The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappa B,a member of the TNFR superfamily

Receptor activator of NF-kappa B (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activate d T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differen tiation. We demonstrate here the interaction of RANK with tumor necrosis fa ctor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro an d in cells. Mapping of the structural requirements for TRAF/RANK interactio n revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be fun ctionally important for the RANK-dependent induction of NF-kappa B and c-Ju n NH2-terminal kinase activities. Site-directed mutagenesis demonstrated th at these TRAF binding sites exhibited. selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determina nts distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-pr oximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappa B signa ling was completely inhibited while c-Jun NH2-terminal kinase activation wa s partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappa B activation, but failed to affect constitutive si gnaling induced by receptor overexpression,revealing a selective role for T RAF6 in ligand-induced activation events.