Voltar
Artigo Revisado por pares
BIBTEX RIS

Enhanced Activation of Tax-dependent Transcription of Human T-cell Leukemia Virus Type I (HTLV-I) Long Terminal Repeat by TORC3

2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês

10.1074/jbc.m409021200

ISSN

1083-351X

Autores

Hiroshi Koga, Takayuki Ohshima, Kunitada Shimotohno,

Tópico(s)

Animal Disease Management and Epidemiology

Resumo

Tax, a protein encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I), interacts with various host cell transcription factors. Tax activates transcription from the long terminal repeat (LTR) of HTLV-I through association with cyclic AMP-responsive element-binding protein (CREB). Here, we present evidence that transducer of regulated cyclic AMP-response element-binding protein 3 (TORC3), a co-activator of CREB, is involved in Tax-induced transcriptional activation from the HTLV-I LTR. By using a luciferase assay system, we show that TORC3 alone can enhance transcription from the HTLV-I LTR, as well as from a cellular cyclic AMP-response element (CRE). Interestingly, we find that co-expression of TORC3 and Tax dramatically increased transcriptional activation at the HTLV-I LTR. We also show by glutathione S-transferase pull-down and co-immunoprecipitation experiments that TORC3 interacts with Tax. Using deletion mutant analysis, we identify the Tax interaction domain of TORC3 as a region spanning from amino acid 1 to 103, which contains a coiled-coil domain. These results provide important clues toward understanding the molecular mechanism of Tax-dependent transcriptional activation of the HTLV-I LTR. Tax, a protein encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I), interacts with various host cell transcription factors. Tax activates transcription from the long terminal repeat (LTR) of HTLV-I through association with cyclic AMP-responsive element-binding protein (CREB). Here, we present evidence that transducer of regulated cyclic AMP-response element-binding protein 3 (TORC3), a co-activator of CREB, is involved in Tax-induced transcriptional activation from the HTLV-I LTR. By using a luciferase assay system, we show that TORC3 alone can enhance transcription from the HTLV-I LTR, as well as from a cellular cyclic AMP-response element (CRE). Interestingly, we find that co-expression of TORC3 and Tax dramatically increased transcriptional activation at the HTLV-I LTR. We also show by glutathione S-transferase pull-down and co-immunoprecipitation experiments that TORC3 interacts with Tax. Using deletion mutant analysis, we identify the Tax interaction domain of TORC3 as a region spanning from amino acid 1 to 103, which contains a coiled-coil domain. These results provide important clues toward understanding the molecular mechanism of Tax-dependent transcriptional activation of the HTLV-I LTR. HTLV-I 1The abbreviations used are: HTLV-I, human T-cell leukemia virus type I; TORC, transducer of regulated CREB activity; CRE, cyclic AMP-responsive element; CREB, cyclic AMP-responsive element-binding protein; CBP, CREB-binding protein; TxRE, Tax-responsive element; LTR, long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; siRNA, small intefering RNA; aa, amino acids; RT, reverse transcription; HA, hemagglutinin; luc, luciferase; RSV, Rous sarcoma virus.1The abbreviations used are: HTLV-I, human T-cell leukemia virus type I; TORC, transducer of regulated CREB activity; CRE, cyclic AMP-responsive element; CREB, cyclic AMP-responsive element-binding protein; CBP, CREB-binding protein; TxRE, Tax-responsive element; LTR, long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; siRNA, small intefering RNA; aa, amino acids; RT, reverse transcription; HA, hemagglutinin; luc, luciferase; RSV, Rous sarcoma virus. is a retrovirus that causes adult T-cell leukemia (1Yoshida M. Seiki M. Yamaguchi K. Takatsuki K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2534-2537Crossref PubMed Scopus (737) Google Scholar) and has been implicated in the development of other diseases such as HTLV-I-associated myelopathy, tropical spastic paraparesis, and uveitis (2Osame M. Usuku K. Izumo S. Ijichi N. Amitani H. Igata A. Matsumoto M. Tara M. Lancet. 