; 11: 1389-1396Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar).Nuclear Targeting Function of the N-terminal Region of CtBP2—The four members of the CtBP family, CtBP1, CtBP2, CtBP1-S, and RIBEYE share a high degree of sequence identity except at the N-terminal region (Fig. 2A). The N-terminal 20-amino-acid region of CtBP2 contains several basic amino acid residues that may potentially constitute a nuclear localization signal. To examine the potential nuclear localization signal function of this sequence, the N-terminal 21 residues of CtBP2 were replaced with Met-Ser-Gly-Val residues (Fig. 2B). The resulting CtBP2 mutant (ΔN-CtBP2), bears the N-terminal residues of CtBP1-S/BARS (Fig. 2A). An immunofluorescence analysis of the mutant, ΔN-CtBP2 revealed its distribution throughout the cells, in sharp contrast to the wt CtBP2 (Fig. 2C). A similar deletion of the N-terminal residues of CtBP1 generated a CtBP1 mutant, ΔN-CtBP1, which is identical to CtBP1-S/BARS (Fig. 2B). This deletion did not affect the partial nuclear and cytoplasmic localization of CtBP1 (Fig. 2C). These results suggest that the N-terminal region of CtBP2 contains a key determinant for the nuclear localization of CtBP2.FIGURE 2Nuclear localization function of the N-terminal region of CtBP2. A, N-terminal sequences of the CtBP family proteins. Numbers denote the position of amino acid residues. The A domain of RIBEYE is unrelated to CtBP and is fused to CtBP2 as a result of differential RNA splicing of a transcript generated from a tissue-specific promoter. B, substitution mutants of CtBP1 and CtBP2. Numbers denote the positions of amino acid residues. In N2-CtBP1, the N-terminal 21 residues of CtBP2 were fused to residue 16 of CtBP1. All constructs carry an N-terminal Flag-HA tag. C, subcellular localization of CtBP N-terminal mutants. HeLa cells were transfected with various mutant plasmids and examined for CtBP proteins by immunofluorescent staining with the FLAG antibody conjugated to Cy3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine whether the CtBP2 N-terminal region is the major determinant for the differential subcellular localization of CtBP1 and CtBP2, the N-terminal 21 residues of CtBP2 were substituted for the N-terminal 15 residues of CtBP1 (Fig. 2B). The resulting chimeric protein, N2-CtBP1, was detected exclusively in the nucleus (Fig. 2C), in a fashion identical to that of wt CtBP2. Thus, the distinct nuclear localization pattern of CtBP2 appears to be determined by its unique N-terminal sequence. Further, it could also confer a predominant nuclear localization pattern to CtBP1.Lys-10 Is Critical for the Nuclear Retention of CtBP2—There are three Lys residues in the N-terminal region of CtBP2 (Fig. 3A). Because Lys residues are frequent targets for post-translational modifications, we decided to examine the effect of these residues in nuclear localization of CtBP2. To examine their roles, they were mutated together or singly to Arg residues (Fig. 3A) so that the overall charge of the mutant CtBP2 would be comparable with that of wt CtBP2. The mutant plasmids were transfected into HeLa cells, and the cells were stained with the FLAG antibody. As shown in Fig. 3B, although K6R and K8R mutations had no effects on the exclusive nuclear localization of CtBP2, mutation of all three Lys residues (3KR) as well as mutation of Lys-10 (K10R) caused CtBP2 to be localized predominantly in the cytoplasm. Thus, Lys-10 plays a critical role in exclusive nuclear localization of CtBP2.FIGURE 3Role of N-terminal Lys residues in nuclear localization of CtBP2. A, N-terminal sequence of CtBP2 and Lys mutations. The basic residues within the N-terminal region are indicated in italics. B, subcellular localization of N-terminal mutants of CtBP2. HeLa cells were transfected and CtBP2 proteins were detected by immunofluorescent staining with the FLAG antibody conjugated to Cy3. C, effect of leptomycin B (LMB) on subcellular localization of 3KR and K10R mutants. The transfected cells were either mock-treated or treated with leptomycin B (7.5 ng/ml for 4 h), and the immunofluorescence analysis was performed as in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The predominant cytoplasmic localization of 3KR-CtBP2 and K10R-CtBP2 (with nuclear exclusion) is in sharp contrast to the partial nuclear localization of ΔN-CtBP2, which totally lacks the N-terminal region (Fig. 2). It appears that the mutations 3KR and K10R confer a dominant cytoplasmic localization pattern to CtBP2. This function may be because of cytoplasmic retention of 3KR-CtBP2 and K10R-CtBP2 by cytosolic factors or because of enhanced nuclear export of these mutant proteins. To examine whether nuclear export accounts for the predominant cytoplasmic localization of K10R-CtBP2, transfected cells were treated with the nuclear export inhibitor, leptomycin B, which specifically inhibits nuclear export mediated by CRM-1 (37.Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1731) Google Scholar). As shown in Fig. 3C, the leptomycin B treatment resulted in partial nuclear localization of mutants 3KR or K10R in majority of cells. A similar treatment of cells transfected with CtBP1 has no detectable effect on CtBP1 localization (not shown). These results suggest that K10R-CtBP2 is capable of nuclear localization but is efficiently exported from the nucleus.N-terminal Lys Residues of CtBP2 Are Acetylated—Our results (Fig. 3) suggest that the Lys-10 is important for nuclear retention of CtBP2 and a mutation of this residue (K10R) does not significantly impede with nuclear import. Because the mutation (K10R) does not cause a significant charge change in CtBP2, we decided to investigate the potential acetylation of the Lys residues. For this purpose, a panel of CtBP proteins was immunoprecipitated from transfected cells and analyzed by Western blotting using a monoclonal pan Ac-Lys antibody. As shown in Fig. 4A, both the wt CtBP2 (lane 4) and a mutant (A58E) defective in PXDLS-binding (17.Nardini M. Spano S. Cericola C. Pesce A. Massaro A. Millo E. Luini A. Corda D. Bolognesi M. EMBO J. 2003; 22: 3122-3130Crossref PubMed Scopus (126) Google Scholar) (lane 6), were acetylated well. Deletion of the N-terminal 20 residues (ΔN) of CtBP2 (lane 5), or mutation of all three N-terminal Lys residues together (mutant 3KR, lane 7), abolished acetylation of CtBP2 almost completely. The acetylation of various single Lys → Arg mutants (K6R, K8R, and K10R) was also severely reduced (lanes 8–10). Among the three mutants, K6R and K10R exhibited a higher level of reduction in acetylation than the mutant K8R. In contrast to CtBP2, acetylation of CtBP1 was not detectable (compare lane 2 with lane 4). However, fusion of the N-terminal region of CtBP2 to CtBP1 rendered CtBP1 to be acetylated (N2-CtBP1, lane 3), albeit at a lower level than CtBP2 (lane 4). In all CtBP-containing lanes except for ΔN-CtBP2 (lane 5), a band of ∼50 kDa was also present, which does not correspond to Flag-HA-tagged CtBP proteins. Its identity is not known.FIGURE 4Acetylation of CtBP2. A, effect of CtBP mutations. Lysates of transfected cells were immunoprecipitated with the FLAG antibody and examined by Western blot using the FLAG antibody (lower panel) or an anti-Acetyl-Lys (Ac-Lys) mAb (upper panel). B, specificity of the CtBP2 peptide antibody. Flag-HA-tagged CtBPs were immunoprecipitated with the FLAG antibody and the Western blot (WB) was probed with the FLAG antibody (upper panel) or the CtBP2 peptide antibody (lower panel). C, acetylation of endogenous CtBP2. Endoge
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