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• W1966097798 abstract "In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor κB (NF-κB) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-κB-mediated transactivation induced by physiological stimuli such as tumor necrosis factor α, interleukin-1β, and overexpression of NF-κB-inducing kinase. TLS augmented NF-κB-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-β gene. These results suggest that TLS acts as a coactivator of NF-κB and plays a pivotal role in the NF-κB-mediated transactivation. In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor κB (NF-κB) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-κB-mediated transactivation induced by physiological stimuli such as tumor necrosis factor α, interleukin-1β, and overexpression of NF-κB-inducing kinase. TLS augmented NF-κB-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-β gene. These results suggest that TLS acts as a coactivator of NF-κB and plays a pivotal role in the NF-κB-mediated transactivation. Nuclear factor κB (NF-κB)1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including cytokines, cell adhesion molecules and human immunodeficiency virus (1Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 3Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar, 4Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). The members of the NF-κB family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved 300-amino acid region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-κB (1Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 4Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). In most cells, Rel family members form hetero- and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-κB family, whereas p50 and p52 primarily serve as DNA binding subunits (1Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 4Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). These proteins play fundamental roles in immune and inflammatory responses and in the control of cell proliferation (4Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar,6Kajino S. Suganuma M. Teranishi F. Takahashi N. Tetsuka T. Ohara H. Itoh M. Okamoto T. Oncogene. 2000; 19: 2233-2239Crossref PubMed Scopus (34) Google Scholar, 7Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Yang J.P. Hori M. Takahashi N. Kawabe T. Kato H. Okamoto T. Oncogene. 1999; 18: 5177-5186Crossref PubMed Scopus (104) Google Scholar). A common feature of the regulation of NF-κB is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as IκBs (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 10Sun S.C. Ganchi P.A. Beraud C. Ballard D.W. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1346-1350Crossref PubMed Scopus (162) Google Scholar). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor α (TNF-α) results in phosphorylation, ubiquitination, and degradation of the IκB proteins (5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar, 11Mercurio F. Manning A.M. Curr. Opin. Cell Biol. 1999; 11: 226-232Crossref PubMed Scopus (446) Google Scholar, 12Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (303) Google Scholar). The degradation of IκB proteins exposes the nuclear localization sequence in the remaining NF-κB dimers, followed by the rapid translocation of NF-κB to the nucleus where it activates the target genes by binding to the DNA regulatory element (1Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar, 4Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar, 5Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4585) Google Scholar). The protein regions responsible for the transcriptional activation (called “transactivation domain”) of p65, RelB, and c-Rel have been mapped in their unique C-terminal regions. p65 contains at least two independent transactivation domains within its C-terminal 120 amino acids (Fig. 1 A) (13Moore P.A. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1993; 13: 1666-1674Crossref PubMed Google Scholar, 14Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 15Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Crossref PubMed Scopus (144) Google Scholar). One of these transactivation domains, TA1, is confined to the C-terminal 30 amino acids of p65. The second transactivation domain, TA2, is located within the N-terminally adjacent 90 amino acids and contains TA1-like domain and leucine-rich regions. Since the nuclear translocation and DNA binding of NF-κB were not sufficient for gene induction (17Yoza B.K. Hu J.Y.Q. McCall C.E. J. Biol. Chem. 1996; 271: 18306-18309Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 18Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), it was suggested that interactions with other protein molecules through the transactivation domain (15Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar, 20Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (711) Google