1986; 328: 1031-1032Abstract Scopus (1906) Google Scholar, 3Gessain A. Barin F. Vernant J.C. Gout O. Maurs L. Calender A. de The G. Lancet. 1985; 326: 407-410Abstract Scopus (2399) Google Scholar, 4Okamoto T. Ohno Y. Tsugane S. Watanebe S. Shimoyama M. Tajima K. Miwa M. Shimotohno K. Jpn. J. Cancer Res. 1989; 80: 191-195Crossref PubMed Scopus (173) Google Scholar, 5Watanabe T. Int. J. Hematol. 1997; 66: 257-278Crossref PubMed Google Scholar). The onset of adult T-cell leukemia is preceded by a long latent period, suggesting that the process of leukemogenesis depends on multiple steps involving various factors, including viral proteins (4Okamoto T. Ohno Y. Tsugane S. Watanebe S. Shimoyama M. Tajima K. Miwa M. Shimotohno K. Jpn. J. Cancer Res. 1989; 80: 191-195Crossref PubMed Scopus (173) Google Scholar, 6Matsuoka M. Oncogene. 2003; 22: 5131-5140Crossref PubMed Scopus (160) Google Scholar).The viral oncoprotein Tax is encoded by the env-pX gene (8Seiki M. Hattori S. Hirayama Y. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3618-3622Crossref PubMed Scopus (1135) Google Scholar) and plays a principal role in the regulation of the proliferation and transformation of HTLV-I-infected T cells. Tax is a pleiotropic factor that interacts with many cellular proteins to regulate gene expression. For example, Tax regulates the NF-κB pathway using a variety of mechanisms. Tax is not only able to activate NF-κB signaling by binding to NF-κB, IκB, and IKKδ (9Jeang K.T. Cytokine Growth Factor Rev. 2001; 12: 207-217Crossref PubMed Scopus (119) Google Scholar, 10Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 25: 17402-17405Abstract Full Text Full Text PDF Scopus (198) Google Scholar, 11Suzuki T. Hirai H. Yoshida M. Oncogene. 1994; 9: 3099-3105PubMed Google Scholar) but also activates transcription of cellular genes with NF-κB-binding sites in their promoter regions (12Ballard D.W. Bohnlein E. Lowenthal J.W. Wano Y. Franza B.R. Greene W.C. Science. 1988; 241: 1652-1655Crossref PubMed Scopus (348) Google Scholar, 13Leung K. Nabel G.J. Nature. 1988; 333: 776-778Crossref PubMed Scopus (356) Google Scholar), such as Bcl-xL, interleukin-2, and interleukin-2 receptor, to regulate proliferation and transformation of HTLV-I-infected cells (14Akagi T. Shimotohno K. J. Virol. 1993; 3: 1211-1217Crossref Google Scholar, 15Yamaoka S. Tobe T. Hatanaka M. Oncogene. 1992; 3: 433-437Google Scholar). Furthermore, Tax also directly binds to p16ink4a, a member of the INK4 family of cyclin-dependent kinase inhibitors, and suppress its function (16Haller K. Wu Y. Derow E. Schmitt I. Jeang K.T. Grassmann R. Mol. Cell. Biol. 2002; 22: 3327-3338Crossref PubMed Scopus (119) Google Scholar, 17Suzuki T. Kitao S. Matsushima H. Yoshida M. EMBO J. 1996; 15: 1607-1614Crossref PubMed Scopus (247) Google Scholar, 18Iwanaga R. Ohtani K. Hayashi T. Nakamura M. Oncogene. 2001; 20: 2055-2067Crossref PubMed Scopus (111) Google Scholar), which leads to aberrant cell cycle progression.In addition to regulating expression of host cell genes, Tax also regulates transcription of viral genes by interacting directly with the cellular transcriptional factor CREB to activate the HTLV-I-LTR (19Giebler H.A. Loring J.E. van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar). The Tax/CREB complex binds to the Tax-responsive element (TxRE) in the HTLV-I LTR, promoting viral transcription through the recruitment of co-activators such as CBP/p300 and p300/CREB-binding protein-associated factor, a process that does not require phosphorylation of CREB (20Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar, 21Jiang H. Lu H. Schiltz R.L.C. A. Ogryzko V.V. Nakatani Y. Brady J.N. Mol. Cell. Biol. 1999; 19: 8136-8145Crossref PubMed Scopus (132) Google Scholar, 22Harrod R. Tang Y. Nicot C. Lu H.S. Vassilev A. Nakatani Y. Giam C.Z. Mol. Cell. Biol. 1998; 18: 5052-5061Crossref PubMed Scopus (158) Google Scholar). The HTLV-I LTR is composed of three tandem repeats of TxRE, which contains a cyclic AMP-responsive element (CRE) flanked by GC-rich sequences. Although the Tax/CREB complex appears to be recruited predominantly to the TxRE, it is not recruited to the cellular CRE as to a functional complex, suggesting that the CRE-flanking sequence provides specificity for binding of various activation complexes (23Tie F. Adya N. Greene W.C. Giam C.Z. J. Virol. 