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• W2040251321 abstract "PML-RARα, a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα), causes acute promyelocytic leukemias (APL). Although the role of nuclear PML-RARα has been extensively studied, a significant amount of PML-RARα is in the cytoplasm. The role cytoplasmic PML-RARα plays in leukemogenesis is unknown. Here we report that PML-RARα induces the N-CoR accumulation in the endoplasmic reticulum (ER), leading to the induction of ER stress and the processing of activating transcription factor 6 (ATF6), the unfolded protein response. PML-RARα stimulates the ubiquitylation of N-CoR via Ubc6 that is involved in the protein quality control. This ER-associated degradation (ERAD) of N-CoR reduces the soluble N-CoR protein levels in the nucleus. The two N-CoR-interacting sites in PML-RARα are required for the ERAD of N-CoR, suggesting the aberrant binding of PML-RARα to N-CoR may induce the ERAD of N-CoR. Overexpression of N-CoR induces the differentiation of APL-derived NB4 cells, suggesting that the low levels of N-CoR in the nucleus may contribute at least partly to PML-RARα-mediated leukemogenesis. PML-RARα, a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα), causes acute promyelocytic leukemias (APL). Although the role of nuclear PML-RARα has been extensively studied, a significant amount of PML-RARα is in the cytoplasm. The role cytoplasmic PML-RARα plays in leukemogenesis is unknown. Here we report that PML-RARα induces the N-CoR accumulation in the endoplasmic reticulum (ER), leading to the induction of ER stress and the processing of activating transcription factor 6 (ATF6), the unfolded protein response. PML-RARα stimulates the ubiquitylation of N-CoR via Ubc6 that is involved in the protein quality control. This ER-associated degradation (ERAD) of N-CoR reduces the soluble N-CoR protein levels in the nucleus. The two N-CoR-interacting sites in PML-RARα are required for the ERAD of N-CoR, suggesting the aberrant binding of PML-RARα to N-CoR may induce the ERAD of N-CoR. Overexpression of N-CoR induces the differentiation of APL-derived NB4 cells, suggesting that the low levels of N-CoR in the nucleus may contribute at least partly to PML-RARα-mediated leukemogenesis. PML-RARα is a fusion protein of PML 1The abbreviations used are: PML, promyelocytic leukemia; RARα, retinoic acid receptor-α; APL, acute promyelocytic leukemias; ATF6, activating transcription factor 6; ER, endoplasmic reticulum; ERAD, ER-associated degradation; NB(s), nuclear body(ies); RA, retinoic acid; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; RIPA, radioimmune precipitation assay buffer; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; PDI, protein-disulfide isomerase. and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL) (1Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1292) Google Scholar, 2de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). The molecular mechanism of leukemogenesis by PML-RARα has been the focus of intensive research in the past decade. PML belongs to a family of Ring finger proteins characterized by the presence of the RBCC motif (3Jensen K. Shiels C. Freemont P.S. Oncogene. 2001; 20: 7223-7233Crossref PubMed Scopus (382) Google Scholar). In PML, this motif consists of a C3HC4-type zinc finger domain known as the Ring finger, two cysteine-rich regions termed B-boxes, and a leucine-rich heptad repeat known as the coiled-coil domain. Although exact roles these domains play in PML function are still poorly understood, it is believed that they serve as protein-protein interacting interfaces. PML negatively regulates cellular proliferation (4Mu Z.M. Chin K.V. Liu J.H. Lozano G. Chang K.S. Mol. Cell Biol. 1994; 14: 6858-6867Crossref PubMed Scopus (294) Google Scholar, 5Wang Z.G. Delva L. Gaboli M. Rivi R. Giorgio M. Cordon-Cardo C. Grosveld F. Pandolfi P.P. Science. 