Scholar) as well as its modification by phosphorylation (16Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Crossref PubMed Scopus (144) Google Scholar) might play a critical role. It has been previously reported that transcriptional activation of NF-κB requires multiple coactivator proteins including CREB-binding protein (CBP)/p300 (19Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar, 20Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (711) Google Scholar), CBP-associated factor, and steroid receptor coactivator 1 (21Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar). These coactivators have histone acetyltransferase activity to modify the chromatin structure and also provide molecular bridges to the basal transcriptional machinery. Recently, p65 was also found to interact specifically with a newly identified coactivator complex, activator-recruited cofactor/vitamin D receptor-interacting protein, which potentiated chromatin-dependent transcriptional activation by NF-κBin vitro (22Naar A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (371) Google Scholar). In addition to general coactivators, the transcriptional activation of gene-specific activators can be mediated by basal transcription factors through direct interaction with the activation domain. In the case of NF-κB, the association of p65 with basal transcription factors such as TFIIB, TAFII105, and TBP has been demonstrated (15Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Xu X. Prorock C. Ishikawa H. Maldonado E. Ito Y. Gelinas C. Mol. Cell. Biol. 1993; 13: 6733-6741Crossref PubMed Scopus (96) Google Scholar, 24Blair W.S. Bogerd H.P. Madore S.J. Cullen B.R. Mol. Cell. Biol. 1994; 14: 7226-7234Crossref PubMed Scopus (102) Google Scholar, 25Kerr L.D. Ransone L.J. Wamsley P. Schmitt M.J. Boyer T.G. Zhou Q. Berk A.J. Verma I.M. Nature. 1993; 365: 412-419Crossref PubMed Scopus (131) Google Scholar, 26Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (69) Google Scholar, 27Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). It is thus postulated that specific protein-protein interactions with NF-κB determine its transcriptional competence: up-regulation of the NF-κB transcriptional activity is mediated by interaction with basal factors and coactivators, and its down-regulation is mediated by interaction with inhibitors and corepressors at multiple levels. In our previous studies, yeast two-hybrid screen yielded two novel regulators of NF-κB. RelA-associated inhibitor was found to interact with the central region of p65 (RelA) and block DNA binding in the nucleus, similar to the action of cytoplasmic inhibitors IκBs (8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The other proteins found to interact with p65 belong to the Grg (Groucho-related genes) family, including amino-terminal enhancer of split (AES) and transducin-like enhancer of split (TLE1) (7Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), previously known as nuclear corepressors (28Parkhurst S.M. Trends Genet. 1998; 14: 130-132Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 29Fisher A.L. Caudy M. Genes Dev. 1998; 12: 1931-1940Crossref PubMed Scopus (255) Google Scholar). Translocated in liposarcoma (TLS), also known as FUS, was originally identified through its fusion to CHOP, a member of the CCAAT/enhancer-binding protein family of transcription factors, in human myxoid liposarcoma with the t(12;16) chromosomal translocation (30Crozat A. Aman P. Mandahl N. Ron D. Nature. 1993; 363: 640-644Crossref PubMed Scopus (750) Google Scholar, 31Rabbitts T.H. Forster A. Larson R. Nathan P. Nat. Genet. 1993; 4: 175-180Crossref PubMed Scopus (476) Google Scholar). TLS has high homology to hTAFII68/RBP56, EWS, andDrosophila protein SARFH (collectively called the “TET” family (32Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (217) Google Scholar)). These genes were found to be involved in carcinogenesis through chromosomal translocation with other genes of transcription factors; normally the N-terminal region of these proteins provides a transcriptional activator domain, and the moiety of counterpart proteins provides a DNA-binding domain (DBD), thus making these fusion proteins constitutively active for their transcriptional activities (33Prasad D.D. Ouchida M. Lee L. Rao V.N. Reddy E.S. Oncogene. 1994; 9: 3717-3729PubMed Google Scholar, 34Bertolotti A. Bell B. Tora L. Oncogene. 1999; 18: 8000-8010Crossref PubMed Scopus (70) Google Scholar, 35Bailly R.A. Bosselut R. Zucman J. Cormier F. Delattre O. Roussel M. Thomas G. Ghysdael J. Mol. Cell. Biol. 1994; 14: 3230-3241Crossref PubMed Scopus (320) Google Scholar). The C-terminal half of TLS spanning the ribonucleoprotein consensus sequence domain is usually excluded by translocation, and tumorigenic transformation is associated with the fusion of the N-terminal portion to the DNA-binding domain of a given transcription factor (30Crozat A. Aman P. Mandahl N. Ron D. Nature. 