1996; 70: 8368-8374Crossref PubMed Google Scholar). Indeed, protease sensitivity analysis demonstrated that the conformation of CREB differs according to the specific context of the CRE to which it is bound, indicating that the conformation of CREB is regulated by CRE-flanking sequences (24Connor L.M. Marriott S.J. Virology. 2000; 270: 328-336Crossref PubMed Scopus (10) Google Scholar). Specifically, the weak association of CREB with the CRE sequence of TxRE appears to expose its Tax-binding domain, allowing easy access of Tax to CREB. Binding of Tax may then tighten the ternary complex composed of CREB, Tax, and TxRE. Analysis of this ternary complex suggested that the GC-rich sequence flanking the CRE element in TxRE is crucial for interaction with Tax since this sequence was protected by Tax in a manner that depended on the dose of CREB (25Lundblad J.R. Kwok R.P.S. Laurance M.E. Huang M.S. Richards J.P. Brennan R.G. Goodman R.H. J. Biol. Chem. 1998; 273: 19251-19259Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Moreover, a drug that binds the minor groove of DNA was able to disrupt Tax binding to the CRE-flanking sequence, suggesting that the interaction of Tax with nucleotides in the minor groove of the flanking GC-rich sequence plays an important role in establishing the stable ternary complex. Interaction of protein factors with the flanking sequences in TxRE in HTLV-I-LTR in vivo has been also demonstrated by DNA protection assays (26Datta S. Kothari N.H. Fan H. J. Virol. 2000; 74: 8277-8285Crossref PubMed Scopus (22) Google Scholar).However, these results do not fully explain the mechanism of Tax-dependent transcription of HTLV-I LTR. First of all, the physiological relevance of the ternary complex reconstituted by in vitro synthesized CREB and Tax may be questioned since no evidence has been presented that this complex has any biological function. Furthermore, protein complexes interacting with the flanking sequence in vivo may contain additional factors besides CREB and Tax. For example, we previously identified a cellular protein, TAXREB803/SRL300, which interacts with the 3′-flanking region of TxRE (27Youn H.G. Matsumoto J. Tanaka Y. Shimotohno K. J. Virol. 2003; 77: 10015-10027Crossref PubMed Scopus (4) Google Scholar). This protein enhances Tax-dependent transcription and CREB binding to TxRE in cooperation with Tax. Thus, the precise mechanism of how Tax activation of CREB leads to transcriptional activation of the LTR remains to be elucidated.Recently, TORCs were identified as a family of CREB coactivators that bind to CREB and enhance CRE-mediated transcription in a phosphorylation-independent manner (28Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 29Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12147-12152Crossref PubMed Scopus (310) Google Scholar). This transcriptional activation is facilitated by recruitment of the TATA-binding protein-associated factor 130 (TAF130) component of transcription factors IID (TFIID) to CREB. One member of the family, TORC3, is expressed at high levels in B and T lymphocytes (29Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12147-12152Crossref PubMed Scopus (310) Google Scholar). Based on these studies, we hypothesized that TORC3 might be involved in the transcriptional activation of the HTLV-I LTR by Tax. In this study, we examine the effect of TORC3 on Tax-mediated transcription regulated by the HTLV-I LTR. We find that TORC3 dramatically enhances Tax-mediated viral promoter activity. Furthermore, we show that TORC3 interacts with Tax directly via the N-terminal region of TORC3, which contains a coiled-coil domain.MATERIALS AND METHODSPlasmid Construction—cDNA encoding human TORC3 (accession number NM_022769) was obtained by RT-PCR from HEK-293T