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• W1997407816 abstract "Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-κB activation pathway that depends upon the inducible processing of NF-κB2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of Epstein-Barr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52·RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-κB-signaling pathway that triggers phosphorylation and degradation of IκBα. Interestingly, LMP1 activation of these two NF-κB pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of IκBα phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of IκBα phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function. Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-κB activation pathway that depends upon the inducible processing of NF-κB2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of Epstein-Barr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52·RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-κB-signaling pathway that triggers phosphorylation and degradation of IκBα. Interestingly, LMP1 activation of these two NF-κB pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of IκBα phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of IκBα phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function. Epstein-Barr virus (EBV) 1The abbreviations used are: EBVEpstein-Barr virusIKKIκB kinaseLMPlatent membrane proteinEBNAEBV nuclear antigenLPleader proteinGRRglycine-rich regiondndominant negativeLCLlymphoblastoid cell lineCTARC-terminal-activating regionTNFtumor necrosis factorLTβ-Rlymphotoxin β receptorHAhemagglutininNIKNF-κB inducing kinasemAbmonoclonal antibodytTAtetracycline-regulated transactivatorMEFmouse embryonic fibroblastRIPAradioimmune precipitation assay bufferEMSAelectrophoretic mobility shift assayEVempty vectorTettetracyclineHTLVhuman T-cell lymphotrophic virus. is a ubiquitous human herpes virus that infects B-lymphocytes and certain epithelial cells (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Virology. Raven Press, Ltd., New York1996: 2397-2446Google Scholar, 2Borza C.M. Hutt-Fletcher L.M. Nat. Med. 2002; 8: 594-599Crossref PubMed Scopus (366) Google Scholar, 3Thorley-Lawson D.A. Nat. Rev. Immunol. 2001; 1: 75-82Crossref PubMed Scopus (750) Google Scholar). EBV is the causative agent of infectious mononucleosis and is also implicated in the etiology of both epithelial (nasopharyngeal carcinoma) and lymphoid malignancies (Burkitt's lymphoma and Hodgkin's disease). Infection of primary human B lymphocytes in vitro with EBV leads to their immortalization and the establishment of lymphoblastoid cell lines (LCLs). Latent membrane protein (LMP) 1 is one of five latent genes shown to be essential for EBV-induced transformation of B cells (4Kaye K.M. Izumi K.M. Mosialos G. Kieff E. J. Virol. 1995; 69: 675-683Crossref PubMed Google Scholar). LMP1 is required for both LCL establishment and continued proliferation (4Kaye K.M. Izumi K.M. Mosialos G. Kieff E. J. Virol. 1995; 69: 675-683Crossref PubMed Google Scholar, 5Mattia E. Chichiarelli S. Hickish T. Gaeta A. Mancini C. Cunningham D. van Renswoude J. Oncogene. 1997; 24: 489-493Crossref Scopus (14) Google Scholar, 6Kilger E. Kieser A. Baumann M. Hammerschmidt W. EMBO. 1998; 17: 1700-1709Crossref PubMed Scopus (377) Google Scholar). The transforming potential of LMP1 has been confirmed both in vitro in fibroblast transformation assays (7Wang D. Liebowitz D. Kieff E. Cell. 1985; 43: 831-840Abstract Full Text PDF PubMed Scopus (992) Google Scholar) and in vivo in transgenic mice (8Kulwichit 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 (335) Google Scholar). Significantly, LMP1 is expressed in the majority of EBV-associated human malignancies (1Rickinson A.B. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Virology. Raven Press, Ltd., New York1996: 2397-2446Google Scholar). Epstein-Barr virus IκB kinase latent membrane protein EBV nuclear antigen leader protein glycine-rich region dominant negative lymphoblastoid cell line C-terminal-activating region tumor necrosis factor lymphotoxin β receptor hemagglutinin NF-κB inducing kinase monoclonal antibody tetracycline-regulated transactivator mouse embryonic fibroblast radioimmune precipitation assay buffer electrophoretic mobility shift assay empty vector tetracycline human T-cell lymphotrophic virus. LMP1 is an integral membrane protein with six hydrophobic transmembrane domains that mediate its constitutive oligomerization and targeting to plasma membrane lipid rafts (9Hatzivassiliou E. Mosalios G. Front. Biosci. 