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• W2010339276 abstract "Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-κB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-κB in different NF-κB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IκB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-κB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-κB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-κB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-κB through distinct signaling pathways. Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-κB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-κB in different NF-κB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IκB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-κB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-κB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-κB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-κB through distinct signaling pathways. Latent membrane protein-1 (LMP1) 1The abbreviations used are: LMP1latent membrane protein 1CTARcarboxyl-terminal activation regionIKKIκB kinaseNEMONF-κB essential modulatorNIKNF-κB inducing kinaseTNFtumor necrosis factorTRAFtumor necrosis factor receptor-associated factorTRADDtumor necrosis factor receptor 1-associated death domainIL-1interleukin-1LPSlipopolysaccharideHTLV-Ihuman T-cell leukemia virus type ILT-βlymphotoxin-βBAFFB cell-activating factorRACE5′-rapid amplification of cDNA endsRTreverse transcriptionwtwild typeEMSAelectrophoretic mobility shift assayRIPA bufferradioimmune precipitation assay bufferGSTglutathione S-transferase. is an oncogenic transmembrane protein encoded by Epstein-Barr virus (1Mosialos G. Cytokine Growth Factor Rev. 2001; 12: 259-270Crossref PubMed Scopus (29) Google Scholar, 2Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (187) Google Scholar, 3Lam N. Sugden B. Cell. Signal. 2002; 15: 9-16Crossref Scopus (102) Google Scholar) that is known to activate NF-κB (4Hammarskjold M.L. Simurda M.C. J. Virol. 1992; 66: 6496-6501Crossref PubMed Google Scholar, 5Laherty C.D. Hu H.M. Opipari A.W. Wang F. Dixit V.M. J. Biol. Chem. 1992; 267: 24157-24160Abstract Full Text PDF PubMed Google Scholar), the c-Jun NH2-terminal kinase pathway (6Eliopoulos A.G. Young L.S. Oncogene. 1998; 16: 1731-1742Crossref PubMed Scopus (251) Google Scholar), and its downstream transcription factors such as AP-1 (6Eliopoulos A.G. Young L.S. Oncogene. 1998; 16: 1731-1742Crossref PubMed Scopus (251) Google Scholar, 7Kieser A. Kilger E. Gires O. Ueffing M. Kolch W. Hammerschmidt W. EMBO J. 1997; 16: 6478-6485Crossref PubMed Scopus (284) Google Scholar, 8Hatzivassiliou E. Miller W.E. Raab-Traub N. Kieff E. Mosialos G. J. Immunol. 1998; 160: 1116-1121PubMed Google Scholar), Janus-activating tyrosine kinase 3 and signal transducer and activator of transcription (9Gires O. Kohlhuber F. Kilger E. Baumann M. Kieser A. Kaiser C. Zeidler R. Scheffer B. Ueffing M. Hammerschmidt W. EMBO J. 1999; 18: 3064-3073Crossref PubMed Scopus (292) Google Scholar), and p38 mitogen-activated protein kinase (10Eliopoulos 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). Previous studies demonstrated that NF-κB activation by LMP1 plays a pivotal role in its transforming activity (11Kaye K.M. Izumi K.M. Mosialos G. Kieff E. J. Virol. 1995; 69: 675-683Crossref PubMed Google Scholar, 12Izumi K.M. Kieff E.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12592-12597Crossref PubMed Scopus (359) Google Scholar, 13He Z. Xin B. Yang X. Chan C. Cao L. Cancer Res. 2000; 60: 1845-1848PubMed Google Scholar). LMP1 is a constitutively active tumor necrosis factor (TNF) receptor family member-like molecule that consists of an NH2-terminal short cytoplasmic domain, six membrane-spanning domains, and a long carboxyl-terminal cytoplasmic domain. LMP1 oligomerizes in the plasma membrane without ligand binding, which results in signals emanating from carboxyl-terminal activation region (CTAR) 1, CTAR2, and CTAR3 (9Gires O. Kohlhuber F. Kilger E. Baumann M. Kieser A. Kaiser C. Zeidler R. Scheffer B. Ueffing M. Hammerschmidt W. EMBO J. 1999; 18: 3064-3073Crossref PubMed Scopus (292) Google Scholar, 14Huen D.S. Henderson S.A. Croom-Carter D. Rowe M. Oncogene. 