1998; 279: 1547-1551Crossref PubMed Scopus (456) Google Scholar) and localizes to nuclear dot-like structures known as nuclear bodies (NBs) or the PML oncogenic domain (POD) (6Dyck J.A. Maul G.G. Miller Jr., W.H. Chen J.D. Kakizuka A. Evans R.M. Cell. 1994; 76: 333-343Abstract Full Text PDF PubMed Scopus (724) Google Scholar, 7Weis K. Rambaud S. Lavau C. Jansen J. Carvalho T. Carmo-Fonseca M. Lamond A. Dejean A. Cell. 1994; 76: 345-356Abstract Full Text PDF PubMed Scopus (620) Google Scholar). PML-RARα expression disrupts NB structures in promyelocytic cells. This event is believed to be linked to transformation because reorganization of NBs always occurs when leukemic cells are induced to differentiate by retinoid (RA) treatment. PML is involved in multiple activities, including Mad- and Rb-mediated transcriptional repression (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Zhong S. Pandolfi P.P. Ishii S. J. Biol. Chem. 2001; 276: 43491-43494Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), RARα-mediated transcriptional activation (10Zhong S. Delva L. Rachez C. Cenciarelli C. Gandini D. Zhang H. Kalantry S. Freedman L.P. Pandolfi P.P. Nat. Genet. 1999; 23: 287-295Crossref PubMed Scopus (115) Google Scholar), and apoptosis (11Quignon F. De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar, 12Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (100) Google Scholar). PML-RARα blocks all of these functions in a dominant negative manner. For instance, PML binds via its coiled-coil region to multiple corepressors (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), including N-CoR, which mediates the transcriptional repression imposed by unliganded nuclear hormone receptors and Mad (13Hörlein A.J. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1712) Google Scholar, 14Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1084) Google Scholar). The binding of PML to the corepressors causes the corepressors to be recruited to the NBs (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In Pml-deficient cells, the transcriptional repression mediated by Mad or Rb is impaired, suggesting that the PML-mediated localization of the corepressors to NBs is an important event needed for the proper function of the corepressors (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Zhong S. Pandolfi P.P. Ishii S. J. Biol. Chem. 2001; 276: 43491-43494Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). When PML-RARα is expressed, Mad- and Rb-mediated transcriptional repression is inhibited. Mapping studies show that this activity depends on the presence of the two N-CoR-interacting sites of PML-RARα, namely, the coiled-coil domain in the PML part and the CoR-box located on the RAR moiety (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Zhong S. Pandolfi P.P. Ishii S. J. Biol. Chem. 2001; 276: 43491-43494Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This suggests that PML-RARα may bind aberrantly to the N-CoR molecule via these two sites. Such binding may induce the N-CoR protein to adopt an abnormal conformation. PML is also involved in p53 acetylation (15Pearson M. Carbone R. Sebastiani C. Cioce M. Fagioli M. Saito S. Higashimoto Y. Appella E. Minucci S. Pandolfi P.P. Pelicci P.G. Nature. 2000; 406: 207-210Crossref PubMed Scopus (1135) Google Scholar) and the recruitment of DNA methyltransferase (16Di Croce L. Raker V.A. Corsaro M. Fazi F. Fanelli M. Faretta M. Fuks F. Lo Coco F. Kouzarides T. Nervi C. Minucci S. Pelicci P.G. Science. 2002; 295: 1079-1082Crossref PubMed Scopus (693) Google Scholar). In addition, the oncogenicity of PML-RARα has been found to involve the silencing the genes of the RA signaling pathway by actively recruiting the histone deacetylase complex (17Lin R.J Nagy L. Inoue S. Shao W. Miller Jr., W.H. Evans R.M. Nature. 