2002; 7: 319-329Crossref PubMed Scopus (54) Google Scholar). Constitutive oligomerization allows LMP1 to function as a ligand-independent receptor and is essential for its transforming potential in both fibroblasts and B cells. The 200-amino acid cytoplasmic C terminus of LMP1 is also required for cell transformation (10Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (188) Google Scholar). This contains two subdomains, termed C-terminal-activating regions (CTARs) 1 and 2, which act as docking sites for complexes of signaling proteins that trigger activation of the transcription factors NF-κB and activator protein-1 (10Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (188) Google Scholar). NF-κB activation is absolutely required to block apoptosis of EBV-transformed LCLs (11Cahir-McFarland E.D. Davidson D.M. Schauer S.L. Duong J. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6055-6060Crossref PubMed Scopus (236) Google Scholar) and is also required for LMP1 transformation of fibroblasts (12He Z. Xin B. Yang X.C.C L. C. Cancer Res. 2000; 60: 1845-1848PubMed Google Scholar). Mammalian cells express five NF-κB proteins: RelA, RelB, c-Rel, NF-κB1 p50, and NF-κB2 p52, which combine to form homo- and heterodimers that regulate genes involved in immune responses, apoptosis, and development (13Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4631) Google Scholar). NF-κB dimers are retained in the cytoplasm of unstimulated cells by interaction with a family of inhibitory proteins (IκBs), which includes IκBα. Activation of the canonical NF-κB-signaling pathway by agonists such as tumor necrosis factor (TNF) α and interleukin 1 induces IκBα phosphorylation, ubiquitination, and subsequent proteolysis by the proteasome. NF-κB dimers, which are predominantly p50-RelA heterodimers, are thereby released to translocate into the nucleus and modulate gene expression. Signal-induced phosphorylation of IκBα is mediated by the IκB kinase (IKK) complex, which comprises two catalytic subunits, IKK1 (IKKα) and IKK2 (IKKβ), and a structural subunit NEMO (IKKγ) (14Sylla B.S. Hung S.C. Davidson D.M. Hatzivassiliou E. Malinin N.L. Wallach D. Gilmore T.D. Kieff E. Mosalios G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10106-10111Crossref PubMed Scopus (142) Google Scholar, 15Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Genetic studies indicate that IKK2 is essential for IκBα phosphorylation triggered by TNFα or interleukin 1, whereas IKK1 is largely dispensable (15Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Recently, an “alternative” NF-κB activation pathway has been described (16Pomerantz J.L. Baltimore D. Mol. Cell. 2002; 10: 693-701Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). This pathway triggers proteasome-mediated processing of the NF-κB2 precursor p100 to produce p52. In contrast to the canonical NF-κB pathway, which is triggered by multiple different stimuli (15Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar), only three cellular receptors have so far been shown to stimulate p100 processing; namely, B cell-activating factor receptor (17Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Crossref PubMed Scopus (579) Google Scholar, 18Kayagaki N. Yan M. Seshasayee D. Wang H. Lee W. French D.M. Grewal I.S. Cochran A.G. Gordon N.C. Yin J. Starovasnik M.A. Dixit V.M. Immunity. 2002; 10: 515-524Abstract Full Text Full Text PDF Scopus (411) Google Scholar), lymphotoxin β receptor (LTβ-R), (19Dejardin 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 (781) Google Scholar) and CD40 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). Receptor activation of this pathway is critically dependent on the mitogen-activated protein 3-kinase NIK and IKK1 (21Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar, 22Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Functionally, LMP1 resembles an activated CD40 receptor, with both molecules promoting B cell survival and proliferation and regulating a highly overlapping spectrum of activation