cells. The expression plasmid for TORC3 was constructed by inserting the full-length cDNA into EcoRI and XhoI sites of the epitope-tagged form of pcDNA3 (Invitrogen). The primers used for the PCR were 5′-aaagaattcATGGCCGCCTCGCCGGGCTCG-3′ and 5′-aaactcgagTCACAGTCTGTCAGCTCGAAA-3′. Lowercase letters indicate a linker sequence containing EcoRI and XhoI sites, respectively. A reporter plasmid, pTxRE-luc, which drives luciferase expression under a promoter with five copies of the Tax-responsive element, TxRE, was used. To construct plasmids expressing GST fusion TORC3 and its deletion mutants, pGST-TORC3, pGST-TORC3 (1–103 aa), pGST-TORC3 (95–620 aa), pGST-TORC3 (1–298 aa), and pGST-TORC3 (298–620 aa), the corresponding DNA fragments were amplified by PCR using their respective primers. The fragments were then subcloned into EcoRI and XhoI sites in pGEX-6P-1 (Amersham Biosciences). The sequences of the primers used are the following: for GST-TORC3, 5′-aaagaattcATGGCCGCCTCGCCGGGCTCG-3′ and 5′-aaactcgagTCACAGTCTGTCAGCTCGAAA-3′; for GST-TORC3 (1–103 aa), 5′-aaagaattcATGGCCGCCTCGCCGGGCTCG-3′ and 5′-tttctcgagGAAGCGGTTCCTGGATGGCCT-3′; for GST-TORC3 (95–620 aa), 5′-aaagaattcGTGGAGAGGCCATCCAGGAAC-3′ and 5′-aaactcgagTCACAGTCTGTCAGCTCGAAA-3′; for GST-TORC3 (1–298 aa), 5′-aaagaattcATGGCCGCCTCGCCGGGCTCG-3′ and 5′-tttctcgagTGCCAAAGTGGTGGTCGGTGG-3′; and for GST-TORC3 (298–620 aa), 5′-aaagaattcGGCAGTATGAGTGTGGGGAAT-3′ and 5′-aaactcgagTCACAGTCTGTCAGCTCGAAA-3′. The expression plasmid for GAL4 fusion Tax was constructed by inserting full-length Tax into BamHI and SalI sites in pGAL4-luc. Full-length Tax was amplified by PCR using 5′-aaaggatccATGGCCCATTTCCCAGGGT-3′ and 5′-aaactcgagTCAGACTTCTGTTTCTCGGAA-3′ as primers and pcDNA3-Tax (30Ariumi Y. Kaida A. Lin J.Y. Hirota M. Masui O. Yamaoka S. Taya Y. Shimotohno K. Oncogene. 2000; 19: 1491-1499Crossref PubMed Scopus (110) Google Scholar) as a template. Lowercase letters indicate a linker sequence containing EcoRI and XhoI sites, respectively.Cell Culture—HEK-293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and were cultured at 37 °C in a 5% CO2 humidified chamber.Plasmid Transfection—HEK-293T cells were grown to 50% confluence in 60-mm dishes or 24-well dishes in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were transfected with expression vectors or corresponding empty vector constructs using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions.Antibodies—Rat anti-HA (3F10; Roche Applied Science) and mouse anti-Myc (9E10; Santa Cruz Biotechnology) antibodies were purchased commercially. A rabbit polyclonal antibody specific for Tax was raised against the bacterially expressed GST-Tax (233–353 aa) fusion protein. Horseradish peroxidase-linked goat antibodies to rat IgG were from Jackson ImmunoReseach Laboratories. Horseradish peroxidase-linked goat antibodies to mouse or rabbit IgG were from Amersham Biosciences.GST Pull-down Assay—GST fusion proteins were expressed in Escherichia coli and isolated by an affinity column with glutathione-Sepharose-4B beads (Amersham Biosciences). GST fusion proteins bound to the resin were then incubated with [35S]methionine-labeled Tax, which was synthesized by in vitro translation using the TnT-coupled transcription-translation system (Promega). The binding reactions were carried out in 600 ml of GST binding buffer (20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5% glycerol, 1% Nonidet P-40, 0.5 mm EDTA, and 1 mm dithiothreitol, supplemented with a Complete protease inhibitor mixture tablet (Roche Applied Science)) for 2 h at 4 °C. The beads were washed three times with 1 ml of GST binding buffer. The bound proteins were eluted by adding 20 ml of sample buffer and resolved by SDS-PAGE and then analyzed by autoradiography.Co-immunoprecipitation Assay—Co-immunoprecipitation assays were performed using HEK-293T cells. The cells were lysed with radioimmune precipitation buffer (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, and 1 mm dithiothreitol, supplemented with a Complete protease inhibitor mixture tablet). Cell debris was removed by centrifugation for 5 