Abstract
Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappaB 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-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappaB 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-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.
Highlights
Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor 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 previously reported that TRAF2, TRAF5, and TRAF6 interact with RANK [16] and that overexpression of RANK in 293 cells activates the NF-B and JNK pathways [1, 16]
To identify which region of the cytoplasmic domain of RANK is necessary for binding TRAF2, TRAF5, and TRAF6, we constructed a series of deletion mutants of the cytoplasmic domain of RANK encompassing the various putative TRAF-binding domains (Fig. 1A)
Summary
Cell Lines, and Antibodies—Human embryonic kidney 293 cell line were obtained from the American Type Culture Collection (Rockville, MD) and cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. Expression plasmids encoding Myc-tagged TRAF2 and FLAGtagged C-terminal truncated versions of RANK (RANK616, -530, -427, and -330) were prepared as described previously [16]. To generate deletion mutants of the cytoplasmic domain of RANK as glutathione S-transferase (GST) fusion proteins, specific 5Ј and 3Ј primers containing EcoRI and SalI sites, respectively, were used in PCR reactions with pSPORT3.0-TR8 [16]. Due to the subcloning at the EcoRI site, all FLAG-tagged RANK deletion mutants contained homologous amino acid substitutions (i.e. Ala241-Leu242 to Gly241-Ile242) These substitutions, did not effect the ability of RANK to activate signaling cascades (see “Results”). For Western blot analysis whole cell lysates (15–30 g) or proteins from GST affinity precipitations were separated by 8.5% SDS-PAGE, electroblotted onto nitrocellulose membranes (Bio-Rad), and incubated with the indicated antibodies. The NF-B SEAP reporter used in these assays was shown to be activated by TNF and by overexpression of TNFR2.2 Similar to the case in previously published reports [7, 9, 20], the specificity was established of this reporter system by the fact that TNF-induced NF-B SEAP activity was inhibited by overexpression of either an IB␣ mutant lacking Ser32/36, a kinaseinactive NIK, or a dominant negative TRAF2 mutant.
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