1998; 391: 811-814Crossref PubMed Scopus (981) Google Scholar, 18Grignani F. De Matteis S. Nervi C. Tomassoni L. Gelmetti V. Cioce M. Fanelli M. Ruthardt M. Ferrara F.F. Zamir I. Seiser C. Grignani F. Lazar M.A. Minucci S. Pelicci P.G. Nature. 1998; 391: 815-818Crossref PubMed Scopus (942) Google Scholar). Thus, the role of nuclear PML-RARα in leukemogenesis has been extensively studied. Significant levels of PML-RARα are also found in the cytoplasm (6Dyck J.A. Maul G.G. Miller Jr., W.H. Chen J.D. Kakizuka A. Evans R.M. Cell. 1994; 76: 333-343Abstract Full Text PDF PubMed Scopus (724) Google Scholar). However, the roles such cytoplasmic PML-RARα molecules may play in leukemogenesis are much less well understood than those played by the nuclear-localized molecules. In this study, we found that the cytoplasmic PML-RARα molecules may recruit the newly synthesized N-CoR molecules to the endoplasmic reticulum (ER) by directing them into the protein quality control system of the cell. This system is a post-translational process whereby newly synthesized polypeptides that fail to attain the proper structure are either refolded or degraded (19Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (916) Google Scholar). The mutated proteins or the accidentally generated proteins by various mechanisms such as chromosome rearrangement are somehow recognized as misfolded and degraded by the ubiquitin-proteasome system (20Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar). A key site for protein quality control is the ER as it is one of the major sites of protein synthesis and the portal of entry for proteins into the secretory pathway. When misfolded proteins accumulate in the ER, they are degraded via the proteasome in a process termed ER-associated degradation (ERAD) (21Fewell S.W. Travers K.J. Weissman J.S. Brodsky J.L. Annu. Rev. Genet. 2001; 35: 149-191Crossref PubMed Scopus (264) Google Scholar). The accumulation of misfolded protein in the ER also induces the proteolytic processing of the bZIP transcription factor ATF6 into its active form, which then leads to the transcriptional induction of ER chaperones and folding enzymes (22Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). The latter process is termed the unfolded protein response (UPR). Here we report that PML-RARα induces the accumulation of insoluble N-CoR protein primarily in the ER, which leads to decreased levels of soluble N-CoR protein in the nucleus. Since N-CoR protein in the nucleus is vital for the transcriptional repression mediated by the tumor suppressor Mad (14Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1084) Google Scholar, 23Jepsen K. Hermanson O. Onami T.M. Gleiberman A.S. Lunyak V. McEvilly R.J. Kurokawa R. Kumar V. Liu F. Seto E. Hedrick S.M. Mandel G. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2000; 102: 753-763Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar), this reduction of soluble N-CoR protein levels in the nucleus may be at least partly responsible for uncontrolled growth and transformation that is responsible for APL. Plasmids and Vectors—The chicken β-actin promoter containing plasmids to express N-CoR, PML-RARα, and PML, and the GST-PML expression vectors encoding various forms of PML were described previously (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The PCR-based method was employed to construct various PML-RARα mutant expression plasmids. In the PML-RARα RF (Ring finger point mutant) construct, the cysteine residues at 57 and 60 of the Ring finger domain of PML were substituted with serine. In PML-RARΔC-C, the whole coiled-coil domain (residues 228-365) was deleted. Similarly, in the B-boxes deletion mutant, the deleted region was the two B-boxes and intervening region, extending from residue 130 to 227. In the PML-RARα (AHT) mutant construct, the A, H, and T residues at 223, 224, and 227 