The C-terminal Activating Region 2 of the Epstein-Barr Virus-encoded Latent Membrane Protein 1 Activates NF-κB through TRAF6 and TAK1
2005; Elsevier BV; Volume: 281; Issue: 4 Linguagem: Inglês
10.1074/jbc.m505903200
ISSN1083-351X
AutoresLiming Wu, Hiroyasu Nakano, Zhenguo Wu,
Tópico(s)Histiocytic Disorders and Treatments
ResumoEpstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-κB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-κB activation, with CTAR2 being the main NF-κB activator. Although the CTAR1-mediated NF-κB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-κB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-κB pathways, the CTAR2-mediated NF-κB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-κB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-κB: a major one through CTAR2/TRAF6/TAK1/IKKβ (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKα (noncanonical pathway). Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-κB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-κB activation, with CTAR2 being the main NF-κB activator. Although the CTAR1-mediated NF-κB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-κB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-κB pathways, the CTAR2-mediated NF-κB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-κB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-κB: a major one through CTAR2/TRAF6/TAK1/IKKβ (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKα (noncanonical pathway). Various mammalian cell viruses exert their cytotoxic effects by interfering with the function of specific host factors and the cellular signaling pathways associated with these host factors, eventually leading to abnormal cellular processes. Thus, elucidation of the molecular mechanisms by which viral proteins engage specific host factors and modulate cellular signaling pathways is the key to the understanding of viral-mediated diseases. Epstein-Barr virus (EBV) 2The abbreviations used are: EBV, Epstein-Barr virus; MEF, mouse embryonic fibroblast; TNF, tumor necrosis factor; LMP, latent membrane protein; CTAR, C-terminal activating region; NPC, nasopharyngeal carcinoma; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; IKK, IκB kinase; NIK, NF-κB-inducing kinase; MAP3K, MAPK kinase kinase; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; TRAF, TNF receptor-associated factor; IL, interleukin; IL-1R, IL-1β receptor; TLR, toll-like receptor; TRADD, TNF receptor-associated death domain; MyD88, myeloid differentiation factor 88; IRAK, IL-1R-associated kinase; GFP, green fluorescent protein; siRNA, small interfering RNA; WCE, whole cell extract(s); HA, hemagglutinin; GST, glutathione S-transferase. 2The abbreviations used are: EBV, Epstein-Barr virus; MEF, mouse embryonic fibroblast; TNF, tumor necrosis factor; LMP, latent membrane protein; CTAR, C-terminal activating region; NPC, nasopharyngeal carcinoma; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; IKK, IκB kinase; NIK, NF-κB-inducing kinase; MAP3K, MAPK kinase kinase; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; TRAF, TNF receptor-associated factor; IL, interleukin; IL-1R, IL-1β receptor; TLR, toll-like receptor; TRADD, TNF receptor-associated death domain; MyD88, myeloid differentiation factor 88; IRAK, IL-1R-associated kinase; GFP, green fluorescent protein; siRNA, small interfering RNA; WCE, whole cell extract(s); HA, hemagglutinin; GST, glutathione S-transferase. is known to be causally linked to a number of human malignancies including Burkitt's lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC) (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven, Philadelphia1996: 2397-2446Google Scholar). The incidence of NPC in southern China including Guangdong Province and Hong Kong is among the highest in the world (2Lee A.W. Foo W. Mang O. Sze W.M. Chappell R. Lau W.H. Ko W.M. Int. J. Cancer. 