markers (23Mosialos G. Cytokine Growth Factor Rev. 2001; 12: 259-270Crossref PubMed Scopus (29) Google Scholar). Indeed, activation of CD40 can compensate for the lack of LMP1 expression and promote short term growth of EBV-transformed LCLs (6Kilger E. Kieser A. Baumann M. Hammerschmidt W. EMBO. 1998; 17: 1700-1709Crossref PubMed Scopus (377) Google Scholar, 24Zimber-Strobl U. Kempkes B. Marschall G. Zeidler R. van Kooten C. Banchereau J. Bornkamm G.W. Hammerschmidt W. EMBO J. 1996; 15: 7070-7078Crossref PubMed Scopus (97) Google Scholar). It is firmly established that LMP1 activates the canonical NF-κB pathway that regulates IκBα proteolysis (9Hatzivassiliou E. Mosalios G. Front. Biosci. 2002; 7: 319-329Crossref PubMed Scopus (54) Google Scholar). In the present study evidence is presented that in B cells and 293 epithelial cells LMP1 also induces NF-κB2 p100 processing to p52, similar to CD40 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). The potential importance of the alternative NF-κB signaling pathway for cell transformation by LMP1 is discussed. cDNA Constructs, Antibodies, and Reagents—The following plasmids have been described previously: pSG5-tCD2 (tCD2, extracellular and transmembrane domains of rat CD2), pSG5-CD2-LMP 192, pSG5-CD2-LMP1 (25Floettmann J.E. Rowe M. Oncogene. 1997; 15: 1851-1858Crossref PubMed Scopus (107) Google Scholar), pcDNA3-Myc-p100, pcDNA3-p100S866A,S870A, pcDNA3-Myc-p100ΔGRR (residues 346-377 deleted), and pcDNA3-Myc-NIKΔN (residues 624-947 of NIK) (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). The tetracycline-regulated pJEF vector constructs encoding EBNA1, EBNA2, LMP1, LMP2 (26Floettmann J.E. Ward K. Rickinson A.B. Rowe M. Virology. 1996; 223: 29-40Crossref PubMed Scopus (119) Google Scholar), and EBNA LP (27Nitsche F. Bell A. Rickinson A.B. J. Virol. 1997; 71: 6619-6628Crossref PubMed Google Scholar) have been described elsewhere. The following plasmids were generous gifts from the originating laboratories: pSV-LMP1, pSV-LMP1Y384G, pSV-LMP1AAA (residues P204A,Q206A,T208A), pSV-LMP1AAA/Y384G (28Kieser A. Kaiser C. Hammerschmidt W. EMBO J. 1999; 18: 2511-2521Crossref PubMed Scopus (104) Google Scholar), pCMV HA-ubiquitin (29Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar), pCR-3-FLAG IKK1S176A,S180A (IKK1.dn), pCR-3-FLAG IKK2S177A,S181A (IKK2.dn) (30Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (473) Google Scholar), pcDNA3-PK-IκBαS32A,S36A (31Roff M. Thompson J. Rodriguez M.S. Jacque J.-M. Baleux F. Arenzana-Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), and pSG5-Tax (32Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cell. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). Purified anti-CD2 mAb (1 μg/ml) from the OX34 murine hybridoma (33Jefferies W.A. Green J.R. Williams A.F. J. Exp. Med. 1985; 162: 117-127Crossref PubMed Scopus (319) Google Scholar) cross-linked with goat anti-mouse antibody (5 μg/ml; Sigma) was used to stimulate transfected 293 cells expressing rat CD2 chimeric proteins. Endogenous human p100/p52 was detected in Western blots using a commercial anti-p100 mAb (UBI 05-361). Anti-mouse p100N and anti-human p100N were used for detection of murine p100/p52 and immunoprecipitation of human p100/p52, respectively (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). Commercial antibodies purchased from Santa Cruz were used to detect the Myc epitope tag (sc-789), IκBα (sc-21), RelB (sc-226), and SAM68 (sc-333) on Western blots. Anti-LMP1-blotting antibody, CS.1-4 (34Rowe 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), was obtained from DAKO (M0897). CD2, EBNA2, EBNA LP, and LMP2A were detected in Western blots with OX34, PE2 (35Young L. Alfieri C. Hennessy K. Evans H. O'Hara C. Anderson K.C. Ritz J. Shapiro R.S. Rickinson A. Kieff E. Cohen J.I. N. Engl. J. Med. 1989; 321: 1080-1085Crossref PubMed Scopus (611) Google Scholar), JF186 (36Finke J. Rowe M. Kallin B. Ernberg I. Rosen A. Dillner J. Klein G. J. Virol. 1987; 61: 3870-3878Crossref PubMed Google Scholar), and 14B7 (37Fruehling S. Lee S.K. Herrold R. Frech B. Laux G. Kremmer E. Grasser F.A. Longnecker R. J. Virol. 