sites, which are required for interaction with the N-CoR molecule, were replaced by G, G, and A residues, respectively. The HA-ATF6 expression vector (pCGN-ATF6) was described previously (22Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). To express Ubc5 or Ubc6 in 293T cells, the pcDNA3.1 vector (Invitrogen) was used. In Ubc5(CS) and Ubc6(CS) expression vectors, the active cysteine residue was replaced with serine. Immunocytochemistry—293T cells were co-transfected with pact-N-CoR-FLAG (1 μg) together with pact-HA-PML-RARα or pact-HA-PML (1 μg), fixed with 4% paraformaldehyde or methanol, and stained with either anti-PML (Santa Cruz Biotechnology), anti-HA (Santa Cruz Biotechnology), and anti-PDI (Stress Gene), anti-BiP (N-20, Santa Cruz Biotechnology), or anti-FLAG (M2, Sigma) antibodies. For visualization of Golgi signals, cells were transfected with Ds-Red-Golgi (0.5 μg) expression plasmid. Staining of NB4 cells was performed under similar conditions using anti-PML (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), anti-N-CoR (C-20, Santa Cruz Biotechnology), anti-Golgin-97 (Molecular Probes), and anti-PDI antibodies, and signals were visualized by confocal microscopy or deconvolution. DNA was stained with TO-PRO (Molecular Probes). Subcellular Fractionation—293T cells co-transfected with N-CoR and PML-RARα plasmids or NB4 cells, were harvested in 2 ml of ice-cold homogenizing buffer (10 mm Tris-HCl, pH 7.4, 250 mm sucrose, 5 mm EDTA, and protease inhibitor mixture), homogenized by passing 50 times in a Potter-Teflon homogenizer on ice. Post-nuclear supernatant was obtained (3,000 rpm, 10 min, 4 °C), and Nycodenz gradient fractionation was performed essentially as described (24Tran K. Thorne-Tjomsland G. DeLong J.C. Cui Z. Shan J. Burton L.C. Jamieson J. Yao Z. J. Biol. Chem. 2002; 277: 31187-31200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). A step gradient was created in Beckman SW41 centrifuge tubes by loading top to bottom 2.5 ml of 10, 14.66, 19.33, and 24% of Nycodenz solution in saline buffer. The solutions were prepared from 27.6% Nycodenz stock solution and 0.75% NaCl (both in 10 mm Tris-HCl, pH 7.4, 3 mm KCl, 1 mm EDTA, 0.02% NaN3). The tube was placed vertically for 45 min at room temperature followed by centrifugation (37,000 rpm, 4 h, 15 °C, SW41 rotor). 2 ml of the post-nuclear supernatant was layered on top of the gradient and fractionated by centrifugation (37,000 rpm, 1.5 h, 15 °C). After centrifugation, fractions (0.8 ml each) were collected from top of the tube using. Aliquots (40 μl) of each fraction was resolved by SDS-PAGE and probed with antibodies specific for marker proteins of various subcellular compartments. Protease Sensitivity of N-CoR—NB4 cells were seeded at 20 × 104 cells/ml and treated with RA (1 μm) for 3 days. Cells were harvested and disrupted with hypotonic buffer (10 mm Tris-HCl, pH 8.0, 10 mm KCl, 1 mm EDTA, 1 mm dithiothreitol) and homogenizer. After centrifugation, the supernatant was kept, and nuclei were extracted with NETN buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, 150 mm NaCl). Nuclei extract and the cytoplasm supernatant were mixed and used as whole cell lysates. Cell lysates were treated with indicated amount of proteinase K or trypsin for 30 min on ice. The degradation products were analyzed with SDS-PAGE, followed by Western blotting with anti-N-CoR antibodies. ATF6 Processing—NB4 cells were treated with RA (1 μm) or vehicle for 72 h, and lysates were prepared in RIPA buffer. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane and blotted with the anti-ATF6 antibody (rabbit anti-B03N) (22Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1552) Google Scholar). To detect the processing of exogenous protein, HeLa cells were co-transfected with LipofectAMINE (Invitrogen) with the HA-ATF6 expression vector and the plasmids expressing N-CoR and PML-RARα or PML. Cells were treated with tunicamycin (2.5 μg/ml) or dithiothreitol (5 mm) for the indicted time before the preparation of lysates. Twenty-four hours after transfection, lysates were prepared as described above and various forms of HA-ATF6 were detected by using the anti-HA antibody. Protein Solubility, Fractionation, and Western Blotting—293T cells were transfected by LipofectAMINE with the N-CoR expression plasmid pact-N-CoR-FLAG (3 μg) and the plasmid expressing various forms of PML-RARα or the control blank DNA (3 μg), and the internal control pact-β-gal (0.5 μg). Forty hours after transfection, cells were passively lysed in NET buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5% Nonidet P-40, protease inhibitor mixture) containing 300 mm NaCl and centrifuged. The soluble fraction was harvested, and the insoluble fraction was solubilized by sonication in SDS sample buffer, after which fragmented DNA was removed by Sepharose beads. The soluble and insoluble fractions were used for Western blotting with anti-FLAG monoclonal antibody and ECL detection reagents (Amersham Biosciences). The amount of lysate used for Western blotting was normalized on the basis of the β-galatosidase activity. NB4 cells were treated with vehicle, MG132 (5 μm) for 8 h or with RA (1 μm) for 72 h. The soluble fraction of NB4 cells was prepared as described above. To determine the total endogenous N-CoR levels in NB4 cells, lysates were prepared in highly stringent RIPA buffer (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.5% Nonidet P-40, 0.5% SDC, 0.5% SDS, and protease inhibitor mixture) by passing though a syringe 10 times with a 21-gauge needle followed by 3 times with a 26-gauge needle. For each lane in Western blotting, 100 μg of protein was used to detect the N-CoR protein with the anti-N-CoR antibody. As a loading control, aliquots of sample were analyzed by SDS-PAGE and stained with Coomassie Blue. Pulse-chase Experiments—293T cells were transfected with the N-CoR-FLAG (3 μg) expression vector together with the PML-RARα expression plasmid or the control DNA (3 μg), and pact-β-gal (0.5 μg). Twenty-four hours after transfection, cells were labeled with [35S]methionine and [35S]cysteine for 6 h, and then the radioactivity was chased. At various times, lysates were prepared using the NET buffer containing 300 mm NaCl (to prepare soluble N-CoR) or the lysis buffer (10 mm Tris-HCl, pH 8.0, 500 mm NaCl, 2% SDS) (to prepare both the soluble and insoluble N-CoR), heated for 10 min at 95 °C and immunoprecipitated by anti-FLAG antibody after 10-fold dilution and brief sonication. The immunocomplexes were analyzed by SDS-PAGE followed by autoradiography. In Vivo Ubiquitination Assays—293T cells were transfected with 2 μg of pact-N-CoR-FLAG expression vector, 2 μg of pcDNA3-Myc-Ub expression vector, and 2 μg of pact-HA-PML-RARα or empty actin vector. Thirty hours after transfection, the cells were treated with MG132 (10 μm) for 12 h or with RA (2 μm) for 30 h. The cells were then scraped into 100 μl of lysis buffer (10 mm Tris-HCl, pH 8.0, 500 mm NaCl, 2% SDS), heated for 10 min at 95 °C, diluted with buffer lacking SDS to reduce the SDS concentration to 0.2%, and then sonicated mildly on ice. After pre-adsorption with protein G- Sepharose, anti-FLAG M2 monoclonal antibody was used to precipitate the immunocomplexes, which were then used for Western blotting with anti-Myc antibody (MBL). For the ubiquitination assay of N-CoR in NB4 Cells, NB4 cells (4 × 107) were treated with MG132 (5 μm, 12 h) or RA (1 μm, 3 days), and lysates were prepared as described above, immunoprecipitated with anti-N-CoR antibody, and blotted with antiubiquitin antibody (FL-76, Santa Cruz Biotechnology). To examine the effect of the dominant negative form of Ubc6, 293T cells was transfected with 1.5 μg of pact-N-CoR-FLAG, 