min. The lysates were first cleared with protein G beads for 15 min and then incubated with 2 μg of anti-Tax for 2 h at 4 °C and incubated for another 1 h following the addition of 20 ml of protein G bead slurry. The beads were then washed three times with radioimmune precipitation buffer, and immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot.Luciferase Reporter Assay—Luciferase assays were performed with the dual luciferase assay system (Promega) according to the manufacturer's instructions. Luciferase activities in cell lysates were normalized relative to the Renilla luciferase activity derived from co-transfected pRL-RSV-luc (31Ohshima T. Koga H. Shimotohno K. J. Biol. Chem. 2004; 279: 29551-29557Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). All reporter assays were performed in triplicate, and standard errors (S.E.) were denoted by the bars in the figures.RNA Interference—A duplex of 21-nucleotide small interfering RNA (siRNA) specific to mRNA for TORC3, including two nucleotides of deoxythymidine at the 3′ end, was synthesized (Qiagen) and annealed according to the manufacturer's protocol. The sense sequence of the duplex was 5′-CACCAUCCUGCCAGAAGACdTdT, and the antisense sequence was 3′-GUCUUCUGGCAGGAUGGUGdTdT. HEK-293T cells were transfected with 25 μl of 20 μm duplex RNA per 6-cm dish with Oligofectamine (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, the medium was replaced with the normal culture medium. For the RNA analysis, total RNA was isolated from cells 24 h after siRNA transfection and used as a template for RT-PCR. Primers used were 5′-AAAGAATTCGCCTTCGAGCAGCTCATGACC-3′ for the forward orientation and 5′-TTTCTCGAGGAAGCGGTTCCTGGATGGCCT-3′ for the reverse orientation.RNA Isolation, RT-PCR, and Reporter Assay—Total RNA was extracted by ISOGEN (Nippon Gene) according to the manufacturer's protocol. To obtain TORC3 cDNA from the isolated RNA, reverse transcription was carried out using oligo(dT)12–18 as a primer in a reaction volume of 20 μl, in which 1 μg of total RNA was present. The cDNA was subjected to PCR with Taq polymerase (Promega) using 5′-AAAGAATTCGCCTTCGAGCAGCTCATGACC-3′ as a forward primer and 5′-TTTCTCGAGGAAGCGGTTCCTGGATGGCCT-3′ as a reverse primer. PCR was run for 25 cycles of denaturation (94 °C for 30 s), annealing (52 °C for 30 s), and extension (72 °C for 1 min). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control for the RT-PCR. The primers used to amplify GAPDH mRNA were 5′-ATGGGGAAGGTGAAGGTCGG-3′ and 5′-TGGAGGGATCTCGCTCCTGG-3′. PCR for GAPDH was conducted for 20 cycles.For the reporter assay, at 24 h after the transfection with siRNA, the reporter plasmids were transfected together with pcDNA3-Tax by using FuGENE 6. At 24 h after the transfection of the reporter plasmids, expression of firefly luciferase from pGL3-LTR-luc and Renilla luciferase from pRL-RSV-luc was evaluated with the dual luciferase reporter assay system.RESULTSTORC3 Activates Transcription at the HTLV-I LTR as Well as Cellular CRE-containing Promoters—Since TORC3 is a co-activator of the transcription factor CREB that enhances CRE-mediated transcription of target genes, we explored the effect of TORC3 on Tax-dependent transcription from the CRE-containing HTLV-I LTR (Fig. 1A). We first performed a luciferase assay using a pLTR-luc reporter plasmid. We found that luciferase activity was enhanced by expression of either Tax or TORC3 (Fig. 1B) and was further enhanced by co-expression of Tax and TORC3. To determine whether TORC3 can activate CREB-dependent transcription of genes whose promoters contain a cellular CRE sequence, we transfected TORC3 into HEK-293T cells carrying the luciferase reporter plasmid, pCRE-luc, which has four repeats of the CRE consensus sequence (Fig. 1C). TORC3 alone activates pCRE-luc ∼200-fold above untransfected control cells, whereas Tax was unable to activate transcription from pCRE-luc (Fig. 1D). However, co-expression of Tax and TORC3 in HEK-293T cells greatly enhanced the expression of luciferase from pCRE-luc (Fig. 1D), suggesting a synergistic interaction between these two factors that