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• W2169896429 abstract "The aryl hydrocarbon receptor nuclear transporter (ARNT) is a member of the basic helix-loop-helix/PAS (Per-ARNT-Sim) family of transcription factors, which are important for cell regulation in response to environmental conditions. ARNT is an indispensable partner of the aryl hydrocarbon receptor (AHR) or hypoxia-inducible factor-1α. This protein is also able to form homodimers such as ARNT/ARNT. However, the molecular mechanism that regulates the transcriptional activity of ARNT remains to be elucidated. Here, we report that ARNT is modified by SUMO-1 chiefly at Lys245 within the PAS domain of this protein, bothin vivo and in vitro. Substitution of the target lysine with alanine enhanced the transcriptional potential of ARNT per se. Furthermore, green fluorescent protein-fused ARNT tended to form nuclear foci in ∼20% of the transfected cells, and the foci partly colocalized with PML nuclear bodies. PML, one of the well known substrates for sumoylation, was found to augment the transcriptional activities of ARNT. ARNT bound AHR or PML, whereas the sumoylated form of ARNT associated with AHR, but not with PML, resulting in a reduced effect of PML on transactivation by ARNT. Our data suggest that the sumoylation of ARNT modulates its transcriptional role through affecting the ability of ARNT to interact with cooperative molecules such as PML. This exemplifies a crucial role of protein sumoylation in modulating protein-protein interactions. The aryl hydrocarbon receptor nuclear transporter (ARNT) is a member of the basic helix-loop-helix/PAS (Per-ARNT-Sim) family of transcription factors, which are important for cell regulation in response to environmental conditions. ARNT is an indispensable partner of the aryl hydrocarbon receptor (AHR) or hypoxia-inducible factor-1α. This protein is also able to form homodimers such as ARNT/ARNT. However, the molecular mechanism that regulates the transcriptional activity of ARNT remains to be elucidated. Here, we report that ARNT is modified by SUMO-1 chiefly at Lys245 within the PAS domain of this protein, bothin vivo and in vitro. Substitution of the target lysine with alanine enhanced the transcriptional potential of ARNT per se. Furthermore, green fluorescent protein-fused ARNT tended to form nuclear foci in ∼20% of the transfected cells, and the foci partly colocalized with PML nuclear bodies. PML, one of the well known substrates for sumoylation, was found to augment the transcriptional activities of ARNT. ARNT bound AHR or PML, whereas the sumoylated form of ARNT associated with AHR, but not with PML, resulting in a reduced effect of PML on transactivation by ARNT. Our data suggest that the sumoylation of ARNT modulates its transcriptional role through affecting the ability of ARNT to interact with cooperative molecules such as PML. This exemplifies a crucial role of protein sumoylation in modulating protein-protein interactions. The aryl hydrocarbon receptor nuclear transporter (ARNT) 1The abbreviations used for: ARNT, aryl hydrocarbon receptor nuclear transporter; bHLH, basic helix-loop-helix; AHR, aryl hydrocarbon receptor; HIF1α, hypoxia-inducible factor-1α; XRE, xenobiotic-responsive element; GST, glutathioneS-transferase; HRE, hypoxia-responsive element; E3, protein isopeptide ligase; NBs, nuclear bodies; HA, hemagglutinin; PBS, phosphate-buffered saline; WT, wild-type; DBD, DNA-binding domain; GFP, green fluorescent protein. belongs to the basic helix-loop-helix (bHLH)/PAS (Per-ARNT-Sim) family of proteins. These transcription factors are required for cell regulation to respond to various environmental conditions (1Crews S.T. Genes Dev. 1998; 12: 607-620Crossref PubMed Scopus (302) Google Scholar, 2Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (858) Google Scholar). The bHLH/PAS proteins include the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor-1α (HIF1α). ARNT is an indispensable partner of these proteins for the formation of heterodimers such as AHR/ARNT and HIF1α/ARNT. Both transcription pathways are not only biologically significant, but represent remarkable regulatory mechanisms in vivo. First, polycyclic aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene are exogenous ligands for AHR and induce the formation of the AHR·ARNT complex (3Rowlands J.C. Gustafsson J. Crit. Rev. Toxicol. 