2003; 103: 680-685Crossref PubMed Scopus (146) Google Scholar). EBV mainly infects two cell types: peripheral B cells and nasopharyngeal epithelial cells (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven, Philadelphia1996: 2397-2446Google Scholar, 3Thorley-Lawson D.A. Nat. Rev. Immunol. 2001; 1: 75-82Crossref PubMed Scopus (735) Google Scholar). Although it is difficult for EBV to infect nasopharyngeal epithelial cells in vitro, EBV readily infects resting human B cells in vitro and converts them to immortalized lymphoblastoid cell lines (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven, Philadelphia1996: 2397-2446Google Scholar). Three forms of latency have been recognized for various EBV-associated diseases (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven, Philadelphia1996: 2397-2446Google Scholar, 4Moss D.J. Schmidt C. Elliott S. Suhrbier A. Burrows S. Khanna R. Adv. Cancer Res. 1996; 69: 213-245Crossref PubMed Google Scholar). In latency I, as exemplified by endemic Burkitt's lymphoma, only one EBV-encoded protein, EBNA1, is expressed. In latency II as exemplified by NPC, three EBV-encoded proteins, namely EBNA1, LMP1, and LMP2, are expressed. In latency III, as exemplified by lymphoblastoid cell lines and infectious mononucleosis, a total of nine EBV-encoded proteins are expressed. Among all of the latent viral proteins expressed in the three different latencies, LMP1 is the only one known to be capable of transforming rodent fibroblasts in vitro (5Wang D. Liebowitz D. Kieff E. Cell. 1985; 43: 831-840Abstract Full Text PDF PubMed Scopus (988) Google Scholar). It is also indispensable for EBV-mediated B cell transformation (6Kaye K.M. Izumi K.M. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9150-9154Crossref PubMed Scopus (654) Google Scholar). In addition, transgenic mice expressing LMP1 under a lymphocyte-specific promoter display a much higher incidence of lymphoma (7Kulwichit W. Edwards R.H. Davenport E.M. Baskar J.F. Godfrey V. Raab-Traub N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11963-11968Crossref PubMed Scopus (330) Google Scholar). LMP1 is a 386-amino acid membrane-localized viral protein with six transmembrane domains (Fig. 1A). Two subdomains in the 200-amino acid cytoplasmic carboxyl tail of LMP1, namely CTAR1 (amino acids 194-232) and CTAR2 (amino acids 351-386), are crucial for interacting with specific cellular factors and for engaging key cellular signaling pathways. Among them are the NF-κB pathway and two mitogen-activated protein kinase (MAPK) pathways (i.e. c-Jun N-terminal kinase (JNK) and p38 pathways) (8Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (185) Google Scholar, 9Mosialos G. Cytokine Growth Factor Rev. 2001; 12: 259-270Crossref PubMed Scopus (28) Google Scholar). Although CTAR2 is solely responsible for the LMP1-induced JNK pathway, both CTAR1 and CTAR2 contribute to NF-κB activation, with CTAR2 accounting for 70% of the total NF-κB activity induced by LMP1 (8Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (185) Google Scholar, 9Mosialos G. Cytokine Growth Factor Rev. 2001; 12: 259-270Crossref PubMed Scopus (28) Google Scholar). NF-κB, a dimeric transcription factor, plays a key role in activation of certain viral genes during viral infection and in host immune response to various microbial infections (10Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar, 11Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar, 12Santoro M.G. Rossi A. Amici C. EMBO J. 2003; 22: 2552-2560Crossref PubMed Scopus (309) Google Scholar). NF-κB is normally sequestered in cytosol by the IκB family proteins. In response to cytokine stimulation or microbial infection, IκB is first phosphorylated by the IκB kinase (IKK) complex, which consists of three core members IKKα, IKKβ, and IKKγ, and subsequently degraded in a ubiquitin/proteasome-dependent manner (10Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar, 11Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar). Two distinct NF-κB pathways have been recognized; one is the canonical pathway, which