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Activation of NF-κB by RANK Requires Tumor Necrosis Factor Receptor-associated Factor (TRAF) 6 and NF-κB-inducing Kinase

1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês

10.1074/jbc.274.12.7724

ISSN

1083-351X

Autores

Bryant G. Darnay, Jian Ni, Paul A. Moore, Bharat B. Aggarwal,

Tópico(s)

Immune Response and Inflammation

Resumo

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor κB (NF-κB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-κB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-κB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-κB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565–568 and 606–611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340–358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-κB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-κB activation. However, RANK-mediated JNK activation required a distal portion (427–603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-κB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-κB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK. Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor κB (NF-κB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-κB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-κB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-κB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565–568 and 606–611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340–358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-κB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-κB activation. However, RANK-mediated JNK activation required a distal portion (427–603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-κB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-κB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK. receptor activator of NF-κB tumor necrosis factor nuclear factor κB RANK ligand TNF receptor-associated factor TNF-related activation-induced cytokine c-Jun N-terminal kinase polyacrylamide gel electrophoresis interleukin-1 receptor-associated kinase glutathione S-transferase polymerase chain reaction NF-κB-inducing kinase RANK1 (forreceptor activator ofNF-κB), a new member of the tumor necrosis factor (TNF) receptor superfamily, is a 616-amino acid receptor that includes a 383-amino acid intracellular domain with no significant homology to other members of this family (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar). Although RANK is ubiquitously expressed in human tissues, its cell surface expression is limited to dendritic cells, the CD4+ T cell line MP-1, and foreskin fibroblasts (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar, 2Wong B.R. Josien R. Lee S.W. Sauter B. Li H.-L. Steinman R.M. Choi Y. J. Exp. Med. 1997; 186: 2075-2080Crossref PubMed Scopus (748) Google Scholar). Human RANK ligand (RANKL/TRANCE/OPGL/ODF), a type II transmembrane protein with an approximate molecular mass of 45 kDa, is expressed primarily on primary T cells, T cell lines, and lymphoid tissue (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar, 3Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar, 4Lacey D.L. Timms E. Tan H.-L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4582) Google Scholar, 5Yasuda H. Shima N. Nakagawa N. Yamaguchi Y. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3526) Google Scholar). Like other ligands of the TNF superfamily, RANKL has been demonstrated to activate nuclear factor κB (NF-κB) (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar) and c-Jun-terminal kinase (JNK) (3Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar). Furthermore, stimulation of dendritic cells with RANKL up-regulates the expression of the anti-apoptotic protein Bcl-XL, suggesting a potential role for RANK/RANKL in dendritic cell survival (2Wong B.R. Josien R. Lee S.W. Sauter B. Li H.-L. Steinman R.M. Choi Y. J. Exp. Med. 1997; 186: 2075-2080Crossref PubMed Scopus (748) Google Scholar). Moreover, RANKL has been demonstrated to play an essential role in osteoclast differentiation and activation (4Lacey D.L. Timms E. Tan H.-L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4582) Google Scholar, 5Yasuda H. Shima N. Nakagawa N. Yamaguchi Y. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3526) Google Scholar).Many of the TNF receptor superfamily members interact with a family of adaptor proteins referred to as TNF receptor-associated factors (TRAFs), which are characterized by a ring and zinc finger motif in their N termini and C-terminal domains that appear to be responsible for self- and non-self associations (6Arch R.H. Gedrich R.W. Thompson C.B. Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (511) Google Scholar). Of the six known TRAF family members, only TRAF2, TRAF5, and TRAF6 activate NF-κB and JNK (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar), and only TRAF2 has been demonstrated to activate p38 kinase (8Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 9Liu Z.