1997; 27: 109-134Crossref PubMed Scopus (437) Google Scholar). In the absence of ligands, AHR is generally found in the cytoplasm in association with hsp90 and other molecules (3Rowlands J.C. Gustafsson J. Crit. Rev. Toxicol. 1997; 27: 109-134Crossref PubMed Scopus (437) Google Scholar, 4Kazlauskas A. Sundström S. Poellinger L. Pongratz I. Mol. Cell. Biol. 2001; 21: 2594-2607Crossref PubMed Scopus (169) Google Scholar). Upon the binding of a ligand, AHR is converted to a functional DNA-binding species in a multistep process involving nuclear translocation, dissociation from the hsp90-containing complex, and dimerization with ARNT. The resulting AHR·ARNT complex binds a specific cis-acting regulatory DNA sequence, termed the xenobiotic-responsive element (XRE), upstream of its target genes encoding drug-metabolizing enzymes such as cytochrome P450 (CYP1A1 and others), quinone reductase, and the glutathioneS-transferase (GST) Ya subunit. Xenobiotic-activated AHR is then degraded by the ubiquitin/proteasome system after being exported from the nucleus to the cytoplasm (5Ma Q. Baldwin K.T. J. Biol. Chem. 2000; 275: 8432-8438Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Mice deficient in the AHR gene demonstrate dioxin-induced cytotoxicities, including developmental defects and tumorigenesis, depending on the AHR/ARNT pathways (6Gonzalez F.J. Fernandez-Salguero P. Drug Metab. Dispos. 1998; 26: 1194-1198PubMed Google Scholar, 7Shimizu Y. Nakatsuru Y. Ichinose M. Takahashi Y. Kume H. Mimura J. Fujii-Kuriyama Y. Ishikawa T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 779-782Crossref PubMed Scopus (539) Google Scholar). Second, ARNT, also known as HIF1β, forms a HIF1α/ARNT heterodimer in response to oxygen tension in the cells. Under hypoxic conditions, it activates the transcription of a number of target genes whose promoters contain the binding motif termed the hypoxia-responsive element (HRE) (8Semenza G.L. Trends Mol. Med. 2001; 7: 345-350Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar). These include genes encoding erythropoietin, vascular endothelial growth factor, glycolytic enzymes, tyrosine hydroxylase, inducible nitric-oxide synthase, and heme oxygenase-1, all of which allow the cells to cope with lower oxygen levels. In addition, HIF1α/ARNT controls the gene expression involved in iron metabolism, pH regulation, cell proliferation and apoptosis, and tumorigenesis. The HIF1α protein is rapidly degraded by the ubiquitin/proteasome pathway under normoxic conditions (9Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4150) Google Scholar, 10Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). In a hypoxic state, it becomes stable and translocates into the nucleus. It then dimerizes with ARNT to activate transcription. Thus, bHLH/PAS family members have significant features of their regulatory mechanisms, and ARNT is implicated in many signaling pathways mediated by AHR or HIF1α as its partner. Increasing numbers of cellular proteins have been found to be covalently conjugated with SUMO-1 (smallubiquitin-related modifier-1). Sumoylation is thought to control the function of substrate proteins through affecting their interaction with cooperative molecules and subcellular localization (11Müller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (650) Google Scholar, 12Seeler J.S. Dejean A. Oncogene. 