mainly utilizes the catalytic activity of IKKβ to phosphorylate IκBα and induces its degradation resulting in generation of free p50/p65 dimer and their translocation into the nucleus, and the other is the noncanonical (or alternative) pathway, which mainly involves IKKα leading to processing of p100 (i.e. NF-κB2) and generation of free p52/RelB dimer (10Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar, 11Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar, 13Senftleben U. Karin M. Crit. Care Med. 2002; 30: S18-S26Crossref Scopus (264) Google Scholar). Both IKKα and β need to be further phosphorylated by upstream kinases to achieve maximal activities. Based on genetic and biochemical evidence, NF-κB-inducing kinase (NIK), a member of the MAP3K superfamily, was found to function mainly in the noncanonical pathway (14Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar), whereas a number of MAP3Ks including MEKK1, MEKK3, and TAK1, have been implicated in the canonical pathway (see “Discussion”). A major breakthrough in our understanding of the molecular mechanisms underlying the LMP1-induced NF-κB activation came after the discovery that LMP1 specifically recruits tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) through a conserved TRAF-binding motif (i.e. 204PQQAT208) in CTAR1 (Fig. 1A) (15Brodeur S.R. Cheng G. Baltimore D. Thorley-Lawson D.A. J. Biol. Chem. 1997; 272: 19777-19784Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Devergne O. Hatzivassiliou E. Izumi K.M. Kaye K.M. Kleijnen M.F. Kieff E. Mosialos G. Mol. Cell. Biol. 1996; 16: 7098-7108Crossref PubMed Google Scholar, 17Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kieff E. Cell. 1995; 80: 389-399Abstract Full Text PDF PubMed Scopus (903) Google Scholar). The TRAF family proteins consist of six members (i.e. TRAF1-6) and mainly function as adaptors in various cytokine-mediated NF-κB and MAPK pathways (18Bradley J.R. Pober J.S. Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (514) Google Scholar, 19Chung J.Y. Park Y.C. Ye H. Wu H. J. Cell Sci. 2002; 115: 679-688Crossref PubMed Google Scholar). Among them, TRAF2/5 mainly function in the TNFα-mediated NF-κB pathway, whereas TRAF6 preferentially acts in the interleukin-1β receptor (IL-1R)/toll-like receptor (TLR)-mediated NF-κB pathway (18Bradley J.R. Pober J.S. Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (514) Google Scholar, 19Chung J.Y. Park Y.C. Ye H. Wu H. J. Cell Sci. 2002; 115: 679-688Crossref PubMed Google Scholar). Interestingly, different TRAFs are selectively recruited to distinct cytokine receptors through unique adaptor molecules. Whereas TNF receptor-associated death domain (TRADD) protein specifically recruits TRAF2/5 and receptor-interacting kinase 1 to TNF receptor, myeloid differentiation factor 88 (MyD88) selectively links IL-1R-associated kinases 1 and 4 (IRAK1/4) and TRAF6 to IL-1R/TLR (18Bradley J.R. Pober J.S. Oncogene. 2001; 20: 6482-6491Crossref PubMed Scopus (514) Google Scholar, 19Chung J.Y. Park Y.C. Ye H. Wu H. J. Cell Sci. 2002; 115: 679-688Crossref PubMed Google Scholar, 20Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6623) Google Scholar). Recent studies by several groups showed that the CTAR1 domain of LMP1 activates NF-κB mainly through the noncanonical pathway (21Atkinson P.G. Coope H.J. Rowe M. Ley S.C. J. Biol. Chem. 2003; 278: 51134-51142Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Eliopoulos A.G. Caamano J.H. Flavell J. Reynolds G.M. Murray P.G. Poyet J.L. Young L.S. Oncogene. 2003; 22: 7557-7569Crossref PubMed Scopus (94) Google Scholar, 23Luftig M. Yasui T. Soni V. Kang M.S. Jacobson N. Cahir-McFarland E. Seed B. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 141-146Crossref PubMed Scopus (147) Google Scholar, 24Saito N. Courtois G. Chiba A. Yamamoto N. Nitta T. Hironaka N. Rowe M. Yamaoka S. J. Biol. Chem. 2003; 278: 46565-46575Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). It remains unclear how CTAR2 activates NF-κB. Although several earlier reports suggested that CTAR2 utilizes TRADD and TRAF2 to activate NF-κB based on overexpression studies (25Izumi K.M. Kaye K.M. Kieff E.