-G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar). TRAF1, TRAF2, and TRAF5 interact with a characteristic TRAF binding motif, PXQXT, in the cytoplasmic domain of several members of the TNF receptor family (10Hsu H. Solovyev I. Colombero A. Elliott R. Kelley M. Boyle W.J. J. Biol. Chem. 1997; 272: 13471-13474Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 11Ishida T. Mizushima S. Azuma S. Kobayshi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 12Marsters S.A. Ayers T.M. Skubatch M. Gray C.L. Rothe M. Ashkenazi A. J. Biol. Chem. 1997; 272: 14029-14032Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 13Boucher L.-M. Marengere L.E.M. Lu Y. Thukral S. Mak T.W. Biochem. Biophys. Res. Commun. 1997; 233: 592-600Crossref PubMed Scopus (89) Google Scholar, 14Miller W.E. Cheshire J.L. Raab-Traub N. Mol. Cell. Biol. 1998; 18: 2835-2844Crossref PubMed Scopus (76) Google Scholar, 15Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). TRAF6 interacts with the cytoplasmic domain of RANK (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and with CD40 via a distinct 16-amino acid region (residues 230–245) (11Ishida T. Mizushima S. Azuma S. Kobayshi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Furthermore, TRAF6 interacts with interleukin-1 receptor-associated kinase 1 and 2 (IRAK1 and IRAK2) (17Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (973) Google Scholar, 18Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 19Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1111) Google Scholar).Besides TRAFs, the activation of NF-κB is also mediated through a recently identified novel member of the mitogen-activated protein kinase kinase kinase family termed NF-κB-inducing kinase (NIK) (20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar). NIK was originally identified as a TRAF2-interacting protein (20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar) and subsequently was found to interact with all TRAF molecules, except TRAF4 (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar). When overexpressed in cultured cells, NIK, but not a kinase-inactive mutant (NIK-KM), activates NF-κB (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar, 15Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar) and JNK (15Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 21Karin M. Delhase M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9067-9069Crossref PubMed Scopus (204) Google Scholar). Furthermore, overexpression of NIK-KM inhibits NF-κB activation by TNF, interleukin-1, CD27, human T-cell leukemia virus type 1 TAX, and Epstein-Barr virus-transforming protein latent infection membrane protein 1 (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar, 15Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar, 22Geleziunas R. Ferrell S. Lin X. Mu Y. Cunningham E.T. Grant M. Connelly M.A. Hambor J.E. Marcu K.B. Greene W.C. Mol. Cell. Biol. 1998; 18: 5157-5165Crossref PubMed Google Scholar, 23Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S.-C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 24Sylla B.S. Hung S.C. Davidson D.M. Hatzivassliiou 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). Consequently, the activation of NF-κB by NIK is mediated through its interaction with the IκBα kinase (IKKα and IKKβ) complex (25Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (267) Google Scholar, 26Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 27Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (444) Google Scholar, 28Nakano 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 (471) Google Scholar), which results in the phosphorylation and degradation of IκBα.Previous studies from our laboratory showed that the cytoplasmic domain of RANK interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-κB and JNK pathways (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). However, it is not known whether these TRAFs bind to the same region of RANK or which TRAF or TRAFs are necessary for activation of NF-κB and JNK. Similarly, it is not known whether NIK is involved in RANK-induced NF-κB activation. In addressing the role of various TRAFs and NIK in NF-κB and JNK activation mediated by RANK, we now demonstrate that RANK activates NF-κB by interacting with TRAF6 via a novel TRAF6 interaction motif and TRAF6 potentially activates NIK, leading to NF-κB activation, whereas RANK activates JNK through a TRAF2-interacting region in RANK.RESULTSIn previous studies, we found that the intracellular domain (residues 234–616) of RANK contains three putative TRAF binding motifs of the sequence PXQXT: two located at the C terminus and one localized in the middle of the cytoplasmic domain (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). We also previously reported that TRAF2, TRAF5, and TRAF6 interact with RANK (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) and that overexpression of RANK in 293 cells activates the NF-κB and JNK pathways (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1922) Google Scholar, 16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Therefore, in the present study, we sought to identify more specifically which regions of RANK are responsible for the activation of NF-κB and JNK and, furthermore, to define which TRAF molecules are responsible for these signaling pathways.Different Regions of RANK Are Responsible for Binding TRAF2, TRAF5, and TRAF6As