2001; 20: 7243-7249Crossref PubMed Scopus (136) Google Scholar, 13Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). The conjugation by SUMO-1 in some cases antagonizes ubiquitination at the identical target lysine of substrates, resulting in the stabilization of sumoylated proteins (14Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar,15Hochstrasser M. Nat. Cell. Biol. 2000; 2: E153-E157Crossref PubMed Scopus (368) Google Scholar). Like the ubiquitin conjugation system, SUMO-1 is activated in an ATP-dependent manner by a Sua1/UBA2 heterodimer SUMO-1-activating enzyme); then transferred to a SUMO-1-conjugating enzyme, UBC9; and subsequently attached to the ε-amino group of target lysines of substrates (11Müller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (650) Google Scholar, 12Seeler J.S. Dejean A. Oncogene. 2001; 20: 7243-7249Crossref PubMed Scopus (136) Google Scholar, 13Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). Sumoylation mostly targets the consensus sequence ΨKXE, where Ψ and K are a hydrophobic residue and the SUMO-1 acceptor lysine in substrate proteins, respectively (16Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-994Crossref PubMed Scopus (327) Google Scholar, 18Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 19Sampson D.A. Wang M. Matunis M.J. J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). These include Ran GTPase-activating protein-1 (20Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 21Saitoh H. Sparrow D.B. Shiomi T. Pu R.T. Nishimoto T. Mohun T.J. Dasso M. Curr. Biol. 1998; 8: 121-124Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), Sp100 (16Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 22Seeler J.S. Marchio A. Losson R. Desterro J.M. Hay R.T. Chambon P. Dejean A. Mol. Cell. Biol. 2001; 21: 3314-3324Crossref PubMed Scopus (107) Google Scholar), p53 (23Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Crossref PubMed Scopus (438) Google Scholar, 24Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Crossref PubMed Scopus (561) Google Scholar, 25Kwek S.S. Derry J. Tyner A.L. Shen Z. Gudkov A.V. Oncogene. 2001; 20: 2587-2599Crossref PubMed Scopus (113) Google Scholar), IκBα (14Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar), c-Jun (26Müller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 27Schmidt D. Müller S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2872-2877Crossref PubMed Scopus (371) Google Scholar), c-Myb (28Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), heat shock factor-1 (29Hong Y. Rogers R. Matunis M.J. Mayhew C.N. Goodson M. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 40263-40267Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), heat shock factor-2 (30Goodson M.L. Hong Y. Rogers R. Matunis M.J. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 18513-18518Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), lymphoid enhancer factor-1 (31Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (464) Google Scholar), and PML (32Müller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (580) Google Scholar, 33Kamitani T. Kito K. Nguyen H.P. Wada H. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 26675-26682Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 34Duprez E. Saurin A.J. Desterro J.M. Lallemand-Breitenbach V. Howe K. Boddy M.N. Solomon E. de The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; 112: 381-393Crossref PubMed Google Scholar). Although it was unknown whether sumoylation requires E3 enzymes with SUMO-1 ligase activities, SUMO E3-like factors have recently been identified in mammals, including members of the PIAS (protein inhibitor of activatedSTAT, where STAT is signaltransducer and activator oftranscription) family of proteins and Ran-binding protein-2 (27Schmidt D. Müller S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2872-2877Crossref PubMed Scopus (371) Google Scholar, 31Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (464) Google Scholar, 35Kahyo T. Nishida T. Yasuda H. Mol. Cell. 2001; 8: 713-718Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 36Pichler A. Gast A. Seeler J.S. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). Furthermore, some sumoylated proteins are found to localize to specified nuclear domains called nuclear bodies (NBs), also referred to as nuclear domain 10 or PML oncogenic domains (29Hong Y. Rogers R. Matunis M.J. Mayhew C.N. Goodson M. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 40263-40267Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 30Goodson M.L. Hong Y. Rogers R. Matunis M.J. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 18513-18518Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 31Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (464) Google Scholar, 32Müller S. Matunis M.J. Dejean A. EMBO J. 1998; 17: 61-70Crossref PubMed Scopus (580) Google Scholar, 33Kamitani T. Kito K. Nguyen H.P. Wada H. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 26675-26682Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 34Duprez E. Saurin A.J. Desterro J.M. Lallemand-Breitenbach V. Howe K. Boddy M.N. Solomon E. de The H. Hay R.T. Freemont P.S. J. Cell Sci. 1999; 112: 381-393Crossref PubMed Google Scholar). The SUMO-1 conjugation of PML enables it to target to NBs, and PML is indispensable in the formation of NBs. Proteins such as Sp100 and p53 are similarly modified by SUMO-1, but their translocation into NBs does not need SUMO-1 modifications (16Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 25Kwek S.S. Derry J. Tyner A.L. Shen Z. Gudkov A.V. Oncogene. 2001; 20: 2587-2599Crossref PubMed Scopus (113) Google Scholar). Recently, some transcription factors and their co-regulators were identified to associate with PML, emphasizing the transcriptional roles of PML-NBs (37Zhong S. Salomoni P. Pandolfi P.P. Nat. Cell. Biol. 2000; 2: E85-E90Crossref PubMed Scopus (490) Google Scholar, 38Hatta M. Fukamizu A. Science's STKE. 2001; (http://www.stke.org/cgi/content/full.OC sigtrans;2001/96/pel)PubMed Google Scholar). Thus, the SUMO-1 conjugation system may cooperate with PML-NBs in transcriptional control. In addition to the heterodimerization with AHR or HIF1α, ARNT is likely to form a homodimer with itself to bind the E-box core sequence CACGTG with high specificity and affinity, suggesting a physiological role of the ARNT·ARNT complex (3Rowlands J.C. Gustafsson J. Crit. Rev. Toxicol. 1997; 27: 109-134Crossref PubMed Scopus (437) Google Scholar, 39Huffman J.L. Mokashi A. Bachinger H.P. Brennan R.G. J. Biol. Chem. 2001; 276: 40537-40544Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Furthermore, the t(1,12)(q21;p13) translocation of human acute myeloblastic leukemia results in a fusion protein containing the amino-terminal part of TEL (translocated ETS leukemia, also known as ETV6) and almost all of ARNT (40Salomon-Nguyen F. Della-Valle V. Mauchauffe M. Busson-Le Coniat M. Ghysdael J. Berger R. Bernard O.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6757-6762Crossref PubMed Scopus (75) Google Scholar). The activity of ARNT may directly contribute to leukemogenesis. It is therefore of considerable interest to study the function of ARNT and the mechanism that regulates the transcriptional role of this protein. During the investigation of ARNT in cultured mammalian cells, we discovered that ARNT is modified by SUMO-1 chiefly at Lys245 within the PAS domain, which is required for forming the complexes with bHLH/PAS and other molecules (3Rowlands J.C. Gustafsson J. Crit. Rev. Toxicol. 1997; 27: 109-134Crossref PubMed Scopus (437) Google Scholar, 41Kobayashi A. Sogawa K. Fujii-Kuriyama Y. J. Biol. Chem. 1996; 271: 12310-12316Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 42Pongratz I. Antonsson C. Whitelaw M.L. Poellinger L. Mol. Cell. Biol. 1998; 18: 4079-4088Crossref PubMed Scopus (91) Google Scholar). ARNT associated with PML or PML-NBs, and the sumoylation of ARNT inhibited both the ability of ARNT to interact with PML and the positive effect of PML on the transactivation by ARNT. These data suggest the importance of the SUMO-1 conjugation system in modulating ARNT-mediated gene expression. Rabbit anti-ARNT (H-172) and rabbit anti-SUMO-1 (FL-101) polyclonal antibodies (Santa Cruz Biotechnology), goat anti-GST polyclonal antibodies (Amersham Biosciences), mouse