1993; 363: 640-644Crossref PubMed Scopus (750) Google Scholar, 31Rabbitts T.H. Forster A. Larson R. Nathan P. Nat. Genet. 1993; 4: 175-180Crossref PubMed Scopus (476) Google Scholar, 36Ichikawa H. Shimizu K. Hayashi Y. Ohki M. Cancer Res. 1994; 54: 2865-2868PubMed Google Scholar). Interestingly, TLS was shown to associate with a subpopulation of the TFIID complex in cells (32Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (217) Google Scholar, 37Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (310) Google Scholar). Moreover, SARFH, a Drosophila homologue of TLS, was colocalized with RNA polymerase II at the active chromatin (38Immanuel D. Zinszner H. Ron D. Mol. Cell. Biol. 1995; 15: 4562-4571Crossref PubMed Scopus (50) Google Scholar). In fact, TLS was shown to be associated with RNA polymerase II through its N-terminal domain (39Yang L. Embree L.J. Hickstein D.D. Mol. Cell. Biol. 2000; 20: 3345-3354Crossref PubMed Scopus (112) Google Scholar). In this study, we demonstrate that TLS interacts with NF-κB p65 through the C-terminal transactivation domain and activates NF-κB-mediated transcription. The yeast two-hybrid interaction assay revealed that the region between two core transactivation domains of p65, TA1-like and TA1, is required for the interaction with TLS. We confirmed the interaction between p65 and TLS in vitro using the bacterially expressed fusion proteins and in vivocoimmunoprecipitation/Western blot assay. In transient transfection assays, TLS showed transactivation potential and activated NF-κB-dependent gene expression. These data indicate that TLS mediates the transcriptional activity of NF-κB. Mammalian expression vector plasmids pCMV-TBP, Gal4-Sp1, pCMV-NIK, ICAM-1-luc (positions −339 to −30), and IFN-β-luc were generous gifts from Drs. T. Tamura (Chiba University), S. T. Smale (UCLA School of Medicine), D. Wallach (Weitzmann Institute of Science), L. A. Madge and J. S. Pober (Yale University School of Medicine), and T. Taniguchi (Tokyo University), respectively. pCMV-p65, pGBT-p65-(1–286), pGBT-p65-(286–551), pGBT-p65-(286–521), pGBT-p65-(286–470), pGBT-p65-(286–442), pGBT-p65-(286–442/477–521), and pGBT-p65-(473–522) have been described previously (7Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To generate the mammalian expression plasmid for TLS, the full-length TLS cDNA fragment was excised from pACT2-TLS with the BamHI and XhoI site, and ligated in frame into pcDNA3.1/HisA vector at theBamHI–XhoI site to form pCMV-TLS. To create a dominant negative form of TLS-(274–525), the TLS cDNA was amplified by polymerase chain reaction using pACT2-TLS as a template with oligoncleotides containing the BamHI–XhoI site. These products were digested withBamHI–XhoI and subcloned in frame into pcDNA3.1/HisA vector at the BamHI–XhoI site to form pCMV-TLS-(274–525). To construct pM-p65-(1–551), which expresses the fusion protein of Gal4-DBD and p65, the cDNA of human p65 (amino acids 1–551) was amplified by polymerase chain reaction using pCMV-p65 as a template with oligonucleotides containingBamHI sites (forward, 5′-CCCCCGGATCCCCGGCCATGGACGAACTGTTC-3′; reverse, 5′-ACCAGGGATCCGGGGAGGGCAGGCGTCACCC-3′). This fragment was digested withBamHI and ligated in frame into the BamHI site of pM (CLONTECH). To generate pM-p65-(1–286) and pM-p65-(286–551), p65 cDNA fragments excised from pGBT-p65-(1–286) with EcoRI and pGBT-p65-(286–551) withBamHI/EcoRI were ligated in frame into the corresponding sites of pM. Construction of a luciferase reporter plasmid, 4κBw-luc or 4κBm-luc, containing four tandem copies of the human immunodeficiency virus-κB sequence upstream of minimal SV40 promoter has been described previously (40Sato T. Asamitsu K. Yang J.P. Takahashi N. Tetsuka T. Yoneyama A. Kanagawa A. Okamoto T. AIDS Res. Hum. Retroviruses. 1998; 14: 293-298Crossref PubMed Scopus (38) Google Scholar). The other luciferase reporter plasmid, 5×Gal4-luc (pFR-luc) was purchased from Stratagene. This plasmid contains five tandem copies of the Gal4 binding site upstream of the TATA box. The yeast two-hybrid screening was performed as previously described (7Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 9Yang J.P. Hori M. Takahashi N. Kawabe T. Kato H. Okamoto T. Oncogene. 