we previously reported (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), RANK contains three putative TRAF binding motifs (Fig.1 A) and RANK interacts with TRAF2, TRAF5, and TRAF6. To identify which region of the cytoplasmic domain of RANK is necessary for binding TRAF2, TRAF5, and TRAF6, we constructed a series of deletion mutants of the cytoplasmic domain of RANK encompassing the various putative TRAF-binding domains (Fig.1 A). Each of these deletion mutants were fused in-frame with GST and purified by glutathione-agarose affinity chromatography. We examined the ability of each GST-RANK fusion protein to precipitate epitope-tagged TRAF2, TRAF5, and TRAF6 upon their overexpression in 293 cells (Fig. 1 B). We observed strong interaction of TRAF2 and TRAF5 with GST-RANK fusion proteins containing residues 529–616. However, while TRAF2 was still capable of binding to GST-RANK fusion proteins lacking the last 13 amino acids, TRAF5 was not (Fig.1 B, top and middle). These data suggest that TRAF-binding domain III is responsible for TRAF5 interaction and that both TRAF II and TRAF III binding motifs are required for high-affinity binding of TRAF2, but the TRAF III binding motif is not essential for RANK's interaction with TRAF2. Unlike TRAF2 and TRAF5, TRAF6 did not interact with TRAF-binding domains II and III (Fig. 1 B, bottom). This is consistent with a data indicating that TRAF6 does not bind to the PXQXT motif (11Ishida T. Mizushima S. Azuma S. Kobayshi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. Chem. 1996; 271: 28745-28748Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Surprisingly, TRAF6 did bind to RANK between residues 326 and 427 (Fig. 1 B, bottom). Conversely, GST-RANK deletion mutants that did not contain residues 326–427 did not bind TRAF6 (Fig. 1, A and B). Inspection of the amino acid sequence between residues 326 and 427 revealed a putative TRAF6 binding motif (see below). Hence, the cytoplasmic domain of RANK appears to interact with TRAF2, TRAF5, and TRAF6 molecules using three distinct motifs.A Minimal Region of RANK (Residues 326–427) Activates NF-κBHow interactions of different TRAFs with RANK affect RANK's ability to activate NF-κB and JNK is not known. To examine this, we constructed FLAG-tagged RANK deletion mutants (identical to those deletion mutants used to construct GST-RANK) in pCMVFLAG1 (Fig.2 A). Their expression was determined by transient transfection in 293 cells and Western blotting with anti-FLAG (Fig. 2 B). As expected, all of the FLAG-tagged RANK deletion mutants were expressed similarly in 293 cells.Figure 2Schematic diagram and expression of FLAG-tagged RANK and its deletion mutants. A, diagrammatic representation of RANK and its deletion mutants. All RANK deletion mutants were fused with a FLAG epitope tag at the N terminus using the signal sequence in the expression vector pCMVFLAG1 as described under “Experimental Procedures.” RANK-616, -530, -427, and -330 were previously described (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). The Roman numerals I, II, and III represent putative TRAF-binding domains within the cytoplasmic domain of RANK. ED, extracellular domain; TM, transmembrane domain; CD, cytoplasmic domain. B, expression of FLAG-tagged RANK and its deletions. Human embryonic 293 cells on 6-well plates were transiently transfected with the indicated RANK expression vectors (2.5 μg) using a total of 3 μg of plasmid DNA. After 24 h, cell lysates were prepared and subjected to SDS-PAGE and Western blotting with an anti-FLAG monoclonal antibody as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)Next, we examined the ability of each RANK deletion mutant to activate a NF-κB-dependent SEAP reporter construct. Transient overexpression of RANK616 in 293 cells activated NF-κB-dependent reporter activity (Fig.3 A), which could be inhibited by co-transfection of an IκBα mutant lacking its N-terminal phosphorylation sites (data not shown). Deletion of the C-terminal region up to residue 427 (RANK427) had no effect on NF-κB-dependent reporter activity, but further deletion to residue 330 (RANK330) failed to activate NF-κB (Fig.3 A). Furthermore, when only the C-terminal region was fused to the transmembrane domain of RANK, NF-κB-dependent activity was either very weak (RANK429–616) or failed to respond (RANK529–616) (Fig. 3 A), although each of the deletion mutants RANK429–616 and RANK529–616 interacted strongly with TRAF2 and TRAF5 (Fig. 2). Truncation of the TRAF5-binding region (RANK603 and RANK326–603) did not appear to affect NF-κB-dependent reporter activity. Together, these data indicate that residues 326–427 are responsible for activation of NF-κB. This was further confirmed by transfection of a deletion mutant, containing only residues 326–427 fused to the transmembrane region of RANK, which activated NF-κB-dependent reporter activity similar to that of RANK616 (Fig. 3 A). The observations that some RANK deletion mutants (i.e. R530) activate NF-κB stronger than the full-length suggest that other factors may regulate RANK signaling such as cell surface receptor expression, other receptor-associated factors, and receptor processing. Nevertheless, taken together, these