may promote high levels of transcription in a cellular context.Since Tax potentiates the binding of CREB to the TxRE in the HTLV-I 5′-LTR to initiate viral gene expression, we next performed a reporter assay using pTxRE-luc. This reporter plasmid has in its promoter region five copies of the Tax-responsive element, which contains a core CRE element flanked by GC-rich sequences (Fig. 1C). In HEK293T cells transiently expressing TORC3 alone, transactivation of the HTLV-I LTR reached levels ∼700-fold above background (Fig. 1B). Intriguingly, co-expression of Tax and TORC3 dramatically enhanced pTxRE-luc expression to over 5,000-fold above background (Fig. 1D). These results indicate that although TORC3 alone can enhance transcription from the HTLV-I LTR via the TxRE, Tax is able to synergize with TORC3 to further increase transcription levels.Determination of the Region of TORC3 Important for Enhancement of Tax-mediated Transcription—To identify the region required for Tax-mediated transcription, we generated several deletion mutants of TORC3 (Fig. 2A) and examined the effect of these mutant proteins on Tax-mediated transcription. When HEK-293T cells were co-transfected with TORC3 (1–298 aa) and Tax, the level of the reporter activity remained at two-thirds of that of cells expressing Tax and full-length TORC3 (Fig. 2B). However, when TORC3 (298–620 aa) or TORC3 (95–620 aa), which lack the N-terminal region of TORC3, were co-transfected with Tax, the reporter activity was at levels comparable with those observed in cells expressing Tax alone (Fig. 2B). These results suggest that the N-terminal region of TORC3 is involved in Tax-mediated transactivation via the TxRE element. In fact, TORC3 (1–103 aa), which contains only the N-terminal region, showed significant activity.Fig. 2TORC3 interacts with Tax through its N-terminal region, which contains a coiled-coil domain. A, a schematic representation of TORC3 and its deletion mutants. The N-terminal black bar shows the coiled-coil domain. B, the effects of TORC3 and deletion mutants on Tax-mediated LTR transactivation. HEK-293T cells were co-transfected with 10 ng of pLTR-luc and an empty plasmid (–) or plasmids expressing TORC3 (full-length), TORC3 (1–298 aa), TORC3 (298–620 aa), TORC3 (1–103 aa), or TORC3 (95–620 aa) together with (open bar) or without (solid bar) a plasmid expressing Tax. At 24 h after transfection, luciferase activities in cell lysates were measured. Relative luciferase activity in cell lysates with and without Tax expression was normalized to that of the cell lysate without Tax and TORC3 expression. Expression levels of HA-TORC3 and its mutants were examined by Western blotting with an anti-HA antibody. Average values of three independent experiments are shown, and standard error (S.E.) is denoted by the error bars. C, in vitro-translated full-length [35S]methionine-labeled Tax was incubated with GST and GST-TORC3 derivatives, which were immobilized on glutathione-Sepharose beads. The pull-down complexes were analyzed by SDS-PAGE followed by autoradiography. Input corresponds to 10% of the total amount of the reaction mixture. D, HEK-293T cells were transfected with 2 μg of pcDNA3-Myc-TORC3 together with (lane 2) or without (lane 1) 2 μg of pcDNA3-Tax. At 36 h after transfection, cell lysates were immunoprecipitated (IP) by an anti-Tax antibody, and co-immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by Western blot with an anti-Myc antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TORC3 Interacts with Tax to Promote the Transactivation via the CRE Sequence—The N-terminal region of TORC3 contains a coiled-coil domain (∼70 aa), a motif that has often been implicated in protein-protein interactions. Since TORC3 is a CREB-interacting protein, we hypothesized that TORC3 enhancement of transcription is mediated through its association with Tax. To test this possibility, we performed a GST pull-down assay using bacterially generated GST-TORC3 fusion proteins and in vitro-translated Tax protein. As shown in Fig. 2C, GST-TORC3, but not GST alone, was able to associate with [35S]methionine-labeled Tax (Fig. 2C, lanes 2 and 3). We found that Tax was able to bind to the region of TORC3 containing the coiled-coil domain (Fig. 2C, lanes 4 and 6) but was not able to interact with GST-TORC3 (95–620 aa) or GST-TORC3 (298–620 aa), both of which lacked the coiled-coil domain (Fig. 2C, lanes 5 and 7).Next, to analyze the interaction of TORC3 with Tax in cells, co-immunoprecipitation experiments were performed. HEK-293T cells were co-transfected with expression plasmids encoding Tax and Myc-tagged TORC3. The immunocomplex precipitated with the anti-Tax antibody was resolved by SDS-PAGE and analyzed by Western blot using the anti-Myc antibody. A 78-kDa band corresponding to the expected size of TORC3 was detected (Fig. 2D, lane 2), suggesting that TORC3 does in fact interact with Tax in cells. This result, together with the data shown above, suggests that the interaction of TORC3 with Tax is required to enhance transcription synergistically via CRE promoter sequences.TORC3 Functions as a Co-activator for Tax-mediated Transcriptional Activation—To examine whether TORC3 has effects on Tax-dependent transcription activity, we constructed an expression plasmid encoding a GAL4 DNA-binding domain-Tax fusion protein, GAL4-Tax, as well as a luciferase reporter plasmid, pGL3-luc, which contains five GAL4-binding sites and an SV40 minimal promoter. These plasmids were introduced into HEK-293T cells together with plasmids expressing full-length TORC3 or TORC3 deletion mutants (Fig. 3). Expression of GAL4-Tax alone is able to activate pGL3-luc. However, co-expression of GAL4-Tax and full-length TORC3 induced a 3-fold increase in pGL3-luc activation as compared with GAL4-Tax alone. Co-expression of GAL4-Tax and TORC3 (1–298 aa) also resulted in enhanced luciferase activity, whereas we observed no synergy in cells co-expressing GAL4-Tax and TORC3 (298–620 aa). These results indicate that the interaction between Tax and TORC3 allows TORC3 to function as a co-activator for Tax.Fig. 3Tax-dependent transcription is enhanced by TORC3. HEK-293T cells were cotransfected with 25 ng of GAL4-luciferase reporter plasmid, and 10 ng of the plasmids expressing the GAL4 DNA-binding domain (indicated by GAL4) or the GAL4 DNA-binding domain-Tax fusion protein (indicated by GAL4-Tax) was co-transfected with 100 ng of the plasmid expressing the full-length TORC3, TORC3 (1–298 aa), TORC3 (298–620 aa), or an empty plasmid. At 24 h after transfection, luciferase activity was measured. Relative luciferase activities normalized to those of the cell lysate from cells transfected with pGAL4-luc, an empty plasmid, and pGAL4 are shown. Average values of three independent experiments are shown, and standard error (S.E.) is denoted by the error bars. Cells transfected with an empty plasmid, pcDNA3, are indicated by a minus sign.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gene Silencing of TORC3 by siRNA Reduces Tax-mediated Transcriptional Activation—To examine the function of TORC3 under physiological conditions, we employed siRNA technology, using a synthetic oligonucleotide that forms a duplex RNA encoding nucleotides 1638–1646 of TORC3. Treatment with TORC3 siRNA efficiently reduced the level of TORC3 mRNA in HEK-293T cells to 10% of the control level, whereas the level of GAPDH RNA remained unchanged (Fig. 4A). TORC3 siRNA treatment also resulted in an approximately two-thirds decrease in Tax-mediated transactivation, as measured by pTxRE-luc and pLTR-luc expression (Fig. 4, B and C). The values of luciferase activity after 48 h of transfection were almost same as those observed at 24 h (data not shown). Taken together, these data suggest that TORC3 plays an important role in Tax-mediated transactivation of the HTLV-I LTR.Fig. 4Tax-mediated transcription depends on endogenous TORC3. A, suppression of endogenous TORC3 by siRNA. Duplexes of 21-nucleotide TORC3 siRNA and control siRNA (random 21-nucleotide sequences) were transfected into HEK-293T cells in 6-cm dishes. TORC3 mRNA levels were examined by RT-PCR at 24 h after transfection. B and C, for the

Referência(s)