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1447-1452Crossref PubMed Scopus (188) Google Scholar, 26Kaye K.M. Devergne O. Harada J.N. Izumi K.M. Yalamanchili R. Kieff E. Mosialos G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11085-11090Crossref PubMed Scopus (221) Google Scholar, 27Kieser A. Kaiser C. Hammerschmidt W. EMBO J. 1999; 18: 2511-2521Crossref PubMed Scopus (104) Google Scholar), a few recent reports raised doubts about their involvement (27Kieser A. Kaiser C. Hammerschmidt W. EMBO J. 1999; 18: 2511-2521Crossref PubMed Scopus (104) Google Scholar, 28Xie P. Bishop G.A. J. Immunol. 2004; 173: 5546-5555Crossref PubMed Scopus (44) Google Scholar, 29Xie P. Hostager B.S. Bishop G.A. J. Exp. Med. 2004; 199: 661-671Crossref PubMed Scopus (106) Google Scholar). We showed here that CTAR2 preferentially recruits TRAF6 and TAK1 to activate IKKβ and NF-κB. TRADD, MyD88, TRAF2/5, IRAK1/4, TAB2, and MEKK1 are not essential in the LMP1-mediated NF-κB pathway. Cell Lines, DNA Constructs, and Reagents—HEK293, HEK293T, MEKK1-/-, TAB2-/-, TRAF6-/-, TRAF2/5-/-, IRAK4-/-, MyD88-/-, and the wild type MEF cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 unit/ml of penicillin, and 100 μg/ml of streptomycin at 37 °C in 5% CO2. LMP1(D335), LMP1(G335), HA-IKKβ, HA-TRAF6, and LMP1(ΔC8) were described previously (30Wan J. Sun L. Mendoza J.W. Chui Y.L. Huang D.P. Chen Z.J. Suzuki N. Suzuki S. Yeh W.C. Akira S. Matsumoto K. Liu Z.G. Wu Z. Mol. Cell. Biol. 2004; 24: 192-199Crossref PubMed Scopus (64) Google Scholar, 31Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar). LMP1(3A) mutant (i.e. Pro204-Gln206-Thr208 → Ala204-Ala206-Ala208) was generated by PCR-based mutagenesis using LMP1(D335) as the template. LMP1(3A/ΔC8) was generated from LMP1(3A) by deleting the last 8 amino acids at the C terminus. GFP-p65 was generated by inserting human p65 gene into pEGFPC1 between XhoI and SmaI sites (a generous gift from G. Natoli). IL-1β and TNFα were purchased from R & D Systems. d(-)-Luciferin was purchased from Roche Applied Science. Puromycin, Polybrene, and tetracycline were purchased from Sigma. Plasmid and siRNA Transfection—For plasmid transfection, either Lipofectamine Plus reagents (for HEK293 cells) or Lipofectamine 2000 (for MEFs) (Invitrogen) were used following the manufacturer's instructions. For siRNA transfection, Lipofectamine 2000 was routinely used. In transfection experiments involving both siRNA and plasmids, the cells were first transfected twice with siRNA. 15 h later, the cells were then transfected with plasmids. All of the siRNAs were purchased from Dharmacon Inc (Lafayette, Co). The target sequences were as follows: TAK1, 5′-(AA)GAGAUCGACUACAAGGAGA; TRADD, 5′-(AA)CUGGCUGAGCUGGAGGAUG; IRAK1, 5′-(AA)GUUGCCAUCCUCAGCCUCC; and GFP (control), 5′-(AA)GGUGGCAGUGUACAGGGCU. Cell Lysis—24-36 h after transfection, the cells were lysed in the lysis buffer (50 mm HEPES, pH 7.6, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 150 mm NaCl, 1 mm EGTA, 1.5 mm MgCl2, 100 mm NaF, 20 mm p-nitrophenyl phosphate, 20 mm β-glycerol phosphate, 2 mm dithiothreitol, 50 μm sodium vanadate, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml of aprotinin, 0.5 μg/ml of leupeptin, and 0.7 μg/ml of pepstatin) for 10 min, followed by removal of insoluble debris with a bench top centrifuge at 15,000 × g for 2 min to obtain whole cell extracts (WCE). Luciferase Reporter Assays—HEK293T cells were co-transfected with a luciferase reporter plasmid (3xκB-luc) containing three tandem κB sites (5′-GATCTGGGGATTCCCCA) and other plasmids as indicated. WCE were harvested 36 h after transfection. 