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J Biol Chem, Vol. 274, Issue 12, 7724-7731, March 19, 1999
From the Various members of the tumor necrosis factor
(TNF) receptor superfamily activate nuclear factor
Activation of NF-
B by RANK Requires Tumor Necrosis Factor
Receptor-associated Factor (TRAF) 6 and NF-
B-inducing Kinase
IDENTIFICATION OF A NOVEL TRAF6 INTERACTION MOTIF
,
Cytokine Research Laboratory,
Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and § Human
Genome Sciences, Inc., Rockville, Maryland 20850
B (NF-
B) and
the c-Jun N-terminal kinase (JNK) pathways through their interaction
with TNF receptor-associated factors (TRAFs) and NF-
B-inducing
kinase (NIK). We have previously shown that the cytoplasmic domain of
receptor activator of NF-
B (RANK) interacts with TRAF2, TRAF5, and
TRAF6 and that its overexpression activates NF-
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-
B activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-
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-
B via a novel TRAF6 interaction motif, which then
activates NIK, thus leading to NF-
B activation, whereas RANK most
likely activates JNK through a TRAF2-interacting region in RANK.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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