FEBS 21470 FEBS Letters 443 (1999) 297^302 Receptor activator of NF-UB recruits multiple TRAF family adaptors and activates c-Jun N-terminal kinase Hong-Hee Kima, Da Eun Leea, Jin Na Shina, Yong Soo Leea, Yoo Mi Jeona, Chae-Heon Chunga, Jian Nib, Byoung Se Kwonc, Zang Hee Leea;* aDepartment of Microbiology and Immunology, Chosun University Dental School, 375 Seosuk-Dong, Dong-Ku, Kwangju 501-759, South Korea bHuman Genome Sciences, Inc., Rockville, MD 20850, USA cDepartment of Life Sciences, Ulsan University, Ulsan, South Korea Received 7 December 1998 trabecular bone and lower in spleen and bone marrow [3], Abstract Receptor activator of NF-UB (RANK) is a recently cloned member of the tumor necrosis factor receptor (TNFR) which suggests physiological functions of RANK in the regu- superfamily, and its function has been implicated in osteoclast lation of bone and immune systems. Consistent with this differentiation and dendritic cell survival. Many of the TNFR suggestion, treatment with RANKL/TRANCE was shown family receptors recruit various members of the TNF receptor- to enhance the function and survival of T cells and dendritic associated factor (TRAF) family for transduction of their signals cells [1,5]. In addition, RANKL/ODF has been demonstrated to NF-UB and c-Jun N-terminal kinase. In this study, the to cause the di¡erentiation of hematopoietic precursor cells involvement of TRAF family members and the activation of the into osteoclasts and to stimulate the bone resorbing activity JNK pathway in signal transduction by RANK were investi- of mature osteoclasts [4]. gated. TRAF1, 2, 3, 5, and 6 were found to bind RANK in vitro. The TNF receptor-associated factor (TRAF) family mole- Association of RANK with each of these TRAF proteins was also cules are cytoplasmic adaptor proteins known to mediate sig- detected in vivo. Expression of RANK in cultured cells also induced the activation of JNK, which was blocked by a naling events speci¢cally for members of the TNFR family, dominant-negative form of JNK. Furthermore, by employing which includes TNFR1, TNFR2, CD30, CD40, CD27, Fas, various C-terminal deletion mutants of RANK, the regions 4-1BB, Ox40, TR2, and LMP-1 [6,7]. To date six members of responsible for TRAF interaction and JNK activation were the TRAF family have been identi¢ed. The TRAF2 protein is identified. TRAF5 was determined to bind to the C-terminal 11 recruited to TNFR1 through a death domain-containing mol- amino acids and the other TRAF members to a region N- ecule TRADD, whereas all TRAF proteins except TRAF4, terminal to the TRAF5 binding site. The domain responsible for which is localized to the nucleus, have been shown to directly JNK activation was localized to the same region where TRAF1, bind to several TNFR family receptors [8^17]. TRAF family 2, 3, and 6 bound, which suggests that these TRAF molecules proteins have a similar structural array consisting of an N- might mediate the RANK-induced JNK activation. terminal zinc RING ¢nger, multiple zinc ¢ngers, and C-ter- z 1999 Federation of European Biochemical Societies. minal TRAF-N and TRAF-C domains. The TRAF1 molecule Key words: Receptor activator of neclear factor UB; is unique in that it lacks the RING ¢nger domain [9]. The N- Tumor necrosis factor receptor-associated factor; terminal zinc binding regions of TRAFs serve as e¡ector do- c-Jun N-terminal kinase; Signal transduction mains, whereas the TRAF domain appears to be involved in binding to the cytoplasmic tail of TNFR proteins [12,18,19]. Characterization of binding sites for TRAFs in TNFR family proteins has led to the identi¢cation of two types of sequence 1. Introduction motif: PXQXT/S [20,21] and EED/EEE [15,20,22]. The PXQXT/S motif seems to be capable of binding to TRAFs Receptor activator of NF-UB (RANK) is a recently cloned 1, 2, 3, and 5 and the EED/EEE motif binds TRAFs 1, 2, and member of the tumor necrosis factor receptor (TNFR) family 3. Characteristic cellular responses to the stimulation of [1]. The RANK protein consists of 616 amino acids contain- TNFR family receptors include the activation of the NF-UB ing the cysteine residues conserved in the extracellular domain transcription factor and c-Jun N-terminal kinase (JNK) of the of the TNFR family proteins. Its relatively long cytoplasmic mitogen-activated protein kinase family. The N-terminal zinc domain (383 amino acids) has little homology with other binding motifs of TRAF2, TRAF5, and TRAF6 have been TNFR family members. The ligand for RANK, RANKL shown to be responsible for the NF-UB activation induced by [1], was found to be identical to TRANCE (TNF-related ac- TNFR family proteins [13^15,18,23^25]. Overexpression of tivation-induced cytokine), ODF (osteoclast di¡erentiation TRAF2, 5, or 6 was reported to activate JNK [26], and factor), and OPGL (osteoprotegerin ligand) [2^4]. The expres- TRAF2 has been implicated in the TNFK-induced activation sion level of RANK mRNA is high in thymus, lung, and of JNK [27]. The role of TRAF2 in the TNFR-induced JNK activation was further supported by a study with transgenic mice expressing a dominant-negative TRAF2 [28]. *Corresponding author. Fax: (82) (62) 227-6589. E-mail: [email protected] The activation of NF-UB appears to be involved in RANK signaling as other TNFR family proteins. Ectopic overexpres- Abbreviations: RANK, receptor activator of NF-UB; TNFR, tumor sion of RANK in a cultured cell line and ligation of endog- necrosis factor receptor; TRAF, tumor necrosis factor receptor- enous RANK in T cells pretreated with phytohemagglutinin associated factor; JNK, c-Jun N-terminal kinase; NF-UB, nuclear factor UB; GST, glutathione S-transferase; ICD, intracellular domain; and interleukin (IL)-4 resulted in NF-UB activation [1]. In- SEK, SAPK (JNK)/ERK kinase volvement of JNK in RANK signaling pathways might also 0014-5793/99/$19.00 ß 1999 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(98)01731-1 FEBS 21470 1-2-99 298 H.-H. Kim et al./FEBS Letters 443 (1999) 297^302 be suggested as RANKL/TRANCE stimulation was shown to bu¡er and twice in the kinase reaction bu¡er, and kinase reaction cause a modest increase in the JNK activity in T cells [2]. As performed as previously described [31] with the omission of [Q-32P]ATP. Samples were resolved by SDS-PAGE and Western TRAF2, 5, and 6 were implicated in the activation of NF-UB blotted with an antibody speci¢c for phosphorylated c-Jun (New Eng- and JNK for some TNFR family members, it may be antici- land Biolabs, Cat. #9161S). Alternatively, the activity of endogenous pated that some TRAF molecules mediate the RANK-in- JNK was measured by the solid-phase kinase assay [32] using 3 Wgof duced responses. In support of this, the amino acid sequence GST-c-Jun-77. analysis of the cytoplasmic domain of human RANK reveals several potential TRAF binding sites: four PXQXT/S-like 3. Results and discussion motifs (302PDQGG, 353PSQPT, 569PVQEE, and 607PVQEQ) and three acidic residue triplets (336EED, 346EDE, and 3.1. Interaction of RANK with TRAF family proteins in vitro 481EEE). To gain insight into the mechanism by which Some TNFR family receptors recruit TRAF2, 5, and 6 to RANK transmits its signal to the nucleus, the interaction activate NF-UB [23^25], while this activation appears to be between RANK and TRAFs and the e¡ect of RANK signal- antagonized by TRAF3 binding [17,23,25]. To determine ing on JNK activation were characterized in this study. whether RANK can also utilize TRAF family proteins in signal transduction, the potential interaction between 2. Materials and methods RANK and TRAFs was examined by in vitro binding experi- ments, employing the GST fusion protein of the cytoplasmic 2.1. Cell culture and transfection domain of human RANK. When the GST-RANK protein HeLa cells were cultured in DMEM containing 10% FCS and 293- was incubated with lysates from HeLa or 293-EBNA cells EBNA cells (Invitrogen) were maintained in DMEM containing 10% FCS and 250 Wg/ml G418. For transient transfection, 2U105 cells per expressing Flag-tagged TRAF molecules (Fig. 1A) and the well were plated onto 6-well plates. The next day, transfection was precipitates were subjected to Western blotting with anti- carried out with a mixture of 1^2 Wg DNA and 10 Wl SuperFect Flag, TRAF1, 2, 3, 5, and 6 were found to bind RANK reagent (Qiagen) following the manufacturer's instructions. 40^48 h (Fig. 1B). The binding of TRAF to GST-RANK was speci¢c after transfection, cells were harvested. For co-immunoprecipitation as the GST control and the GST fusion of protein X, which experiments, 1.5U106 cells plated in T-25 £asks were transfected with 4^7 Wg DNA and 20 Wl SuperFect. does not possess any homology to RANK, showed little bind- ing, even though comparable amounts of the GST proteins 2.2. Plasmid constructions were used (data not shown). Although the possibility of the The cDNA encoding the full-length human RANK was obtained involvement of a third cellular protein mediating the interac- from Human Genome Sciences (Rockville, MD). The T7-tagged mammalian expression vector of hRANK, pSRa-RANK-T7, was con- tions between RANK and TRAFs cannot be excluded, given structed by PCR. The sequence of PCR product was veri¢ed by au- tomated DNA sequencing (Perkin Elmer, ABI 310). RANK C-termi- nal deletion mutants, pSRa-RANK-605 and -432, were generated using XhoI (amino acid 605 position) and MscI (amino acid 432 and 513 positions) sites, respectively.