TAK1 Is a Component of the Epstein-Barr Virus LMP1 Complex and Is Essential for Activation of JNK but Not of NF-κB

Abstract

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-κB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-κB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-κB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IκB kinase γ is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-κB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-κB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-κB activation in LMP1 signaling. Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-κB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-κB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-κB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IκB kinase γ is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-κB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-κB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-κB activation in LMP1 signaling. Persistent latent infection with Epstein-Barr virus (EBV), 2The abbreviations used are: EBV, Epstein-Barr virus; LMP1, latent membrane protein 1; JNK, c-Jun N-terminal kinase; TRAF, tumor necrosis factor receptor-associated factor; TNF, tumor necrosis factor; TGF-β, transforming growth factor β; TAK1, TGF-β-activated kinase 1; TAB, TAK1-binding protein; IKK, IκB kinase; CTAR, C-terminal activator region; TRADD, TNF receptor-associated death domain protein; IL-1, interleukin 1; RANK, receptor activator of NF-κB; NIK, NF-κB-inducing kinase; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA; Flag-TfR, Flag-tagged transferrin receptor; MAPKKK, mitogen-activated protein kinase kinase kinase; DTT, dithiothreitol; HA, hemagglutinin; WT, wild type. 2The abbreviations used are: EBV, Epstein-Barr virus; LMP1, latent membrane protein 1; JNK, c-Jun N-terminal kinase; TRAF, tumor necrosis factor receptor-associated factor; TNF, tumor necrosis factor; TGF-β, transforming growth factor β; TAK1, TGF-β-activated kinase 1; TAB, TAK1-binding protein; IKK, IκB kinase; CTAR, C-terminal activator region; TRADD, TNF receptor-associated death domain protein; IL-1, interleukin 1; RANK, receptor activator of NF-κB; NIK, NF-κB-inducing kinase; MEF, mouse embryonic fibroblast; siRNA, small interfering RNA; Flag-TfR, Flag-tagged transferrin receptor; MAPKKK, mitogen-activated protein kinase kinase kinase; DTT, dithiothreitol; HA, hemagglutinin; WT, wild type. a γ herpes virus that is classified as a human DNA tumor virus, is widespread in the human population and can cause the development of malignancies such as Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngel carcinoma. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by EBV and is critically involved in the effective immortalization and proliferation of B-cells latently infected by EBV (1Farrell P.J. Trends Microbiol. 1995; 3: 105-109Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 2Klein G. Cell. 1994; 77: 791-793Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 3Rickinson A. Kieff E. Fields Virology.in: Howley P.M. Knipe D. Lippincott, Philadelphia2001: 2575-2627Google Scholar). LMP1 is a transmembrane protein of 386 amino acids containing a short N-terminal cytoplasmic domain of 24 amino acids, six transmembrane-spanning domains, and a C-terminal cytoplasmic tail of 200 amino acids (Fig. 1A). LMP1 mimics a constitutively activated tumor necrosis factor (TNF) receptor-like molecule, in the absence of ligand binding (4Cahir-McFarland E.D. Izumi K.M. Mosialos G. Oncogene. 1999; 18: 6959-6964Crossref PubMed Scopus (140) Google Scholar, 5Lam N. Sugden B. Cell. Signal. 2003; 15: 9-16Crossref PubMed Scopus (101) Google Scholar). The transmembrane domains of LMP1 mediate spontaneous autoaggregation within plasma membrane, and this aggregation is a prerequisite for LMP1 function (6Coffin W.F. II I Geiger T.R. Martin J.M. J. Virol. 2003; 77: 3749-3758Crossref PubMed Scopus (42) Google Scholar, 7Yasui T. Luftig M. Soni V. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 278-283Crossref PubMed Scopus (58) Google Scholar). Previous studies have identified two domains in the LMP1 C-terminal cytoplasmic tail, called the C-terminal activator regions (CTARs) 1 (amino acids 194–231) and 2 (amino acids 332–386), that are important for the cell transformation activity of LMP1 (4Cahir-McFarland E.D. Izumi K.M. Mosialos G. Oncogene. 1999; 18: 6959-6964Crossref PubMed Scopus (140) Google Scholar, 5Lam N. Sugden B. Cell. Signal. 2003; 15: 9-16Crossref PubMed Scopus (101) Google Scholar, 8Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (186) Google Scholar). Both CTAR1 and CTAR2 participate in the activation of the transcription factor NF-κB. CTAR1 binds to several TNF receptor-associated factors (TRAFs), TRAF1, -2, -3, and -5, through a consensus TRAF-binding motif, while CTAR2 has been shown to bind to the TNF receptor-associated death domain protein (TRADD). However, experiments using gene knock-out and small interfering RNA (siRNA) treatment have determined that neither TRAF2, TRAF5, nor TRADD is essential for LMP1 signaling (9Luftig M. Prinarakis E. Yasui T. Tsichritzis T. Cahir-McFarland E. Inoue J. Nakano H. Mak T.W. Yeh W.C. Li X. Akira S. Suzuki N. Suzuki S. Mosialos G. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15595-15600Crossref PubMed Scopus (110) Google Scholar, 10Wan 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). Therefore, these molecules are likely to act redundantly. In contrast, two independent studies have shown that TRAF6, which was previously established as an essential mediator of interleukin 1 (IL-1) and receptor activator of NF-κB (RANK), is also a critical factor for LMP1-induced activation of NF-κB and JNK (9Luftig M. Prinarakis E. Yasui T. Tsichritzis T. Cahir-McFarland E. Inoue J. Nakano H. Mak T.W. Yeh W.C. Li X. Akira S. Suzuki N. Suzuki S. Mosialos G. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15595-15600Crossref PubMed Scopus (110) Google Scholar, 10Wan 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). Unlike TRAF2 and TRAF5, TRAF6 has not been extensively studied in connection with LMP1. It is still unclear which LMP1 domains are involved in its association with TRAF6. Moreover, the mechanism by which TRAF6 mediates LMP1 signaling has not been well studied.FIGURE 1LMP1 associates with TAK1, TAB2, and TRAF6. A, schematic drawing of wild type LMP1 and mutants. B–D, 293 cells were transfected with expression vectors for various versions of Flag-LMP1 or Flag-TfR together with HA-TAK1 (B), HA-TAB2 (C), or HA-TRAF6 (D). Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE, whole cell extracts; HC, immunoglobulin heavy chain; LC, immunoglobulin light chain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family and is activated by various cytokines, including the family of TGF-β ligands (11Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1174) Google Scholar). TAK1 is also involved in the IL-1 signaling pathway (12Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1018) Google Scholar). Following exposure of cells to IL-1, endogenous TAK1 is recruited to the TRAF6 complex and activated, upon which it stimulates both the JNK and NF-κB pathways. Several lines of evidence indicate that TAK1 is an essential mediator of TAK1 or TAK1 NF-κB activation G. J. Matsumoto K. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, S. H. K. J. M. T. Matsumoto K. Akira S. PubMed Scopus Google a TAK1 activation of NF-κB and JNK J. T. K. T. Matsumoto M. M. M. M. Matsumoto K. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google TAK1 in an to infection S. M. B. 2001; 15: PubMed Scopus Google Scholar). In we have and its that with and are also in a signaling T. G. J. Shibuya H. Matsumoto K. J. 2003; PubMed Scopus Google Scholar, G. S. Hiyama A. K. Shibuya H. Irie K. J. Matsumoto K. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of both and by IL-1 and have is involved in the TRAF6 associates with TRAF6 as well as TAK1, and this facilitates the assembly of a signaling complex of TRAF6, TAB2, and TAK1 in to IL-1 and ligand G. S. Hiyama A. K. Shibuya H. Irie K. J. Matsumoto K. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. G. H. H. J. Matsumoto K. N. Mol. Cell. Biol. PubMed Scopus Google Scholar, G. J. S. Li X. Matsumoto K. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). has that TAK1 is activated by LMP1 and that of TAK1 expression by a in LMP1-induced JNK activation 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). that TAK1 in LMP1 signaling. However, the LMP1, TRAF6, TAK1, and JNK to Here we that TAK1 and are involved in LMP1 signaling. found that TRAF6, TAB2, and TAK1 were a complex with LMP1. of these factors was of JNK and was through the transmembrane region of LMP1. In contrast, activation of NF-κB the C-terminal cytoplasmic region of LMP1 is Furthermore, we found that TAK1 was essential for LMP1-induced JNK activation but for NF-κB These results suggest that the of the complex with LMP1 mediates activation of JNK, while recruited the C-terminal region of LMP1 can activated through that vectors were by Kieff M. Izumi K.M. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). and a or was a expression and were by and The were by expression of C-terminal transferrin were receptor and and HA-TRAF6 were also the expression of TAK1, TAK1 were to of the TAK1 region were the to Cell and and mouse embryonic S. H. K. J. M. T. Matsumoto K. Akira S. PubMed Scopus Google and cells were in with or growth in the 293 cells were in transfected with a of of DNA containing various expression vectors by the and for and and cells were transfected using 6 and and to TRAF6 TAK1 and have been previously (12Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1018) Google Scholar, G. S. Hiyama A. K. Shibuya H. Irie K. J. Matsumoto K. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). and were used for and was used to the of JNK. The TAK1 has been previously J. T. K. T. Matsumoto M. M. M. M. Matsumoto K. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). human and were of Cell cell lines that wild type TAK1, were transfected with together with an expression for were in containing 200 and expression of was by immunoblotting with of Cell for cell lines that TAK1, cells were transfected with together with an expression for were in containing and expression of TAK1 was determined by the gene 293 cells or were or cells were 24 cells were transfected with a and expression vectors as cells were with TNF for was used to gene was used to gene the of the or the of the were used to and were with and in of 2 2 DTT, 1 1 cells were in of 1 1 Cell was by for cell lysates were immunoprecipitated with 1 of various and of protein The were with with 1 and in of the or whole cell lysates were and to The were with various and the were with or mouse using the or were or with TNF for and and for in NF-κB were The binding containing the of cell 1 DTT, of and of in a of were for 1 and to a in 1 1 of or were to the and for were The were and to have previously that TAK1 and its protein important in IL-1 and signaling complex with TRAF6 G. S. Hiyama A. K. Shibuya H. Irie K. J. Matsumoto K. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. G. H. H. J. Matsumoto K. N. Mol. Cell. Biol. PubMed Scopus Google Scholar). it was shown that TRAF6 is an indispensable intermediate in LMP1 signaling (9Luftig M. Prinarakis E. Yasui T. Tsichritzis T. Cahir-McFarland E. Inoue J. Nakano H. Mak T.W. Yeh W.C. Li X. Akira S. Suzuki N. Suzuki S. Mosialos G. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15595-15600Crossref PubMed Scopus (110) Google Scholar, 10Wan 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). the that TAK1 and also participate in LMP1 signaling. this we used in human embryonic 293 cells to LMP1 associates with TAK1, TAB2, and TRAF6 (Fig. 293 cells were transfected with expression vectors for Flag-tagged LMP1 together with TAK1 or TRAF6 Flag-tagged transferrin receptor was used as a membrane Cell were immunoprecipitated with or and were by found that TAK1, TAB2, and TRAF6 were of with LMP1. The N-terminal transmembrane region of LMP1 mediates which is a prerequisite for LMP1 activation (6Coffin W.F. II I Geiger T.R. Martin J.M. J. Virol. 