The Involvement of Multiple Tumor Necrosis Factor Receptor (TNFR)-associated Factors in the Signaling Mechanisms of Receptor Activator of NF-κB, a Member of the TNFR Superfamily

Laurent Galibert,Mark E Tometsko,Dirk M Anderson,David Cosman,William C Dougall

doi:10.1074/jbc.273.51.34120

Abstract

Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events.

Highlights

RANK1 and RANK ligand (RANKL) are a recently described cognate pair of the TNF receptor/ligand superfamilies [1]
Interaction of the RANK Cytoplasmic Domain with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 —In order to define whether TRAFs may bind to RANK, each of the known TRAFs was transcribed and translated in vitro in the presence of [35S]methionine/cysteine and coprecipitation assays were performed to determine the interaction with a GST fusion expressing the full-length RANK cytoplasmic domain
In contrast to the full-length RANK cytoplasmic domain, a deletion construct that lacks the COOH-terminal 72 amino acids (RANK ⌬544) was unable to interact with TRAFs 1, 2, 3, and 5 (Fig. 1), and TRAF6 binding was reduced by approximately 50%

Summary

Introduction

RANK1 and RANK ligand (RANKL) are a recently described cognate pair of the TNF receptor/ligand superfamilies [1]. The same ligand has been identified by screening a T cell hybridoma cell line (termed TRANCE) [2] and as an osteoclast differentiation factor [3] whose activity can be inhibited by a soluble TNF receptor family member, osteoprotegerin [4]. Substantial progress has been made to define the cytoplasmic proteins that function as adaptors (TRAFs 1– 6, TRADD, FADD) or as serine/ threonine kinases (RIP) and link TNF receptor stimulation with the induction of cell death, Jun kinase (JNK) or NF-␬B activation pathways (reviewed in Ref. 6). The amino acid sequences of the human and mouse RANK cytoplasmic domains include multiple sections that show striking homology (64% amino acid identity and 78% similarity) between species [1], suggesting a conserved functional role for these structures. TRAF6 appears to mediate RANK function through unique determinants independent of other TRAF proteins and plays a selective role in RANKL-induced signaling

Methods

Results

Conclusion