1996; 70: 6216-6226Crossref PubMed Google Scholar) mAbs, respectively. Human anti-EBNA1 antiserum was kindly provided by Alan Rickinson (University of Birmingham, Birmingham, UK). Polyclonal antiserum to human T-cell lymphotrophic virus 1 (HTLV) Tax was obtained through the National Institutes of Health AIDS Research and Reference reagent program. The proteasome inhibitor MG132 (Biomol Research Labs, 20 μm) and cycloheximide (Sigma; 10 μg/ml) were added 15-30 min before stimulation with anti-CD2 mAb and goat anti-mouse Ig antibody. Recombinant TNFα (20 ng/ml) was obtained from R & D Systems. Cells—The Ramos tetracycline-regulated transactivator (tTA) and Ramos tTA-LMP1 cell lines have been described previously (38Henriquez N.V. Floettmann E. Salmon M. Rowe M. Rickinson A.B. J. Immunol. 1999; 162: 3298-3307PubMed Google Scholar). Ramos tTA-LMP1 cells are stably transfected with two vectors; pJEF3 encoding a constitutively expressed tTA and pJEF6neo, in which LMP1 is cloned downstream of a promoter containing tTA binding sites. The Ramos tTA cell line is transfected only with pJEF3 vector. Ramos tTA-LMP1 and Ramos tTA cells were cultured in a complete medium comprising RPMI 1640 (Sigma) supplemented with 10% fetal calf serum, 2 mml-glutamine, penicillin (100 units/ml), streptomycin (50 units/ml), 300 μg/ml hygromycin B, and 1 μg/ml tetracycline (Roche Applied Science). For Ramos tTA-LMP1 cells, 1 mg/ml G418 (Invitrogen) was also included in the culture medium to ensure retention of pJEF6neo-LMP1 vector. To induce LMP1 expression, cells were extensively washed in phosphate-buffered saline and then recultured for the indicated times in complete medium without added tetracycline. BL41 cells and their B95.8 EBV-infected derivative BL41+B95 cells were cultured as described previously (39Rowe M. Rooney C.M. Edwards C.F. Lenoir G.M. Rickinson A.B. Int. J. Cancer. 1986; 37: 367-373Crossref PubMed Scopus (74) Google Scholar). Mouse embryonic fibroblasts (MEFs) lacking IKK1 or IKK2 and control wild type MEFs were kindly provided by the originating laboratories (40Takeda 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 (538) Google Scholar, 41Li Q. Van Antwerp D. Mercurio F. Lee K.-F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (857) Google Scholar) and were cultured as described (42Salmeron A. Janzen J. Soneji Y. Bump N. Kamens J. Allen H. Ley S.C. J. Biol. Chem. 2001; 276: 22215-22222Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Protein Analyses—293 cells (5 × 105 cells/60-mm Nunc tissue culture dish) were transiently transfected using LipofectAMINE (Invitrogen). Amounts of plasmids used are indicated in the figure legends. Cells transfected with vectors encoding CD2-LMP 192 or tCD2 were stimulated as described in the figure legends. Whole cell lysates were prepared using buffer A (43Beinke S. Belich M.P. Ley S.C. J. Biol. Chem. 2002; 277: 24162-24168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), which was supplemented with 0.5% deoxycholate and 0.1% SDS (RIPA buffer). p100 ubiquitination experiments were performed as previously described (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). Equal protein loading of whole cell lysates was confirmed by Western blotting for tubulin. Preparation of cytoplasmic and nuclear fractions was performed as described (44Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar). Fractionation efficiency and protein loading were controlled by Western blotting for cytoplasmic (tubulin) and nuclear (SAM68) markers. To facilitate NF-κB subunit detection in Western blots, ∼5-fold more cell equivalents of nuclear extract (10-25 μg) was loaded relative to cytoplasmic extract (30 μg). For immunoprecipitation experiments, nuclear and cytoplasmic extracts were diluted in buffer A (43Beinke S. Belich M.P. Ley S.C. J. Biol. Chem. 2002; 277: 24162-24168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and adjusted to 10% (v/v) glycerol and 150 mm NaCl before incubation with anti-p100N antibody. NF-κB Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs and antibody supershifting were performed as described (44Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar). A radiolabeled double-stranded oligonucleotide corresponding to the NF-κB binding site in the mouse immunoglobulin enhancer (Promega) was used to detect NF-κB complexes. LMP1 Induces NF-κB2 p52 Production in Transfected 293 Epithelial Cells—It has previously been reported that LMP1 expression in epithelial cells is associated with increases in p100 and p52 levels (45Paine E. Scheinman R.I. Baldwin A.S. Raab-Traub N. J. Virol. 