2003; 77: 3749-3758Crossref PubMed Scopus (42) Google Scholar, 7Yasui T. Luftig M. Soni V. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 278-283Crossref PubMed Scopus (58) Google while CTAR1 and CTAR2 domains in the C-terminal cytoplasmic tail are to mediate LMP1 signaling. TRAF2, TRAF5, and TRADD are involved in signaling (4Cahir-McFarland E.D. Izumi K.M. Mosialos G. Oncogene. 1999; 18: 6959-6964Crossref PubMed Scopus (140) Google Scholar, 5Lam N. Sugden B. Cell. Signal. 2003; 15: 9-16Crossref PubMed Scopus (101) Google Scholar, 8Eliopoulos A.G. Young L.S. Semin. Cancer Biol. 2001; 11: 435-444Crossref PubMed Scopus (186) Google Scholar). which domain is involved in the binding of TAK1, TAB2, and TRAF6, we several LMP1 (Fig. 1A). The and by have in essential amino of and M. Izumi K.M. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). In we the and which the C-terminal cytoplasmic tail and both the N-terminal and C-terminal cytoplasmic domains, transfected expression vectors for these various LMP1 together with or HA-TRAF6 (Fig. found that TAK1, TAB2, and TRAF6 were with of LMP1 and These results suggest that the CTAR1 and CTAR2 domains are not involved in the association of LMP1 with TAK1, TAB2, and TRAF6. Furthermore, or TAK1, TAB2, and TRAF6, to with TAK1 and TRAF6. The (Fig. B–D, that TAK1, TAB2, and TRAF6 LMP1 and and These results suggest that TAK1, TAB2, and TRAF6 with LMP1 through the N-terminal transmembrane region. The C-terminal region of LMP1 the assembly of the the of these we the LMP1 and endogenous transfected 293 cells with expression vectors for various versions of Flag-LMP1 and used to to endogenous including TRAF2, TRAF6, TAB2, and TAK1 (Fig. TRAF2 is to with LMP1 through the CTAR1 domain E. Izumi K.M. K.M. Kieff E. Mosialos G. Mol. Cell. Biol. PubMed Google Scholar). with TRAF2 was found to with LMP1 and the but not with the or (Fig. In contrast, we found that TRAF6 was with LMP1 and and mutants. and TAK1 were also with LMP1 and and and with is with the results shown The was in cell LMP1 and cells (Fig. TAK1 LMP1 but These results suggest that LMP1 can TRAF6, TAB2, and TAK1 through its N-terminal transmembrane region and that the C-terminal region to the The N-terminal transmembrane region is essential for the of LMP1. However, the role of the has not been the of expression the activation of JNK and NF-κB (Fig. 293 cells were transfected with expression vectors for various Flag-LMP1 mutants. of JNK was by immunoblotting with JNK The domains of LMP1 have been in JNK activation A.G. M. Young L.S. J. Virol. 1999; PubMed Google Scholar, A. J. 1999; 18: PubMed Scopus Google Scholar). we found that both domains and the of the C-terminal region JNK activation (Fig. However, we found that the N-terminal transmembrane region to JNK it was (Fig. and activation with the of TAK1, TAB2, and TRAF6 the LMP1 which with the and with the of LMP1 activation of JNK and NF-κB. A, 293 cells were transfected with expression vectors for various versions of of vectors were The activated of JNK was with 293 cells were transfected with expression vectors for various versions of The of were the as in A. Cell lysates were to using NF-κB DNA NF-κB binding was by with The of in the cell lysates and expression of LMP1 are also WCE, whole cell 293 cells were transfected with an and together with expression vectors for various versions of Flag-LMP1 or of vectors were activity was determined and to the of activity. to with is 293 cells were transfected with expression vectors for various versions of Flag-LMP1 or Flag-TfR together with Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE, whole cell 293 cells were transfected with expression vectors for together with in the absence or of Flag-LMP1 or Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE, whole cell Large Image Figure ViewerDownload Hi-res image Download (PPT) NF-κB activation was by an NF-κB (Fig. and (Fig. In with studies E. Izumi K.M. K.M. Kieff E. Mosialos G. Mol. Cell. Biol. PubMed Google Scholar, K.M. Kieff E.