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J Biol Chem, Vol. 274, Issue 12, 7724-7731, March 19, 1999

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

Bryant G. DarnayDagger , Jian Ni§, Paul A. Moore§, and Bharat B. AggarwalDagger

From the Dagger  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

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 NIK, thus leading to NF-kappa 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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