1999; 18: 5177-5186Crossref PubMed Scopus (104) Google Scholar). The various portions of the p65 C-terminal regions corresponding to amino acids 286–551, 286–521, 286–470, 286–442, 286–442/477–521, and 473–522 were fused in-frame to Gal4 DNA binding domain (positions 1–147) using the pGBT9 vector (CLONTECH). They were tested for activation of Gal4-dependent lacZ expression (β-galactosidase activity). Among them, pGBT-p65 (286–442/477–521) was chosen as a bait for library screening, since it had undetectable background in the β-galactosidase assay. Yeast strain Y190 was transformed with pGBT-p65-(286–442/477–521), and the human placenta cDNA expression library was fused to the Gal4 transactivation domain in the pACT2 vector (CLONTECH). Approximately one million transformants were screened for the ability to grow on the plates with medium lacking tryptophan/leucine/histidine and containing 25 mm3-aminotriazole. Plasmids were rescued from clones that were positive for β-galactosidase activity and identified by nucleotide sequencing. cDNA sequences and their amino acid sequences were compared with GenBankTM and SwissProt data bases for identification of the interacting proteins. 293 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. Cells were transfected using Fugene-6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. At 48 h post-transfection, the cells were harvested, and the extracts were prepared for luciferase assay. Luciferase activity was measured by the Luciferase Assay System (Promega, Madison, WI) as previously described (8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Transfection efficiency was monitored by Renilla luciferase activity using the pRL-TK plasmid (Promega) as an internal control. The data are presented as the -fold increase in luciferase activities (means ± S.E.) relative to control of three independent transfections. Human recombinant TNF-α and IL-1β were purchased from Roche Molecular Biochemicals. Bacterial expression of GlutathioneS-transferase (GST) fusion proteins utilize pGEX expression vectors. To generate pGEX-TLS-(1–273) and pGEX-TLS-(274–525), which express GST-TLS-(1–273) and GST-TLS-(274–525), the TLS cDNA was amplified by polymerase chain reaction using pACT2-TLS as a template with oligonucleotides containing aBamHI–XhoI site. These products were digested with BamHI–XhoI and subcloned in frame into pGEX-5X-2 vector (Amersham Pharmacia Biotech) at theBamHI–XhoI site. GST fusion proteins were expressed in Escherichia coli strain DH5α and purified as described (8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In vitro protein-protein interaction assays were carried out as described previously (7Tetsuka T. Uranishi H. Imai H. Ono T. Sonta S. Takahashi N. Asamitsu K. Okamoto T. J. Biol. Chem. 2000; 275: 4383-4390Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8Yang J.P. Hori M. Sanda T. Okamoto T. J. Biol. Chem. 1999; 274: 15662-15670Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). p65 and proteins were labeled with [35S]methionine by the in vitrotranscription/translation procedure using the TNT wheat germ extract-coupled system (Promega) according to the manufacturer's protocol. Approximately 20 μg of GST fusion proteins were immobilized on 20 μl of glutathione-Sepharose beads and washed two times with 1 ml of modified HEMNK buffer (20 mm HEPES-KOH (pH 7.5), 100 mm KCl, 12.5 mm MgCl2, 0.2 mm EDTA, 0.3% Nonidet P-40, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). The beads were left in 0.6 ml of HEMNK after the final wash and were incubated with the radiolabeled proteins for 2 h at 4 °C with gentle mixing. The beads were then washed three times with 1 ml of HEMNK buffer and two times with 1 ml of HEMNK buffer containing 150 mm KCl. Bound radiolabeled proteins were eluted with 30 μl of Laemmli sample buffer, boiled for 3 min, and resolved by 10% SDS-PAGE. After transfection of relevant plasmids, 293 cells were cultured for 48 h and then harvested with lysis buffer (25 mm HEPES-NaOH (pH 7.9), 150 mm NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 0.3% Nonidet P-40, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). The lysate was incubated with 1 μg of anti-p65 (NLS) mouse monoclonal antibody (Roche Molecular Biochemicals) or control mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-p65 (C-terminal) rabbit polyclonal antibody (Santa Cruz Biotechnology) or control rabbit polyclonal antibody (Santa Cruz Biotechnology) overnight at 4 °C. 10 μl of protein G-agarose beads were added, and the reaction was further incubated for 1 h. The beads were washed five times with 1 ml of lysis buffer. Antibody-bound complexes were eluted by boiling in 2× Laemmli sample buffer. Supernatants were resolved by 10% SDS-PAGE and transferred on nitrocellulose membrane (Hybond-C; Amersham Pharmacia Biotech). The membrane was incubated with anti-TLS antibody, and immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal; Pierce) as described previously (41Tetsuka T. Daphna-Iken D. Miller B.W. Guan Z. Baier L.D. Morrison A.R. J. Clin. Invest. 