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google of CTAR1 or CTAR2 or NF-κB binding while the NF-κB activation (Fig. The was to NF-κB binding activity. The that the not NF-κB (Fig. these results suggest that of a complex containing TRAF6, TAB2, and TAK1, through with the LMP1 N-terminal transmembrane participate in JNK activation but is not to mediate NF-κB of NF-κB activation by the LMP1 transmembrane region that the signaling complex through the transmembrane region is not to NF-κB. that the not recruited in the complex of LMP1 transmembrane region. is in the LMP1 transfected expression vectors for together with the LMP1 and or (Fig. found that was with LMP1 and and but not with the C-terminal mutants. that the LMP1 through its C-terminal region. Therefore, the transmembrane region is not for of the has to the that it not NF-κB. that of TRAF2, TRAF5, or TRADD, which is recruited through domains, is likely to essential for NF-κB TAK1 and in the LMP1 transfected expression vectors for TAK1, together with the LMP1 or (Fig. TAK1 was not with in the absence of LMP1. of LMP1 but not of TAK1 with These results suggest that TAK1 by not with and that of LMP1 is important for assembly of the signaling complex leading to NF-κB TAK1 is essential for LMP1 signaling by a of TAK1, (Fig. shown previously J. T. K. T. Matsumoto M. M. M. M. Matsumoto K. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google can activation of JNK by TNF (Fig. and IL-1 not LMP1 were with or and JNK activity was LMP1-induced JNK to an to its of the TNF (Fig. the of NF-κB activation using an (Fig. previously J. T. K. T. Matsumoto M. M. M. M. Matsumoto K. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google activation of while of NF-κB-inducing kinase activated NF-κB of found that LMP1-induced NF-κB These results suggest that TAK1 is involved in LMP1 signaling leading to activation of JNK but not of NF-κB. the role of TAK1 in LMP1 we mouse embryonic a of the within the TAK1 kinase domain (amino acids (Fig. cell was by containing TAK1 by with a S. H. K. J. M. T. Matsumoto K. Akira S. PubMed Scopus Google Scholar). The was using the LMP1-induced JNK activation in and transfected with JNK. JNK activity was by of by immunoblotting with (Fig. of the can a that activates JNK. found that activated JNK in both and In contrast, LMP1 activated JNK in but not in the is to the TAK1 we wild type TAK1 (Fig. of TAK1 in LMP1-induced JNK These results that TAK1 is an essential mediator of LMP1 signaling leading to JNK is essential for LMP1-induced JNK A, schematic drawing of and were transfected with expression vectors for and cells were with for to was and the activated of JNK was with WCE, whole cell and were transfected with expression vectors for Cell lysates were to using an NF-κB DNA NF-κB binding was by with of in the cell lysates and expression of LMP1 are WCE, whole cell and were transfected with and together with expression for activity was determined and to the of activity. to with is or TAK1 cells were with and or TAK1 cells were transfected with expression vectors for and cells were with for to was immunoprecipitated and the activated of JNK was with WCE, whole cell or TAK1 cells were transfected with or and together with expression for activity was determined and to the of activity. to with is Large Image Figure ViewerDownload Hi-res image Download (PPT) NF-κB activation in (Fig. and shown that of TAK1 TNF signaling G. J. Matsumoto K. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, S. H. K. J. M. T. Matsumoto K. Akira S. PubMed Scopus Google Scholar). an that NF-κB binding activity was in (Fig. In contrast, we found that LMP1 was to NF-κB binding activity to a or in TAK1 with also activity of an (Fig. found that LMP1 activated both and a of TAK1 that the as this TAK1 has kinase it that the TAK1 still can activation of NF-κB in a of independent of kinase activity. this we used cells TAK1 in which expression of TAK1 is (Fig. of TAK1 protein signaling we JNK activation (Fig. and activity of or (Fig. was used to the of JNK. LMP1 not JNK in the cells TAK1 siRNA, which is with the results TAK1 of was also by TAK1 while activation of NF-κB was not These results that TAK1 is for LMP1-induced NF-κB It is likely that TAK1 to NF-κB with in LMP1 The transmembrane region of LMP1 is essential for LMP1 signaling. through the LMP1 transmembrane region is to signaling (6Coffin W.F. II I Geiger T.R. Martin J.M. J. Virol. 2003; 77: 3749-3758Crossref PubMed Scopus (42) Google Scholar, 7Yasui T. Luftig M. Soni V. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 278-283Crossref PubMed Scopus (58) Google Scholar). However, the role of the N-terminal transmembrane region has not been Here we demonstrate that TRAF6, TAK1, and its binding protein can with LMP1 through the LMP1 N-terminal transmembrane region 1 and a role of LMP1 transmembrane region in the assembly of a signaling complex of TRAF6, TAB2, and TAK1 (Fig. The C-terminal region to this assembly as shown in 1 and also that expression of the N-terminal transmembrane region of LMP1 has the to JNK (Fig. that an LMP1 transmembrane complex containing TRAF6, TAB2, and TAK1 can mediate activation of JNK. LMP1 C-terminal region to the complex and enhances JNK The also revealed that TAK1 is essential for LMP1-induced activation of JNK (Fig. and and assembly of a signaling complex of mediates activation of JNK (Fig. In with we that the N-terminal transmembrane region is to NF-κB (Fig. and that the signaling complex not mediate activation of NF-κB. In IL-1 signaling TAK1, and TRAF6 a signaling complex and However, in this not the complex of but also of this complex is indispensable for activation L. M. Inoue J. Chen Z.J. Nature. 2001; PubMed Scopus Google Scholar, A. Sun L. M. A. L. Chen Z.J. Mol. Cell. 2004; 15: Full Text Full Text PDF PubMed Scopus Google Scholar). LMP1 can found that is recruited by LMP1 but not by (Fig. that LMP1 transmembrane region TRAF6, TAB2, and TAK1 but not the complex in of TAK1 is essential for and activation both of JNK and of NF-κB S. H. K. J. M. T. Matsumoto K. Akira S. PubMed Scopus Google Scholar). In to we found that TAK1 is for NF-κB activation the that TAK1 is recruited the LMP1 that such as NIK, which can function with is in LMP1 signaling A. Sun L. M. A. L. Chen Z.J. Mol. Cell. 2004; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, N. G. A. N. T. N. M. S. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). LMP1 to not TAK1 but also and the leading to activation of JNK and NF-κB. It is that not have to JNK, TAK1 can both JNK and NF-κB. can bind TRAF6 and TAK1 and mediate assembly of a signaling complex J. G. H. H. J. Matsumoto K. N. Mol. Cell. Biol. PubMed Scopus Google Scholar). that TRAF6, TAB2, and TAK1 are also of the LMP1 it is likely that facilitates assembly of the LMP1 complex by binding to both TRAF6 and with studies that is for activation of both JNK and NF-κB in LMP1 signaling (9Luftig M. Prinarakis E. Yasui T. Tsichritzis T. Cahir-McFarland E. Inoue J. Nakano H. Mak T.W. Yeh W.C. Li X. Akira S. Suzuki N. Suzuki S. Mosialos G. Kieff E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15595-15600Crossref PubMed Scopus (110) Google Scholar, 10Wan 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). However, we previously that a of TAB2, plays a role with in the IL-1 and TNF signaling S. M. B. 2001; 15: PubMed Scopus Google Scholar). It is that in knock-out for the function of in LMP1 complex identified LMP1 complex containing TRAF6, TAB2, and TAK1 to important for the activity of LMP1. studies TAK1 the in role of this complex in E. Kieff and E. Nishida for