1995; 69: 4572-4576Crossref PubMed Google Scholar). However, it is unclear from this study whether other EBV-encoded latent proteins can also induce such changes. To address this question, LMP1, LMP2A, EBNA1, EBNA2, EBNA LP (Fig. 1A), and EBNA 3C (data not shown) were individually expressed in 293 epithelial cells. Western blotting of cell lysates indicated that none of the EBNA proteins or LMP2A had any effect on endogenous p100/p52 levels compared with empty vector (EV) control. In contrast, LMP1 expression stimulated a dramatic increase in steady state levels of p52 together with a more modest increase in p100. Therefore, of the panel of latent proteins tested, LMP1 was unique in its ability to increase p52 levels. Previous studies have indicated that signaling from the LMP1 cytoplasmic tail can be induced by cross-linking of chimeric molecules comprising tCD2 fused to the C-terminal 192 amino acids of LMP1 (CD2-LMP 192; Ref. 25Floettmann J.E. Rowe M. Oncogene. 1997; 15: 1851-1858Crossref PubMed Scopus (107) Google Scholar). CD2-LMP 192, transiently expressed in 293 cells, induced an increase in endogenous p52 levels after 3-6 h of extensive cross-linking with anti-CD2 mAb, whereas p100 levels were largely unaffected (Fig. 1B). The time course of the p52 increase was similar to that previously observed with cross-linked CD40 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar) and was markedly slower than IκBα proteolysis induced by CD2-LMP 192, which was detectable at 30 min. (Fig. 1B). Cross-linked tCD2 failed to induce any alteration in p52 levels, whereas tCD2 fused to full-length wild type LMP1 (CD2-LMP1), which signals constitutively (25Floettmann J.E. Rowe M. Oncogene. 1997; 15: 1851-1858Crossref PubMed Scopus (107) Google Scholar), increased p52 levels without anti-CD2 mAb cross-linking. These data suggest that stimulation of p52 levels by the LMP1 requires aggregation of its cytoplasmic tail. LMP1 Induces p52 as a Consequence of Proteasome-mediated p100 Proteolysis—To determine whether NF-κB-dependent gene expression is required for LMP1-induced p52 production, 293 cells were co-transfected with LMP1 vector together with an expression vector encoding a super-repressor mutant of IκBα (IκBαSS/AA), which blocks NF-κB activation (31Roff M. Thompson J. Rodriguez M.S. Jacque J.-M. Baleux F. Arenzana-Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), or EV. Expression of IκBαSS/AA on its own reduced the basal levels of endogenous p100 expression compared with EV alone and blocked the ability of LMP1 to increase the steady state levels of p100. However, LMP1 still clearly induced p52 levels when co-expressed with IκBαSS/AA while concomitantly reducing levels of p100 (Fig. 2A). Thus, LMP1 induces p52 production independently of NF-κB activation. To investigate the role of the proteasome in LMP1-mediated p52 production, 293 cells were transfected with a vector encoding CD2-LMP 192 to facilitate analysis of the acute up-regulation of p52 by LMP1. Increases in p52 induced by cross-linked CD2-LMP 192 were blocked by pretreatment of cells with the proteasome inhibitors MG132 (Fig. 2B) or N-acetyl-leu-leu-norleucinal (data not shown), demonstrating a requirement for proteasome activity for LMP1 induction of p52. Protein ubiquitination is often a prerequisite for proteasome action. Therefore, experiments were conducted to determine whether LMP1 induced the ubiquitination of p100. 293 cells were co-transfected with plasmids encoding Myc-p100 and HA-ubiquitin together with LMP1 or EV. LMP1-induced polyubiquitination of Myc-p100 was clearly demonstrated by the appearance of high molecular weight bands in Western blots of immunoprecipitated p100 probed for HA-ubiquitin (Fig. 2C). NIK expression also induced Myc-p100 ubiquitination (Fig. 2C), as reported previously (22Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). Thus, proteasome-mediated production of p52 induced by LMP1 involves p100 ubiquitination. Furthermore, the observation that LMP induces p52 levels concomitantly with decreases in p100 levels when NF-κB activity is blocked (Fig. 2A) strongly suggests that LMP1 induces p52 production via proteasome-mediated proteolysis of p100. Two motifs in p100 have been previously shown to be required for CD40-induced processing of p100 to p52 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar); namely the glycine-rich region (GRR; residues 346-377) (46Heusch M. Lin L. Geleiunas R. Greene W.C. Oncogene. 