data suggest that the interaction of TRAF2 and TRAF5 with RANK is not required for RANK-induced NF-κB, but that the interaction of TRAF6 with RANK is necessary and sufficient for mediating NF-κB activation by RANK.Figure 3A minimal region of RANK (residues 326–427) activates NF-κB-dependent SEAP reporter activity via NIK. A, localization of a minimal region of RANK for NF-κB activation. Human 293 cells were transiently transfected with 3 μg of total plasmid DNA in duplicate with pNF-κB-SEAP (0.5 μg) and the indicated RANK expression plasmids (2.5 μg) as described under “Experimental Procedures.” At 36 h post-transfection, the conditioned medium was assayed for SEAP activity as described under “Experimental Procedures.” Lysates were prepared and Western blotting with anti-FLAG was performed to verify receptor expression. The results are representative of at least six independent transfection experiments with similar results.B, inhibition of RANK-induced NF-κB activation by NIK-KM. Human 293 cells transiently transfected with 5 μg of total plasmid DNA were transfected in duplicate with pNF-κB-SEAP (0.5 μg), pFLAG-NIK or pFLAG-NIK (KM) (1.5 μg), and the indicated RANK expression plasmids (1 μg) as described under “Experimental Procedures.” At 36 h post-transfection, the conditioned medium and cells were processed as described in A. The data are representative of at least three independent transfection experiments that produced similar results.View Large Image Figure ViewerDownload (PPT)A Kinase-inactive NIK Inhibits NF-κB-dependent SEAP Activity Induced by RANK and RANK326–427When transiently overexpressed in cultured cell lines, NIK, but not a kinase-inactive mutant (NIK-KM), activates NF-κB (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar, 15Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C. Malinin N.L. Wallach D. Yagita H. Okumura K. J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar) (Fig. 3B), while NIK-KM inhibits TNF-induced NF-κB dependent reporter activity (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar, 20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1158) Google Scholar) (data not shown). We thus examined the effect of NIK-KM on RANK616- and RANK326–427-induced NF-κB reporter activity. Co-transfection of NIK-KM with RANK616 or RANK326–427 inhibited NF-κB-dependent reporter activity (Fig. 3 B). Collectively, these data indicate that RANK activates NF-κB via residues 326–427, which interacts with TRAF6. Since TRAF6 has been demonstrated to interact with NIK (7Song H.Y. Regnier C.H. Kirschning C.J. Goeddel D.V. Rothe M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9792-9796Crossref PubMed Scopus (505) Google Scholar), RANK most likely utilizes the TRAF6-NIK pathway for activation of NF-κB.TRAF2-binding Domain of RANK Is Required for JNK ActivationTransient overexpression of RANK in 293 cells (16Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) or treatment of T cells with RANKL (3Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar) has been demonstrated to activate the JNK pathway. We therefore examined the ability of each RANK deletion mutant to activate co-transfected HA-JNK1. RANK616 activated JNK strongly, RANK427 and RANK530 activated JNK marginally, while RANK330 failed to activate JNK (Fig.4 A). Similar to our results in the NF-κB-dependent reporter assay, the C-terminal region of RANK (residues 529–616) failed to activate JNK. Moreover, truncation of the TRAF5-binding domain, residues 604–616 (RANKE603), had no effect on JNK activation, which suggests that RANK's interaction with TRAF5 is not required for JNK activation. The inability of these deletion mutants to activate JNK was not due to a lack of expression of transfected HA-JNK (Fig. 4 B). Furthermore, unlike NF-κB activation by RANK, truncation of the TRAF2-binding domain (i.e. RANK326–427 and RANK326–530) reduced JNK activation by 3-fold when compared with RANKE616 (Fig.4 A). These data suggest that, unlike the TRAF6-binding domain of RANK, which is required for NF-κB activation, the TRAF2-binding domain is required but not sufficient for activation of JNK.Figure 4The region in RANK that activates JNK overlaps the region in RANK necessary for induction of NF-κB. Panels A andB, immune complex kinase assays of HA-JNK1α induced by RANK. Human 293 cells transiently transfected with 5 μg of total plasmid DNA were transfected with pNF-κB-SEAP (0.5 μg), pSRα-HA-JNK1α (0.5 μg), and the indicated RANK expression plasmids (1.5 μg) as described under “Experimental Procedures.” At 36 h post-transfection, the conditioned medium and cells were collected and JNK kinase assays (A) and Western blotting with anti-HA (B) were performed as described under “Experimental Procedures.” The data are representative of at least three independent transfection experiments that produced similar results.View Large Image Figure ViewerDownload (PPT)Identification of a Novel TRAF6 Binding Motif in RANKOf all the members of the TNF receptor superfamily, only CD40 (11Ishida T. Mizushima S. Azuma S. Kobayshi N. Tojo T. Suzuki K. Aizawa S. Watanabe T. Mosialos G. Kieff E. Yamamoto T. Inoue J. J. Biol. 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