15 μl of WCE were mixed with 150 μl of freshly made luciferase reaction buffer (100 mm Tris acetate, pH 7.8, 1 mm EDTA, 10 mm magnesium acetate, 66 μm d(-)-luciferin, and 2 mm ATP). Luciferase activity was determined with an LB 9507 luminometer (Berthold Technologies). Luciferase units were normalized against the total protein amount present in each sample determined by protein assay reagent from Bio-Rad. Antibodies and Western Blotting—Mouse monoclonal antibodies to HA (F-7, sc-7392; Santa Cruz), p100/p52 (05-361; Upstate Biotechnology, Inc.), β-tubulin (T4026; Sigma), rabbit polyclonal antibodies to IKKβ (H470, sc-7607; Santa Cruz) and IRAK1 (H-273, sc-7883; Santa Cruz), and goat polyclonal antibody to TRADD (C-20, sc-1163; Santa Cruz) were used in this study. Monoclonal anti-LMP1 and polyclonal anti-TAK1 antibodies were described previously (32Chan B.C. To K.F. Pang J.C. Chung Y.F. Lo K.W. Tong J.H. Huang D.W. Lim P.L. Chui Y.L. Int. J. Cancer. 2002; 102: 492-498Crossref PubMed Scopus (5) Google Scholar, 33Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar). For Western blot, 20 μg of WCE were first resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Immun-blot PVDF; Bio-Rad), and probed with various antibodies. The proteins were visualized with the enhanced chemiluminescence solution (Pierce). In Vitro Immune Complex Kinase Assays—The transfected or endogenous IKKβ was immunoprecipitated from WCE using anti-HA (F-7, sc-7392) and anti-IKKβ (H470, sc-7607) antibodies, respectively. The immunoprecipitates were extensively washed three times with the lysis buffer and once with the kinase buffer (20 mm HEPES, pH 7.6, 20 mm β-glycerolphosphate, 10 mm p-nitrophenylphosphate, 10 mm MgCl2, 1 mm dithiothreitol, and 50 μm sodium vanadate). The kinase reaction was reconstituted in 20 μl of the kinase buffer containing 1 μg of GST-IκB (1-54), 20 μm of ATP, and 3 μCi of [γ-32P]ATP (3,000 Ci/mmol) and incubated at 30 °C for 30 min. The reaction mixtures were separated by SDS-PAGE, and the protein bands were visualized by autoradiography. Generation of the Wild Type and IRAK4-/- MEFs Stably Expressing LMP1 by Retroviral Infection—The full-length LMP1 cDNA fragment was inserted into the NotI/BamHI sites of pBPSTR1 (34Paulus W. Baur I. Boyce F.M. Breakefield X.O. Reeves S.A. J. Virol. 1996; 70: 62-67Crossref PubMed Google Scholar). LMP1-pBP-STR1 and pCLeco (a packaging vector) were then co-transfected into HEK293T cells for virus production (35Naviaux R.K. Costanzi E. Haas M. Verma I.M. J. Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar). The supernatant containing retrovirus was collected 48 h after transfection, filtered through a 0.45-μm filter (Millipore, Bedford, MA), and stored at -80 °C freezer. The wild type and IRAK4-/- MEFs (50% confluent) were separately infected with the viruses in the presence of polybrene (8 μg/ml) and tetracycline (1.5 μg/ml) for 8 h. The medium was then aspirated and replaced with fresh medium. After culturing for another 48 h, the cells were split into several 10-cm dishes in selection medium containing puromycin (1.5 μg/ml) and tetracycline (2.0 μg/ml). After 7-10 days, the well isolated individual clones were picked and expanded. To induce LMP1 expression, stable cells were grown in 10% fetal bovine serum with puromycin but without tetracycline. Gel Mobility Shift Assays—A double-stranded oligonucleotide probe containing a consensus NF-κB site, 5′-AGTTGAGGGGACTTTCCCAGGC (sense) (Promega, WI), was end-labeled with T4 polynucleotide kinase and [γ-32P]ATP. For each binding reaction, 15 μg of WCE, 1 μg of poly(dI-dC) (Amersham Biosciences), and 20 μg of bovine serum albumin were mixed in the binding buffer (15 mm HEPES, pH 7.6, 40 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, and 5% glycerol) and preincubated on ice for 30 min. The binding reaction was initiated upon addition of the probe (∼20,000 cpm) and incubated at room temperature for another 30 min. The samples were then separated on a 5% native polyacrylamide gel. After electrophoresis, the gel was subsequently dried and visualized by autoradiography. An NPC-associated LMP1 Variant, LMP1(D335), Activates NF-κBas Well as the Prototypic LMP1 Does—Many different LMP1 variants have been found over the years (36Cheung S.T. Leung S.F. Lo K.W. Chiu K.W. Tam J.S. Fok T.F. Johnson P.J. Lee J.C. Huang D.P. Int. J. Cancer. 