1995; 10: 549-560PubMed Google Scholar, 15Mitchell T. Sugden B. J. Virol. 1995; 69: 2968-2976Crossref PubMed Google Scholar). CTAR1 and CTAR2 were reported to mediate induced nuclear translocation of p50, p52, RelA, RelB, or c-Rel depending on the cell types studied (12Izumi K.M. Kieff E.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12592-12597Crossref PubMed Scopus (359) Google Scholar, 16Paine E. Scheinman R.I. Baldwin Jr., A.S. Raab-Traub N. J. Virol. 1995; 69: 4572-4576Crossref PubMed Google Scholar, 17Izumi K.M. Cahir McFarland E.D. Ting A.T. Riley E.A. Seed B. Kieff E.D. Mol. Cell. Biol. 1999; 19: 5759-5767Crossref PubMed Google Scholar, 18Pai S. O'Sullivan B.J. Cooper L. Thomas R. Khanna R. J. Virol. 2002; 76: 1914-1921Crossref PubMed Scopus (29) Google Scholar). However, it is not fully understood how CTAR1 and CTAR2 differentially regulate NF-κB. Prior studies provided evidence that NF-κB activation by LMP1 involved TNF receptor-associated factor 2 (TRAF2), Tpl-2/Cot, an NF-κB-inducing kinase (NIK)-related kinase, IκB kinase 1/α (IKK1/α) and IKK2/β (17Izumi K.M. Cahir McFarland E.D. Ting A.T. Riley E.A. Seed B. Kieff E.D. Mol. Cell. Biol. 1999; 19: 5759-5767Crossref PubMed Google Scholar, 19Mosialos G. Birkenbach M. Yalamanchili R. VanArsdale T. Ware C. Kieff E. 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Virol. 2002; 76: 4567-4579Crossref PubMed Scopus (52) Google Scholar), although these results awaited further assessment in knockout cells. latent membrane protein 1 carboxyl-terminal activation region IκB kinase NF-κB essential modulator NF-κB inducing kinase tumor necrosis factor tumor necrosis factor receptor-associated factor tumor necrosis factor receptor 1-associated death domain interleukin-1 lipopolysaccharide human T-cell leukemia virus type I lymphotoxin-β B cell-activating factor 5′-rapid amplification of cDNA ends reverse transcription wild type electrophoretic mobility shift assay radioimmune precipitation assay buffer glutathione S-transferase. The NF-κB family of transcription factors plays crucial roles in the immune, inflammatory and apoptotic responses (23Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3292) Google Scholar). NF-κB activation is induced by a variety of stimuli including TNF-α, interleukin-1 (IL-1), lipopolysaccharide (LPS), double-stranded RNA, Tax of human T-cell leukemia virus type I (HTLV-I), and LMP1 (24Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3447) Google Scholar). NF-κB is composed of dimers of p50, p52, RelA, RelB, or c-Rel that are endogenously complexed to inhibitor proteins called IκB and NF-κB precursor proteins p105 or p100, which sequester NF-κB in the cytoplasm. In response to various stimuli, inhibitory proteins are phosphorylated at specific serine residues and are rapidly processed by the proteasome after polyubiquitination. This exposes the nuclear localization signal of NF-κB, leading to its nuclear translocation. The phosphorylation step is usually controlled by the IκB kinase (IKK) complex, which contains two catalytic subunits, IKK1/α and IKK2/β, and the regulatory subunit NF-κB essential modulator (NEMO/IKKγ/IKKAP), HSP90, and Cdc37 (25DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar, 26Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1853) Google Scholar, 27Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1588) Google Scholar, 28Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar, 29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 30Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar, 31Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 32Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Although IL-1- or TNF-α-induced nuclear translocation of NF-κB was not impaired in IKK1-deficient cells (33Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (714) Google Scholar, 34Takeda K. Takeuchi O. Tsujimura T. Itami S. Adachi O. Kawai T. Sanjo H. Yoshikawa K. Terada N. Akira S. Science. 1999; 284: 313-316Crossref PubMed Scopus (539) Google Scholar), it was severely impaired in IKK2- or NEMO-deficient cells (35Tanaka M. Fuentes M.E. Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 36Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (856) Google Scholar, 37Makris C. Godfrey V.L. Krahn-Senftleben G. Takahashi T. Roberts J.L. Schwarz T. Feng L. Johnson R.S. Karin M. Mol. Cell. 2000; 5: 969-979Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 38Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Thus, activation of the IKK complex constitutes a converging regulatory step in the NF-κB signaling pathway. Recent studies have revealed two distinct pathways of NF-κB activation. The canonical pathway is triggered by many inflammatory stimuli including TNF-α, IL-1, LPS, and double-stranded RNA; depends on IKK2 and NEMO; and induces specific phosphorylation of IκB proteins. The non-canonical pathway is triggered by a limited number of stimuli including lymphotoxin-β (LT-β), B cell-activating factor (BAFF), and CD40 ligand that function in the development, organization, and proper function of lymphoid tissue (39Matsushima A. Kaisho T. Rennert P.D. Nakano H. Kurosawa K. Uchida D. Takeda K. Akira S. Matsumoto M. J. Exp. Med. 2001; 193: 631-636Crossref PubMed Scopus (178) Google Scholar, 40Saitoh T. Nakano H. Yamamoto N. Yamaoka S. FEBS Lett. 2002; 532: 45-51Crossref PubMed Scopus (56) Google Scholar, 41Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar, 42Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Crossref PubMed Scopus (577) Google Scholar, 43Coope H.J. Atkinson P.G. Huhse B. Belich M. Janzen J. Holman M.J. Klaus G.G. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (368) Google Scholar, 44Muller J.R. Siebenlist U. J. Biol. Chem. 2003; 278: 12006-12012Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). This pathway involves the phosphorylation-dependent processing of NFKB2 p100 to p52, which requires IKK1 and functional NIK, resulting in the nuclear translocation of p52-RelB dimers. A previous study on HTLV-I Tax uncovered a unique mechanism whereby this viral protein persistently activates NF-κB. Tax resides in the cytoplasm associated with the IKK complex and physically interacts with p100, thereby inducing its phosphorylation and processing through recruitment of the IKK complex to p100 (45Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). Although NIK, p52, and RelB were previously implicated in the LMP1 signaling to NF-κB activation (16Paine E. Scheinman R.I. Baldwin Jr., A.S. Raab-Traub N. J. Virol. 1995; 69: 4572-4576Crossref PubMed Google Scholar, 18Pai S. O'Sullivan B.J. Cooper L. Thomas R. Khanna R. J. Virol. 2002; 76: 1914-1921Crossref PubMed Scopus (29) Google Scholar, 20Sylla B.S. Hung S.C. Davidson D.M. Hatzivassiliou E. Malinin N.L. Wallach D. Gilmore T.D. Kieff E. Mosialos G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10106-10111Crossref PubMed Scopus (141) Google Scholar, 21Luftig M.A. Cahir-McFarland E. Mosialos G. Kieff E. J. Biol. Chem. 2001; 276: 14602-14606Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the precise mechanism by which LMP1 persistently activates NF-κB has remained elusive. Our initial study on the LMP1 signaling identified a novel NEMO-independent NF-κB activation pathway that is mediated by CTAR1 and involves aberrant expression of p52 and nuclear translocation of RelB. We further provide genetic evidence of how NIK, IKK1, and IKK2 are involved in this process. Plasmids—Plasmids pSG5, pSG5-LMP1, pSG5-LMP1.Y384G, pSG5-LMP1.349Δ, pSG5-LMP1.AAA, and pSG5-LMP1.Δ187–351 were described previously (14Huen D.S. Henderson S.A. Croom-Carter D. Rowe M. Oncogene. 1995; 10: 549-560PubMed Google Scholar, 46Liebowitz D. Mannick J. Takada K. Kieff E. J. Virol. 1992; 66: 4612-4616Crossref PubMed Google Scholar, 47Floettmann J.E. Rowe M. Oncogene. 1997; 15: 1851-1858Crossref PubMed Scopus (107) Google Scholar, 48Eliopoulos A.G. Blake S.M. Floettmann J.E. Rowe M. Young L.S. J. Virol. 1999; 73: 1023-1035Crossref PubMed Google Scholar). LMP1, LMP1.Y384G, and LMP1.AAA were subcloned into the pMX-puro retrovirus vector (49Kawakami Y. Miura T. Bissonnette R. Hata D. Khan W.N. Kitamura T. Maeda-Yamamoto M. Hartman S.E. Yao L. Alt F.W. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3938-3942Crossref PubMed Scopus (128) Google Scholar), a kind gift of Dr. Toshio Kitamura (University of Tokyo, Tokyo, Japan), to generate pMX-puro-LMP1, pMX-puro-LMP1.Y384G, and pMX-puro-LMP1.AAA, respectively. The plasmid Igκ-ConAluciferase was described previously (50Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). EF1-lacZ is a kind gift of Dr. Sylvie Mémet (Institut Pasteur, Paris, France). pIRES1neo-tax was constructed by subcloning a BamHI fragment of the HTLV-I tax gene into the pIRES1neo vector (Clontech). Plasmids pIgκ2bsrH and pIgκ2tkH were previously described (51Chinanonwait N. Miura H. Yamamoto N. Yamaoka S. FEBS Lett. 2002; 531: 553-560Crossref PubMed Scopus (7) Google Scholar). Plasmids pCn and pCn100 encoding human NFKB2 were described previously (52Yamaoka S. Inoue H. Sakurai M. Sugiyama T. Hazama M. Yamada T. Hatanaka M. EMBO J. 1996; 15: 873-887Crossref PubMed Scopus (179) Google Scholar). Cells—Rat-1 and 5R cells were described previously (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). IKK2–/– and NEMO–/– fibroblasts were kindly provided by Dr. Manolis Pasparakis (EMBL, Rome, Italy), IKK1–/–IKK2–/– fibroblasts were by Dr. Inder M. Verma (Salk Institute, La Jolla, CA), IKK1–/– fibroblasts were by Drs. Michael Karin and Véronique Baud (University of California, San Diego, CA), and NIKaly/aly fibroblasts were by Dr. Mitsuru Matsumoto (Tokushima University, Tokushima, Japan). To generate cells carrying a mutation that results in impaired NF-κB activation, we mutagenized Rat-1 cells stably expressing the HTLV-1 Tax protein to constitutively activate NF-κB and then subjected them to a lethal selection that employs the combination of NF-κB-dependent expression of herpes simplex virus thymidine kinase and ganciclovir. Rat-1 cells were stably transfected with pIRES1neo-tax, pIgκ2bsrH, and pIgκ2tkH, and selected under G418, hygromycin, and blasticidin S. Isolated clones were tested for resistance to blasticidin S and susceptibility to ganciclovir. One clone, D2–19, was found to express Tax, exhibit constitutive NF-κB activity, be resistant to blasticidin S, and be killed completely in the presence of ganciclovir. D2–19 cells were subjected to four rounds of mutagenesis with 10 μg/ml frameshift mutagen ICR191, followed by lethal selection in the presence of 1 μg/ml ganciclovir. One cell clone, N1, did not significantly induce κB-DNA binding activity in response to TNF-α or LPS. This clone was found to have lost Tax expression and resistance to G418. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin) at 37 °C in humidified atmosphere with 5% CO2. PCR, Cloning of Rat nemo, and Southern Blotting—One microgram of total RNA was extracted from ∼1 × 105 Rat-1 cells and reverse-transcribed into minus-strand cDNA in a 50-μl reaction using Superscript II (Invitrogen) under the instructions from the manufacturer. PCR was performed in a reaction mixture containing 5 μl of the above synthesized cDNA. DNA sequence homology between murine and human NEMO at the amino and carboxyl termini enabled us to design primers N2 (5′-GTGCAGCCCAGTGGTGGCCCAG) and C1 (5′-CTACTCTATGCACTCCATGAC) and to amplify part of rat nemo cDNA by PCR. 5′-Rapid amplification of cDNA ends (RACE) was conducted using SMART™ RACE cDNA amplification kit (Clontech) under the instructions from the manufacturer. PCR products were purified by agarose gel electrophoresis, ligated to p-GEM-T Easy (Promega) vector, and sequenced. Based on the sequencing results, primers N0 (5′-CCTAGGAGCTCCGATTCTGC) and C0 (5′-GGAGCTGTCTACCCTAATAGGGG) were used to amplify the entire coding sequence of rat NEMO. Five μl of the initial PCR reaction was subjected to nested PCR with primers Nemo5′ (5′-GGATCCAGCAGGCACCTCTGGAAGA) and Nemo3′ (5′-CTCGAGCTACTCTATGCACTCCATGACATGTATC). DNA fragments were subcloned into the BamHI and XhoI sites of pcDNA3HA, and pcDNA3HA-NEMO-Rat-1 and pcDNA3HA-NEMO-N1 were generated and sequenced. For PCR-Southern analysis, PCR was performed using cDNA with primers N0 and C0, followed by nested PCR with primers Nemo5′ and Nemo3′. PCR products were electrophoresed on a 1% agarose gel, visualized by ethidium bromide staining, and transferred to Hybond-N+ nylon membrane (Amersham Biosciences) by alkaline blotting. Hybridization was carried out in a standard