anti-ARNT (G-3) and mouse anti-PML (PG-M3) monoclonal antibodies (Santa Cruz Biotechnology), mouse anti-GMP-1 monoclonal antibodies (SUMO-1, Zymed Laboratories Inc.), mouse anti-FLAG monoclonal antibodies (M5, Sigma), mouse anti-Myc monoclonal antibodies (PL-14, Medical & Biological Laboratories), and mouse monoclonal antibody 9E10 (Roche Molecular Biochemicals) were used in this study. MCF-7, HeLa, and Hepa-1 cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal calf serum. The full-length cDNA for murine ARNT was PCR-amplified from pBOS-ARNT (43Ema M. Hirota K. Mimura J. Abe H. Yodoi J. Sogawa K. Poellinger L. Fujii-Kuriyama Y. EMBO J. 1999; 18: 1905-1914Crossref PubMed Google Scholar) with a cloned Pfu DNA polymerase (Stratagene) using primers 5′-CCGGTGCCTCGAGCCGAATTCATGGCGGCGACTACAGCTAAC-3′ (forward, containing XhoI and EcoRI sites (underlined)) and 5′-CACAGGCTCGAGCTATTCGGAAAAGGGGGGAAAC-3′ (reverse, containing an XhoI site (underlined)). The PCR fragment was digested with EcoRI and XhoI, cloned into pcDNA3-FLAG, and inserted into the XhoI site of pEGFP-C1. To generate pcDNA3-Gal4-ARNT, the fragment of the Gal4 DNA-binding domain was amplified from pcDNA3-Gal4DBD (44Fujita N. Shimotake N. Ohki I. Chiba T. Saya H. Shirakawa M. Nakao M. Mol. Cell. Biol. 2000; 20: 5107-5118Crossref PubMed Scopus (127) Google Scholar) by primers 5′-CCGGTGGAATTCATGAAGCTACTGTCTTCTATCGAA-3′ (forward, containing an EcoRI site (underlined)) and 5′-CACAGGGAATTCGGATCCCGATACAGTCAACTGTCTTTGAC-3′ (reverse, containingEcoRI and BamHI sites (underlined)) and cloned into the EcoRI site of pcDNA3-FLAG-ARNT. pcDNA3-His-FLAG-ARNT was constructed by inserting a fragment of FLAG-ARNT from pcDNA3-FLAG-ARNT into predigested pcDNA3-His-FLAG-SUMO-1. Next, to construct plasmids expressing ARNT K245A and ARNT K546A, site-directed mutagenesis was performed using oligonucleotides 5′-ACAGTGGCCAAGGAAGGCCAGCAGTCTTCC-3′ and 5′-CTGGCCTTCCTTGGCCACTGTTCCAGTCTT-3′ for K245A and oligonucleotides 5′-AAGCCCCTTGAGGCCTCAGAAGGTCTCTTT-3′ and 5′-GAGACCTTCTGAGGCCTCAAGGGGCTTGCT-3′ for K546A. To generate pcDNA3-FLAG-ARNT K245A/K546A, both pcDNA3-FLAG-ARNT K245A and pcDNA3-FLAG-ARNT K546A were digested with SspI, and the fragment containing FLAG and the region of amino acids 1–395 of ARNT K245A were ligated into predigested pcDNA3-FLAG-ARNT K546A. For plasmids expressing GST-fused ARNT and ARNT K245A (amino acids 145–345), the cDNAs for ARNT and ARNT K245A were amplified primers 5′-TCCTTGGGATCCACTGGCAACACATCTACTGAT-3′ (forward, containing a BamHI site (underlined)) and 5′-GGGAGAAAGCTTTACCTGCAGCCTGCCAATGGC-3′ (reverse, containing a HindIII site (underlined)). The amplified DNAs were cloned into pGEX-2T predigested with BamHI andHindIII. To generate pcDNA3-Myc-AHR, the full-length cDNA for murine AHR was PCR-amplified from pBOS-AHR by primers 5′-CCGGTGCCTCGAGAATATTTAATGAGCAGCGGCGCCAACATC-3′ (forward, containingXhoI and SspI sites (underlined)) and 5′-CACAGGCTCGAGTCAACTCTGCACCTTGCTTAGGA-3′ (reverse, containing an XhoI site (underlined)). The PCR fragment was digested with SspI and XhoI and cloned into theEcoRV and XhoI sites of pcDNA3-Myc. To generate pcDNA3-Myc-PML, PML cDNA was amplified by primers 5′-AAGCTGGAATTCATGGAGCCTGCACCCGCCCGA-3′ (forward, containing an EcoRI site (underlined)) and 5′-CTTTTTCTCGAGAAGCTTCTAAATTAGAAAGGGGTGGGGGTA-3′ (reverse, containingXhoI and HindIII sites (underlined)). The cDNA for SUMO-1 (amino acids 1–97) was amplified from HeLa cDNAs by primers 5′-GCCACCCTCGAGATGTCTGACCAGGAGGCAAAA-3′ (forward, containing an XhoI site (underlined)) and 5′-GCCACCCTCGAGAAGCTTCTAACCCCCCGTTTGTTCCTGATA-3′ (reverse, containingXhoI and HindIII sites (underlined)) to construct pcDNA3-FLAG-SUMO-1. The cDNA for SUMO-1-(1–97) was also cloned into the XbaI site of pCGN-HA. To generate plasmids expressing His6-fused FLAG-SUMO-1, the fragment of FLAG and SUMO-1 was obtained by digesting pcDNA3-FLAG-SUMO-1 withBamHI and HindIII and cloned into pRSET, yielding pRSET-FLAG-SUMO-1. For expressing His6-fused FLAG-SUMO-1, the cDNA for His6-FLAG-SUMO-1 