1998; 76: 399-406Crossref PubMed Scopus (69) Google Scholar). One of them, LMP1(D335), was frequently found in biopsies from NPC patients in Hong Kong (36Cheung S.T. Leung S.F. Lo K.W. Chiu K.W. Tam J.S. Fok T.F. Johnson P.J. Lee J.C. Huang D.P. Int. J. Cancer. 1998; 76: 399-406Crossref PubMed Scopus (69) Google Scholar). To gain insight into the potential pathological impact of this particular LMP1 variant, we first compared it with its prototypic counterpart (i.e. LMP1(G335)) for their ability to activate NF-κB. When separately co-transfected with an NF-κB-dependent reporter gene into 293T cells, both forms of LMP1 significantly activated the reporter to similar extent in a dose-dependent manner (Fig. 1B). Next, we directly compared the two forms of LMP1 for their ability to activate the IKK complex. When HEK293 cells were co-transfected with HA-IKKβ together with either form of LMP1, we showed that both forms of LMP1 significantly activated IKKβ to a similar extent (Fig. 1C), in agreement with the reporter assay above. Our data suggested that LMP1(D335) behaves similarly to its prototypic counterpart in terms of its impact on the NF-κB pathway. CTAR2 Is the Main Contributor to the LMP1-mediated NF-κB Activation—Both CTAR1 and CTAR2 have previously been implicated in NF-κB activation (37Huen D.S. Henderson S.A. Croom-Carter D. Rowe M. Oncogene. 1995; 10: 549-560PubMed Google Scholar, 38Mitchell T. Sugden B. J. Virol. 1995; 69: 2968-2976Crossref PubMed Google Scholar). To reassess their individual contribution to the LMP1-mediated NF-κB activation, we generated three LMP1 mutants: one (3A) with a triple mutation in the conserved TRAF3-binding motif in CTAR1 (i.e. P204QQAT208 to AQAAA), another (ΔC8) with an 8-amino acid deletion at the C terminus of CTAR2, and the third one (3A/ΔC8) with both types of mutations combined. Previous studies have shown that the first two mutations described above abolish the functions of CTAR1 and CTAR2, respectively, whereas the last one inactivates both (15Brodeur S.R. Cheng G. Baltimore D. Thorley-Lawson D.A. J. Biol. Chem. 1997; 272: 19777-19784Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Devergne O. Hatzivassiliou E. Izumi K.M. Kaye K.M. Kleijnen M.F. Kieff E. Mosialos G. Mol. Cell. Biol. 1996; 16: 7098-7108Crossref PubMed Google Scholar, 30Wan J. Sun L. Mendoza J.W. Chui Y.L. Huang D.P. Chen Z.J. Suzuki N. Suzuki S. Yeh W.C. Akira S. Matsumoto K. Liu Z.G. Wu Z. Mol. Cell. Biol. 2004; 24: 192-199Crossref PubMed Scopus (64) Google Scholar, 39Devergne O. Cahir McFarland E.D. Mosialos G. Izumi K.M. Ware C.F. Kieff E. J. Virol. 1998; 72: 7900-7908Crossref PubMed Google Scholar, 40Eliopoulos A.G. Gallagher N.J. Blake S.M. Dawson C.W. Young L.S. J. Biol. Chem. 1999; 274: 16085-16096Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). When co-transfected into HEK293T cells together with an NF-κB reporter, the wild type LMP1 potently activated the reporter activity, whereas LMP1(3A) and LMP1(ΔC8) were about 65 and 29% as active as the wild type, respectively (Fig. 2A). In contrast, LMP1(3A/ΔC8) was almost completely inactive. Using immune complex kinase assays, we also compared LMP1 with its mutant derivatives for their ability to activate the endogenous IKKβ after separately transfecting them into HEK293 cells. Similar to the results from reporter assays, the IKKβ-activating activity of these LMP1 constructs was found to be in the following order: wild type > 3A >> ΔC8, whereas the double mutant (3A/ΔC8) was completely inactive (Fig. 2B). TAK1 Is a Specific MAP3K Involved in the CTAR2-mediated NF-κB Pathway—Several MAP3Ks including MEKK1 and TAK1 have been shown to act upstream of IKK in various cytokine-mediated NF-κB pathways (see “Discussion”). To find out which MAP3K specifically acts in the