protocol, using a probe prepared by PCR with primers Southern F0 (5′-CAGATGCTGAGGGAACGCT) and Southern R0 (5′-AGTTCCCCCAGCAATGATGT). The blot was exposed to an x-ray film (MXJB-1; Eastman Kodak Co.) at –80 °C. Glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified with primers rat-gapdh-F (CGGTGTCAACGGATTTGG) and rat-gapdh-R (GTAGGCCATGAGGTCCACC). For semiquantitative PCRs, 5 μl of cDNA samples used above and serially diluted rat nemo plasmid DNA, ranging from 0.01 to 1011 copies/sample, were used as template. The first step PCR was done for 35 cycles with primers N0 and C0, followed by the second step PCR for 35 cycles with primers Nemo5′ and Nemo3′, using 5 μl of the first PCR products. Transient Transfection and Luciferase assay—Transfection was carried out in six-well plates by the calcium phosphate precipitation method, FuGENE reagent (Roche Molecular Biochemicals), or DEAE-dextran method. Total amount of transfected plasmid DNA was kept constant with pcDNA3HA or pSG5. Where indicated, cells were stimulated with 15 μg/ml LPS or 10 ng/ml TNF-α for 3 h before lysis. The luciferase activities were normalized on the basis of β-galactosidase activity. Experiments were repeated at least three times in duplicate. Retrovirus Infection—Supernatants of transfected Plat-E cells (53Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1361) Google Scholar) were recovered and passed through a 0.45-μm filter every 12 h from 36 to 72 h after transfection, and either immediately used for infection of cells or frozen and stored at –80 °C. For infection, cells on 100-mm dishes were exposed to 4 ml of virus supernatant in the presence of 10 μg/ml Polybrene for 3 h. Rat-1 and 5R cells were harvested at 30 h after infection. Wild type, IKK1–/–, IKK2–/–, NEMO–/–, and NIKaly/aly fibroblasts were harvested at 36 h after infection. N1 cell clones expressing LMP1 were isolated through limiting dilution. Preparation of Cell Extracts and Kinase Assay—Kinase assay was performed in parallel with immunoblot analysis following immunoprecipitation. Cells were lysed in Buffer A (20 mm HEPES, pH 7.8, 0.15 mm EDTA, 0.15 mm EGTA, 10 mm KCl) supplemented with 1 μg/ml aprotinin, 1 μg/ml leupeptin, 0.57 mm phenylmethylsulfonyl fluoride, 100 μm sodium vanadate, and 20 mm β-glycerol phosphate and left on ice for 15 min. Nonidet P-40 was added to 1%, and the cell suspension was incubated for 2 min on ice. After centrifugation at 14,000 rpm for 5 min, supernatants were used as cytoplasmic extracts and pellets were used for extraction of nuclear proteins. Gel filtration was performed with Superdex-200 as described previously (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). Immunoprecipitation was performed as described previously (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). After the last washing, one half of the beads were used to perform kinase assays and the remaining beads were used for immunoblot analysis. Kinase reactions were carried out at 30 °C for 30 min in reaction mix (20 mm HEPES, pH 7.5, 10 mm MgCl2, 50 mm NaCl, 100 μm sodium vanadate, 20 mm β-glycerol phosphate, 2 mm dithiothreitol, 20 nm ATP, [γ-32P]dATP, and 1 μg of GST-IκBα-(1–72)). Recombinant GST-IκBα wild type and GST-IκBα S32A/S36A mutant proteins were described previously (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). Reactions were fractionated on a 12% SDS-acrylamide gel, and phosphorylated GST-IκBα was detected by autoradiography. Where indicated, cells were lysed in RIPA buffer (20 mm Tris-HCl, pH 8.0, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10% glycerol, 1% Nonidet-P40, 0.5% deoxycholate, 0.1% SDS, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 0.57 mm phenylmethylsulfonyl fluoride). Protein concentrations were determined by Bradford assay (Bio-Rad). Antibodies—Antisera 3328, 1263, 1226, and 1319 were kind gifts from Dr. Nancy Rice. Anti-p52 (06-413) serum used for supershift assays was purchased from Upstate Biotechnology, Inc. Anti-LMP1 monoclonal antibody (CS.1–4) and anti-Tax monoclonal antibody (Lt-4), a kind gift from Dr. Yuetsu Tanaka (University of Ryukyus, Okinawa, Japan), were described previously (54Rowe M. Evans H.S. Young L.S. Hennessy K. Kieff E. Rickinson A.B. J. Gen. Virol. 