Activation of NF-kappa B by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappa B-inducing kinase - Identification of a novel TRAF6 interaction motif

Various members of the tumor necrosis factor (TNF) receptor superfamily act ivate nuclear factor kappa B (NF-kappa B) and the c-Jun N-terminal kinase ( JNK) pathways through their interaction with TNF receptor-associated factor s (TRAFs) and NF-kappa B-inducing kinase (NIK). We have previously shown th at the cytoplasmic domain of receptor activator of NF-kappa B (RANK) intera cts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-k appa B and JNK pathways. Through a detailed mutational analysis of the cyto plasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consens us TRAF binding motifs located in the C terminus at positions 565-568 and 6 06-611, respectively. In contrast, TRAF6 interacts with a novel motif locat ed between residues 340 and 358 of RANK. Furthermore, transfection experime nts with RANK and its deletion mutants in human embryonic 293 cells reveale d that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-bindi ng region, is necessary and sufficient for RANK-induced NF-kappa B activati on. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kap pa B activation. However, RANK-mediated JNK activation required a distal po rtion (427-603) of RANK containing the TRAF2-binding domain. Thus, our resu lts indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappa B via a novel TRAF6 interaction motif, which then a ctivates MK, thus leading to NF-kappa B activation, whereas RANK most likel y activates JNK through a TRAF2-interacting region in RANK.