1.5 μg pact-HA-PML-RARα and 1 μg of pcDNA3-Myc-Ubiquitin with an increasing amount of pcDNA3-Ubc6 (CS mutant) or Ubc5 (CS mutant) expression vectors, and ubiquitination assays were performed as described above. GST Pull-down Assays and Co-immunoprecipitation—GST pull-down assays were performed essentially as described (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The GST fusion of full-length PML-RARα or various domains of PML were used with various in vitro-translated Ubc enzymes in a binding buffer containing 10 mm HEPES (pH 6.5), 2.5 mm MgCl2, 50 μm ZnCl2, 0.5 mm dithiothreitol, and 0.01% bovine serum albumin. For co-immunoprecipitation of PML-RARα and Ubc6, lysates from 293T cells transfected with 3 μg of pact-HA- PML-RARα and 3 μg of FLAG-Ubc6 expression vector were prepared by sonication in LSLD buffer (50 mm Hepes, pH 6.5, 50 mm NaCl, 20 μm NaF, 20% glycerol, and a protease inhibitor mixture). Immunoprecipitation was performed with anti-FLAG antibody, and the anti-HA antibody was used for Western blotting. For co-immunoprecipitation between endogenous Ubc6 and PML-RARα, lysate of NB4 cells prepared in above mentioned buffer was immunoprecipitated with UbcH6 antibody (Boston Bio-chemicals) and Western was done with anti-RARα antibody to detect endogenous PML-RARα protein. For co-immunoprecipitation of PML-RARα and PDI, NB4 cells were lysed in PBS-0.1% Nonidet P-40 by mild sonication, immunoprecipitated with the anti-PDI antibody and blotted with the anti-RAR antibody (Santa Cruz Biotechnology). To detect the association of N-CoR with PDI, a cytosolic fraction of NB4 cells was prepared as described below, immunoprecipitated with anti-PDI antibody, washed with PBS, and N-CoR was detected with goat anti-N-CoR antibody. Flow Cytometry—The retroviral expression plasmid for N-CoR was constructed using the MSCV (murine stem cell virus)-based retroviral vector. The plasmid pMSCV-N-CoR encodes both N-CoR and GFP separated by internal ribosome entry site so that they are expressed together. NB4 cells (10 × 106 cells) were transfected with 40 μg of pMSCV-N-CoR or MSCV empty vector using DMRIE-C reagent (Invitrogen) for 24 h, and then complete medium was added and the cells were cultured for an additional 48 h. After that, the cells were collected, washed twice with PBS, 0.5% BSA, and incubated in 500 μl of PBS, 0.5% BSA with RPE-conjugated monoclonal mouse anti-human CD14 antibodies (DAKO) for 60 min on ice. The cells were then washed with PBS-0.5% BSA and analyzed on a FACScan flow cyometer (BD Biosciences). The GFP-positive cells comprised 3-4% of all the NB4 cells. To detect the exogenous N-CoR in NB4 cells, lysate prepared from nuclear fraction of transfected NB4 cells was subjected to Western blotting using anti-FLAG antibody. To investigate the effect of tunicamycin on RA-mediated differentiation of NB4 cells, NB4 cells were treated with RA (1 μm) alone or with RA and tunicamycin (2.5 μg/ml). Tunicamycin was added in RA-treated cells 24 h before harvesting. Cells were incubated with RPE-conjugated monoclonal mouse anti-human CD11b antibody (DAKO), and FACS analysis was done as described above. Luciferase Reporter Assays—293T cells in 6-well plates were co-transfected with the Gal4 site-containing luciferase reporter vector (0.1 μg), the Ubc6(CS) or Ubc5(CS) expression plasmids in increasing amount (0.2-μg increments), PML-RARα (0.2 μg) expression plasmid, and the internal control plasmid pRL-TK (0.01 μg) using LipofectAMINE-Plus reagent. Dual luciferase assays were performed as recommended by the manufacturer. The PML-RARα/N-CoR Complex Is Recruited to the ER—We previously demonstrated that PML-RARα has two sites that directly bind to N-CoR (8Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Zhong S. Pandolfi P.P. Ishii S. J. Biol. Chem. 2001; 276: 43491-43494Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It