1987; 68: 1575-1586Crossref PubMed Scopus (197) Google Scholar, 55Lee B. Tanaka Y. Tozawa H. Tohoku J. Exp. Med. 1989; 157: 1-11Crossref PubMed Scopus (87) Google Scholar). Anti-IKK1 monoclonal antibody (IMG-136) was purchased from Imgenex. Anti-IKK2 (550621) and anti-NEMO (68341A) monoclonal antibodies were purchased from Pharmingen. Anti-IKK1 (H-744), anti-IκBα (C-21) polyclonal antibodies, and anti-p52 (sc-7386), and anti-actin (C-2) monoclonal antibodies were purchased from Santa Cruz Biotechnology. Immunoblot Analysis—Immunoblot analysis was performed under a standard protocol. Immunoreactive bands were visualized by ECL (NEN Life Sciences). When necessary, membranes were incubated in stripping buffer (62.5 mm Tris-HCl, pH 6.8, 100 mm 2-mercaptoethanol, 2% SDS) for 30 min at 50 °C with constant agitation, washed, and reprobed with another antibody. The intensity of the p52 and p100 bands was determined by a computerized analysis (Image Gauge version 3.01, Fuji Film). Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts were prepared as described previously (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar). Nuclear extract proteins (5 μg determined by Bradford) were incubated in binding buffer (10 mm HEPES, pH 7.8, 100 mm NaCl, 1 mm EDTA, 2.5% glycerol, 0.5 μg of poly(dI-dC)) and 0.5 ng of 32P-labeled κB probe (KBF1) (56Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israel A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (602) Google Scholar) for 30 min at room temperature. Samples were fractionated on a 5% polyacrylamide gel in 0.5× TBE and visualized by autoradiography. In supershift assays, antiserum to p50 (1263), RelA (1226), RelB (1319), or p52 (06-413) was added to the binding reaction. LMP1 Activates NF-κB in NEMO-deficient Cells—We used the 5R cell line to examine whether LMP1 can activate NF-κB in a NEMO-independent manner. We demonstrated previously that 5R cells did not express any detectable NEMO protein by Western blot analysis (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar), but its mRNA expression was not explored. For this purpose, the entire coding region of the nemo cDNA expressed in Rat-1 cells was cloned by reverse transcription-polymerase chain reaction (RT-PCR), using primers corresponding to regions highly conserved between the human and murine nemo genes (Fig. 1A). Southern blot analysis of RT-PCR products failed to detect nemo mRNA in 5R cells (Fig. 1B). Semiquantitative PCR analysis revealed more than 1000 copies of nemo cDNA in the sample prepared from 100 Rat-1 cells, whereas none was detected in 100 5R cells (Fig. 1C). Aliquots of the PCR-amplified material were further analyzed for NEMO cDNA by nested PCR. Although 10 copies of nemo cDNA were amplified in the control samples, none was amplified from 5R cells (Fig. 1D). These results indicate 5R cells do not express nemo mRNA, and the assay sensitivity is 10 copies of NEMO cDNA in 100 cells. Fig. 1E shows that stimulation of 5R cells with TNF-α or LPS does not induce any NF-κB-dependent reporter gene activation as previously reported (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar), whereas LMP1 expression in 5R cells resulted in a significant activation that was, however, weaker than that observed in Rat-1 cells (see Fig. 5B). We also tested a previously characterized NEMO-deficient pre-B cell line 1.3E2 (29Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 50Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar) for NF-κB activation by LMP1 (Fig. 1F). Transient expression of LMP1 in 1.3E2 and its parental cell line 70Z/3 caused strong NF-κB-dependent reporter gene activation. These results indicate that LMP1 can activate NF-κB in a NEMO-independent manner.Fig. 5CTAR1 mediates NEMO-independent NF-κB activation.A, structure of wild type and mutant LMP1 proteins. CTAR1 is located between amino acid residues 194 and 232. CTAR2 is between 351 and 386. The hollow circles indicate amino acid substitutions. B, CTAR1, but not CTAR2, LMP1 activates NF-κB in 5R cells. Rat-1 and 5R cells were co-transfected with 0.25 μg of Igκ-ConAlucifer" @default.
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