1996; 97: 2051-2056Crossref PubMed Scopus (155) Google Scholar, 42Tetsuka T. Baier L.D. Morrison A.R. J. Biol. Chem. 1996; 271: 11689-11693Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Polyclonal antibody to human TLS was a generous gift from K. Shimizu and M. Ohki (National Cancer Center Research Institute, Tokyo, Japan). This antibody was raised by immunizing rabbits with purified recombinant GST-TLS (amino acids 8–134). To evaluate the level of exogenous p65 expressed by pCMV-p65 containing the His epitope tag, rabbit polyclonal anti-His6 antibody (Santa Cruz Biotechnology) was used. To identify proteins interacting with the p65 subunit of NF-κB, we performed the yeast two-hybrid screen using the unique C-terminal region of NF-κB p65 as a bait (TableI and Fig.1). As depicted in Fig. 1 A, various portions of p65 (i.e. amino acids 286–551, 286–521, 286–470, 286–442, 473–522, 286–442/477–521, and 473–522) were fused to Gal4 DNA-binding domain (Gal4-DBD) in the pGBT9 vector. Among these clones, pGBT-p65 (286–442/477–521) was chosen as a bait for the screening, since it had no detectable background in β-galactosidase assay (Table I). Yeast strain Y190 was used to screen human placenta cDNA library fused to the Gal4 transcriptional activation domain in the pACT2 vector (CLONTECH). From ∼1.0 × 106 Y190 yeast transformants, 90 colonies grew on selective medium and turned blue when tested in a filter lift β-galactosidase assay. Each plasmid, purified from the positive colonies, was cotransfected with bait plasmid into the yeast to confirm the specific interaction. DNA sequencing and comparison with GenBankTM and SwissProt data bases revealed the gene for TLS (one clone) in addition to Bcl-3 (one clone) and the IκB family including Iκβα/MAD3 (five clones), which were previously shown to interact with p65.Table IYeast two-hybrid interaction assays between p65 and TLSGal4-DBD hybridGal4-AD hybridpACT2pACT2-TLSpACT2-IκBαpGBT9−−−pGBT-p65-(1–286)−−−pGBT-p65-(286–551)+NDNDpGBT-p65-(286–521)+NDNDpGBT-p65-(286–470)+NDNDpGBT-p65-(286–442)−−+pGBT-p65-(286–442/477–521)−++pGBT-p65-(473–522)−+−Yeast Y190 cells were cotransformed with expression vectors encoding various proteins fused to Gal4-DBD and Gal4-AD. pACT2-TLS is a rescued clone that encodes TLS fused to Gal4-AD. pACT2-IκBα encodes full-length IκBα (amino acids 1–317) fused to Gal4-AD. Leu+ Trp+ transformants were streaked on selective medium lacking leucine/tryptophan and allowed to grow for 2 days at 30 °C. At least three colonies of each transformant were tested for β-galactosidase activity using a 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside colony filter assay (Clontech). +, positive for β-galactosidase activity (blue colony) after 2–3 h; −, no β-galactosidase activity (white colony) after 24 h; ND, activity not determined. Open table in a new tab Yeast Y190 cells were cotransformed with expression vectors encoding various proteins fused to Gal4-DBD and Gal4-AD. pACT2-TLS is a rescued clone that encodes TLS fused to Gal4-AD. pACT2-IκBα encodes full-length IκBα (amino acids 1–317) fused to Gal4-AD. Leu+ Trp+ transformants were streaked on selective medium lacking leucine/tryptophan and allowed to grow for 2 days at 30 °C. At least three colonies of each transformant were tested for β-galactosidase activity using a 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside colony filter assay (Clontech). +, positive for β-galactosidase activity (blue colony) after 2–3 h; −, no β-galactosidase activity (white colony) after 24 h; ND, activity not determined. To determine the region of p65 involved in the binding to TLS, various regions of the protein were fused to Gal4-DBD in the pGBT9 vector and cotransfected with pACT2-TLS, encoding TLS fused to Gal4 transcriptional activation domain. Interactions were tested by β-galactosidase activity (Table I) and by growth of yeast cells on plates with medium lacking histidine, leucine, and tryptophan and containing 25 mm 3-aminotriazole (Fig. 1 B). pGBT-p65-(1–286), pGBT-p65-(286–442), pGBT-p65-(286–442/477–521), and pGBT-p65-(473–522) alone did not show any background in the prototrophic selection or in the β-galactosidase assay. Among these, pGBT-p65-(286–442/477–521) and pGBT-p65-(473–522) were shown to be positive for the interaction with pACT2-TLS (Table I and Fig.1 B). These results indicate that the minimal region of p65 responsible for the interaction with TLS resides within the amino acid sequence 477–521. It was previously implicated that the N-terminal portion of TLS might act as a transcriptional activator in fusion with other transcription factors such as CHOP and ERG (33Prasad D.D. Ouchida M. Lee L. Rao V.N. Reddy E.S. Oncogene. 1994; 9: 3717-3729Pub" @default.
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• W1966097798 date "2001-04-01" @default.
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• W1966097798 title "Involvement of the Pro-oncoprotein TLS (Translocated in Liposarcoma) in Nuclear Factor-κB p65-mediated Transcription as a Coactivator" @default.
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• W1966097798 doi "https://doi.org/10.1074/jbc.m011176200" @default.
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