was amplified using primers 5′-GCCACCGGTACCGCCACCATGCGGGGTTCTCATCAT-3′ (forward, containing a KpnI site (underlined)) and 5′-GCCACCTCTAGACTAACCCCCCGTTTGTTC-3′ (reverse, containing an XbaI site (underlined)). The PCR fragment was digested with KpnI and XbaI and cloned into pcDNA3-FLAG, resulting in pcDNA3-His-FLAG-SUMO-1. The cDNA for human UBC9 was amplified from HeLa cDNAs and cloned into theXbaI site of pCGN-HA, yielding pCGN-HA-UBC9. For pGL3-promoter-XRE, oligonucleotides containing six XREs (5′-CTAGATGCGTGTGCGTGTGCGTGTGCGTGTGCGTGTGCGTGT-3′ and 5′-CTAGACACGCACACGCACACGCACACGCACACGCACACGCAT-3′) were equally mixed, heated to 95 °C for 5 min, and then cooled to room temperature. The double-stranded oligonucleotides were ligated into the NheI site of the pGL3-promoter vector (Promega). For pGL3-basic-mCyp1A1, ∼1.5 kb of the promoter sequence of the mousecyp1A1 gene was amplified from genomic DNA by primers 5′-TCCTGGGGTACCTCACAGAGCAGATATCAATGT-3′ (forward, containing a KpnI site (underlined)) and 5′-TCTAGGAAGCTTGGGAGGATCGGGGAAGCTCCA-3′ (reverse, containing a HindIII site (underlined)). The PCR fragment was cloned into the KpnI and HindIII sites of the pGL3-basic vector (Promega). Cell lysates were prepared with SDS sample buffer (60 mm Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.005% bromphenol blue, and 100 mmdithiothreitol). Samples were separated on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose filters with a constant current of 140 mA for 2 h. The filters were blocked with phosphate-buffered saline (PBS) containing 10% skim milk and then incubated with the appropriate antibodies in PBS containing 0.03% Tween 20 for 2 h and washed three times for 7 min with PBS containing 0.3% Tween 20. The filters were incubated with horseradish peroxidase-conjugated secondary antibodies for 40 min, and then specific proteins were detected using the ECL system (AmershamBiosciences). MCF-7 cells (30–50% confluent in six-well plates) were transfected with 1 μg of each of the expression vectors for His6-FLAG-ARNT (wild-type (WT) or K245A) and HA-SUMO-1 per well using FuGENE 6 (Roche Molecular Biochemicals). At 48 h after transfection, the cells were washed with ice-cold PBS, harvested in 200 μl of Gua8 buffer (6m guanidine HCl, 100 mm NaCl, 10 mmTris, and 50 mm NaH2PO4 (pH 8.0)) (22Seeler J.S. Marchio A. Losson R. Desterro J.M. Hay R.T. Chambon P. Dejean A. Mol. Cell. Biol. 2001; 21: 3314-3324Crossref PubMed Scopus (107) Google Scholar) per well, briefly sonicated, and then centrifuged. The cell lysates were incubated for 2 h with 20 μl of ProBond nickel-chelating resin (Invitrogen). Bound proteins were washed twice with Gua8 buffer; three times with buffer containing 8 murea, 100 mm NaCl, and 50 mmNaH2PO4 (pH 6.5)) (22Seeler J.S. Marchio A. Losson R. Desterro J.M. Hay R.T. Chambon P. Dejean A. Mol. Cell. Biol. 2001; 21: 3314-3324Crossref PubMed Scopus (107) Google Scholar) with 10 mmimidazole; and once with cold PBS before being eluted by boiling in SDS sample buffer. Transfected cells in one well were lysed directly with SDS sample buffer as an input sample. All samples were electrophoresed on an 8% SDS-polyacrylamide gel, followed by Western blot analysis. Sf9 insect cells expressing both Sua1 and human UBA2 (45Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (183) Google Scholar) were lysed by sonication in buffer containing 10 mm Tris-HCl (pH 7.4), 3 mmMgCl2, 200 mm NaCl, 0.1% Nonidet P-40, and 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. The lysates were centrifuged at 13,000 rpm for 15 min and used as a source of Sua1/human UBA2. His6-UBC9 and His6-FLAG-SUMO-1-(1–97) were expressed inEscherichia coli BL21(SI) and purified by affinity chromatography using ProBond. GST-ARNT WT and GST-ARNT K245A were expressed in BL21(star) and purified using glutathione-agarose beads (Sigma). GST-ARNT proteins were immobilized on glutathione-agarose beads and then incubated with Sua1/human UBA2, UBC9, and ATP with or without His6-FLAG-SUMO-1 at 25 °C for 120 