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

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappa B (NF-kappa B) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappa B-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappa B (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappa B 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-kappa B activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappa B 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-kappa B via a novel TRAF6 interaction motif, which then activates MK, thus leading to NF-kappa B activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.