LMP1-mediated NF-κB pathway, we first co-transfected LMP1 and HA-IKKβ into MEFs derived from either the wild type or MEKK1 knock-out mice (41Xia Y. Makris C. Su B. Li E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (231) Google Scholar). As shown in Fig. 2A, LMP1 was still able to activate IKKβ in MEKK1-/- MEFs as well as in the wild type MEFs, suggesting that MEKK1 is less likely to be involved in the LMP1-mediated NF-κB pathway. We then turned to TAK1, which was recently shown by us as a key MAP3K involved in the LMP1-mediated JNK pathway (30Wan J. Sun L. Mendoza J.W. Chui Y.L. Huang D.P. Chen Z.J. Suzuki N. Suzuki S. Yeh W.C. Akira S. Matsumoto K. Liu Z.G. Wu Z. Mol. Cell. Biol. 2004; 24: 192-199Crossref PubMed Scopus (64) Google Scholar). HEK293T cells were co-transfected with LMP1 and HA-IKKβ in the presence of either a control or TAK1-specific siRNA. Whereas the control siRNA had no obvious effect, the TAK1-specific siRNA significantly knocked down the expression of the endogenous TAK1 and reduced the LMP1-mediated IKKβ activation (Fig. 3B, lane 3). To find out which CTAR domain preferentially utilizes TAK1, we also tested two CTAR mutants of LMP1, LMP1(3A) and LMP1(ΔC8) (see previous section). In agreement with results in Fig. 2, LMP1(3A), which retains a functional CTAR2, was still able to significantly activate IKK, and this activation could be largely abolished by the TAK1-specific siRNA (Fig. 3B, lanes 4 and 5). In contrast, LMP1(ΔC8), which retains a functional CTAR1, poorly activated IKK (Fig. 3B, lane 6). Importantly, the TAK1-specific siRNA had little effect on the LMP1(ΔC8)-induced IKK activation (Fig. 3B, lane 7). This result suggested that TAK1 is mainly involved in the CTAR2-mediated NF-κB pathway. To further substantiate our claim, we also tested the involvement of TAK1 in the LMP1-induced p100 processing, a process known to be mediated by CTAR1 (21Atkinson P.G. Coope H.J. Rowe M. Ley S.C. J. Biol. Chem. 2003; 278: 51134-51142Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Eliopoulos A.G. Caamano J.H. Flavell J. Reynolds G.M. Murray P.G. Poyet J.L. Young L.S. Oncogene. 2003; 22: 7557-7569Crossref PubMed Scopus (94) Google Scholar, 23Luftig M. Yasui T. Soni V. Kang M.S. Jacobson N. Cahir-McFarland E. Seed B. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 141-146Crossref PubMed Scopus (147) Google Scholar, 24Saito N. Courtois G. Chiba A. Yamamoto N. Nitta T. Hironaka N. Rowe M. Yamaoka S. J. Biol. Chem. 2003; 278: 46565-46575Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The whole cell extracts from the above experiment were subjected to immunoblot to detect the levels of the endogenous p100 and its processed product p52. As shown in Fig. 3C, LMP1 clearly induced increased p52 production in the presence of the control siRNA. Importantly, the TAK1-siRNA had no obvious effect on LMP1-induced p52 production, suggesting that TAK1 is not involved in the CTAR1-mediated p100 processing. Loss of TAB2 Does Not Affect the LMP1-induced IKK Activity—TAB2 is a TAK1-binding protein and is involved in IL-1-mediated NF-κB activation (42Ishitani T. Takaesu G. Ninomiya-Tsuji J. Shibuya H. Gaynor R.B. Matsumoto K. EMBO J. 2003; 22: 6277-6288Crossref PubMed Scopus (218) Google Scholar, 43Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). To test whether TAB2 is absolutely required in the LMP1-mediated NF-κB pathway, we compared the LMP1-induced IKK activity in both the wild type and TAB2-/- MEFs (44Sanjo H. Takeda K. Tsujimura T. Ninomiya-Tsuji J. Matsumoto K. Akira S. Mol. Cell. Biol. 2003; 23: 1231-1238Crossref PubMed Scopus (105) Google Scholar). As shown in Fig. 4, LMP1 activated IKK in TAB2-/- MEFs as well as in the wild type MEFs. This suggested that TAB2 is not essential in the LMP1-mediated NF-κB pathway. CTAR2 Specifically Utilizes TRAF6 for NF-κB Activation—Although it has been convincingly shown that CTAR1 specifically recruits TRAF3 to activate NF-
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