min in buffer containing 50 mm Tris-HCl (pH 7.4), 10 mm ATP, 10 mm MgCl2, and 2 mmdithiothreitol. After the reaction, the beads were washed with 0.2% Triton X-100 in PBS five times and then boiled in SDS sample buffer. MCF-7 cells were plated at a density of 6∼8 × 105 cells/ml in a six-well plate and cultured for 24 h prior to transfection. The cells were introduced with 0.5 μg of reporter plasmid and 0.1 μg of pRL-CMV (Promega), which was used for monitoring the transfection efficiency, together with the indicated expression plasmids by TransFast (Promega). The transfected cells were treated with 10 μm 3-methylcholanthrene (Aldrich) and 200 μm cobalt chloride (Wako Bioproducts) or solvent alone for 12 h during the incubation. At 48 h after transfection, the cells were lysed in the lysis buffer provided by the manufacturer (Promega). The insertless pcDNA3 was used as a mock vector. The luciferase activities were determined with the dual-luciferase reporter assay system. Values are the means ± S.D. of the results from at least three independent experiments. For Gal4 transactivation studies, 1 μg of Gal4-ARNT (WT or K245A) or the Gal4 DNA-binding domain (DBD) vector, 0.5 μg of the pG5luc reporter vector, and 0.1 μg of pRL-CMV were introduced into the cells in each well using TransFast. pCGN-HA-SUMO-1 (the indicated amount), pCGN-HA-UBC9 (1 μg), and pcDNA3-Myc-PML (1 μg) were cotransfected, and luciferase analysis was done at 48 h after transfection. For immunofluorescence analysis, MCF-7 and Hepa-1 cells were transfected with the indicated vectors using FuGENE 6. At 48 h after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing with PBS, the cells were incubated with specific primary antibodies at room temperature for 60 min. The samples were incubated with fluorescein isothiocyanate (BIOSOURCE)- or Cy3 (Amersham Biosciences)-conjugated secondary antibodies for 60 min and visualized with an Olympus confocal laser scanning microscope. To avoid bleed-through effects in the double-staining experiments, each dye was independently excited, and images were electronically merged. MCF-7 or HeLa cells were transfected with the indicated plasmids with FuGENE 6 and lysed at 48 h after transfection with hypotonic buffer (10 mm NaCl, 50 mm Tris-HCl (pH 8.0), 0.05% Nonidet P-40, 5% glycerol, 5 mm N-ethylmaleimide, 100 μm4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 50 μg/ml aprotinin, 10 μg/ml leupeptin, 1.5 μm pepstatin, and 1 mm sodium orthovanadate) on ice for 30 min. After brief sonication and centrifugation (13,000 rpm) for 20 min, the supernatants were incubated with the indicated antibodies at 4 °C for 2 h and then with protein A/G beads (Calbiochem) at 4 °C for 2 h. For immunoprecipitation of FLAG-tagged proteins, anti-FLAG antibody M2-agarose affinity gel (Sigma) was used. The beads were washed five times with buffer containing 150 mm NaCl, 50 mmTris-HCl (pH 8.0) and 0.5% Nonidet P-40. The immunoprecipitates were suspended in SDS sample buffer and separated by SDS-PAGE. The pcDNA3-FLAG-ARNT or mock vector was transfected into MCF-7 cells with or without pCGN-HA-SUMO-1 and pCGN-HA-UBC9 using FuGENE 6. At 48 h after transfection, the cells were lysed with binding buffer (10 mm HEPES (pH 7.5), 50 mm KCl, 2.5 nm MgCl2, 50 μm ZnCl2, 2.5 mm dithiothreitol, 0.025% Nonidet P-40, and 5% glycerol), briefly sonicated, and centrifuged. The supernatants were incubated with GST-PML or GST at 4 °C for 2 h. 25 μl of glutathione-agarose beads as a 50% slurry in binding buffer was added to the mixture. After incubation at 4 °C for" @default.
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• W2169896429 date "2002-11-01" @default.
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• W2169896429 title "The Aryl Hydrocarbon Receptor Nuclear Transporter Is Modulated by the SUMO-1 Conjugation System" @default.
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• W2169896429 doi "https://doi.org/10.1074/jbc.m205987200" @default.
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