is possible that when the N-CoR molecule binds to PML-RARα through these two sites, it adopts an aberrant protein conformation. This may cause N-CoR to become a target of the protein quality control machinery located in the ER. To test this, we assessed whether the PML-RARα/N-CoR complex is bound by protein-disulfide isomerase (PDI) and BiP/GRP78 (immunoglobulin-binding protein/glucose-regulated protein), two of the ER-resident proteins, as they have been shown to mediate ER retention and degradation of poorly assembled and misfolded proteins (25Bottomley M.J. Batten M.R. Lumb R.A. Bulleid N.J. Curr. Biol. 2001; 11: 1114-1118Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 293T cells were cotransfected with the PML-RARα and N-CoR expression vectors and stained with antibodies against either protein together with antibodies that recognize the endogenous PDI and BiP molecule, followed by confocal microscopy analysis. When N-CoR or PML-RARα was expressed alone, the N-CoR signals were localized in the nuclear dot-like structures known as NBs, while PML-RARα was found predominantly in the cytosol like the endogenous PDI and BiP (Fig. 1A). As reported (6Dyck J.A. Maul G.G. Miller Jr., W.H. Chen J.D. Kakizuka A. Evans R.M. Cell. 1994; 76: 333-343Abstract Full Text PDF PubMed Scopus (724) Google Scholar), the microspeckled nuclear PML and PML-RARα signals were very weak compared with the cytosolic signals of overexpressed PML-RARα signals. When N-CoR was co-expressed with PML-RARα, both proteins colocalized with the endogenous PDI or BiP molecules in the ER (Fig. 1B). The signal of PDI and BiP proteins in 293T cells co-expressing PML-RARα/N-CoR was much stronger than those of non-transfected cells, suggesting that co-expression of PML-RARα/N-CoR may induce expression of those proteins (Fig. 1, A and B). When PML-RARα/N-CoR were co-expressed with Golgi-ds-Red, a vector containing Golgi-targeting sequence, some fraction of the PML-RARα/N-CoR signals colocalized with the Golgi signal (Fig. 1C). RA treatment of transfected cells co-expressing PML-RARα/N-CoR resulted in some degree of nuclear relocation of both PML-RARα and N-CoR, and PML NBs formation (Fig. 1D). To confirm the localization of the PML-RARα/N-CoR complex in the ER, we performed a Nycodenz gradient fractionation of the cytoplasmic lysates prepared from 293T cells ectopically expressing N-CoR and PML-RARα (Fig. 1E). Subcellular compartments were fractionated by Nycodenz gradient, and each fraction was probed with antibodies specific to marker protein of the compartment. Fractions 10-16 were designated ER by their possession of either PDI or BiP, and fractions 4-8 were designated Golgi because they contained Golgin-97, the integral membrane protein localized on the cytoplasmic face of the Golgi apparatus (26Barr F.A. Curr. Biol. 1999; 9: 381-384Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). N-CoR was found predominantly in the ER fraction and to some extent, also in the Golgi fraction. PML-RARα was known to form a dimer or oligomer (27Minucci S Maccarana M. Cioce M. De Luca P. Gelmetti V. Segalla S. Di Croce L. Giavara S. Matteucci C. Gobbi A. Bianchini A. Colombo E. Schiavoni I. Badaracco G. Hu X. Lazar M.A. Landsberger N. Nervi C. Pelicci P.G. Mol. Cell. 2000; 5: 811-820Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 28Lin R.J. Evans R.M. M R. Mol. Cell. 2000; 5: 821-830Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), and we could detect both monomer and dimer under standard SDS-PAGE conditions, probably due to its very strong capacity to form a dimer. Both the dimeric and monomeric forms of PML-RARα were found in the ER fraction, while the monomeric PML-RARα was also abundant in the Golgi fraction. Since it was recently shown that the ER-Golgi traffic is a p" @default.
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