1999; 18: 6201-6208Crossref PubMed Scopus (97) Google Scholar) and two serines (Ser-866 and Ser-870) in the C-terminal PEST region of p100 that are thought be phosphorylated by IKK1 (21Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1144) Google Scholar). To investigate whether the GRR and serines 866/870 are important in LMP1-triggered p52 production, 293 cells were co-transfected with plasmids encoding LMP1 or EV and either wild type Myc-p100, Myc-p100ΔGRR, or Myc-p100S866A,S870A. LMP1 co-transfection with wild type Myc-p100 significantly increased Myc-p52 levels compared with EV control (Fig. 2D). However, LMP1 failed to induce Myc-p52 from either Myc-p100ΔGRR or Myc-p100S866A,S870A (Fig. 2D). Therefore, both the GRR and serines 866 and 870 in the PEST region of p100 are required for LMP1-induced p52 production from p100. These results further support the conclusion that LMP1 induces p52 as a consequence of p100 processing. LMP1 Induction of p52 Requires Protein Synthesis—Both CD40 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar) and B cell-activating factor receptor (17Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Crossref PubMed Scopus (579) Google Scholar) have a requirement for de novo protein synthesis to trigger p100 processing. The requirement for protein synthesis in CD2-LMP1 192-induced p52 production, which occurs over several hours (Fig. 1B), was therefore investigated. 293 cells were transfected with an expression vector encoding CD2-LMP 192 and cycloheximide was added 30 min before CD2-LMP 192 cross-linking with anti-CD2 mAb. Cycloheximide treatment inhibited p52 production induced by cross-linked CD2-LMP 192 (Fig. 2B). Cycloheximide also blocked CD2-LMP 192 induction of p52 when added simultaneously with anti-CD2 mAb but not when added 2 h after CD2 cross-linking (data not shown). These results, which show a striking similarity to those reported for CD40 (20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar), demonstrate that de novo protein synthesis is required during the first 2 h of cross-linked CD2-LMP 192 signaling to induce p100 processing efficiently. LMP1 Induces the Nuclear Translocation of p52 and RelB in 293 Epithelial Cells—p100 is the major IκB for RelB in the cell (47Solan N.J. Miyoshi H. Bren G.D. Paya C.V. J. Biol. Chem. 2001; 277: 1405-1418Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Therefore, one predicted outcome of LMP1-mediated p100 processing is the nuclear translocation of p52·RelB complexes. To initially investigate this, cytoplasmic/nuclear fractions were prepared from 293 cells transfected with expression vectors encoding CD2-LMP 192 or tCD2, stimulated with anti-CD2 mAb, or left unstimulated. Efficient separation of the cytoplasmic and nuclear fractions was demonstrated for this (Fig. 3A) and subsequent experiments by Western blotting for cytoplasmic (tubulin) and nuclear (SAM68) markers. In unstimulated or control tCD2-transfected cells, very little endogenous p52 or RelB was detected in the nuclear fraction (Fig 3A). Cross-linking of CD2-LMP 192 for 30 min resulted in a small increase in nuclear p52 but had no effect on nuclear RelB levels. This small increase in nuclear p52 at 30 min is likely to be due to the release of pre-existing p52 bound to small IκBs, including IκBα (Fig. 1B; Ref. 20Coope H.J. Atkinson P.G.P. Huhse B. Belich M.P. Janzen J. Holman M. Klaus G.G.B. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (370) Google Scholar). However, marked increases in both nuclear p52 and RelB were detected after 6 h of cross-linking of CD2-LMP 192, although cytoplasmic RelB levels were unaffected. Moreover, immunoprecipitation of nuclear extracts with anti-p100N antibody demonstrated that the translocated nuclear p52 and RelB are associated with one another" @default.
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• W1997407816 title "Latent Membrane Protein 1 of Epstein-Barr Virus Stimulates Processing of NF-κB2 p100 to p52" @default.
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