MEMBRANES AND BIOENERGETICS
Activation of NF-κB by RANK Requires Tumor Necrosis Factor Receptor-associated Factor (TRAF) 6 and NF-κB-inducing Kinase: IDENTIFICATION OF A NOVEL TRAF6 INTERACTION MOTIF*

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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.

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This work was supported by the Clayton Foundation for Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.