Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-B. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-B pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-B. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-B-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-␤-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.The pro-inflammatory cytokines interleukin-1 (IL-1) 2 and tumor necrosis factor (TNF) elicit a critical function in the innate immune response. Following receptor activation, these cytokines induce a cascade of signaling events leading to the activation of transcription factors such as NF-B and AP1 through upstream kinases, including IB kinase (IKK) and the mitogen-activated protein kinases (MAPKs; JNK, p38, and ERK). These events culminate in the expression and regulation of numerous pro-inflammatory genes (1, 2).TNF receptor-associated factors (TRAFs) constitute a family of seven known adaptor proteins, and most of them participate in activation of the transcription factor NF-B and members of the MAPKs (3-5). Several TRAF proteins interact directly with the intracellular regions of various members of the TNF receptor family through a highly conserved motif at their C terminus termed the TRAF domain. In contrast, the N-terminal domain of the TRAFs is less well conserved but consists of zinc finger motifs and in some TRAFs a RING (really interesting new gene) domain, which has been identified in a number of E3 ubiquitin ligases (6).Recently, the N-terminal RING domain of TRAF6 has been included in a growing fami...

Transforming growth factor ␤-activated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the transforming growth factor ␤-signaling pathway, but recent evidence has emerged implicating TAK1 in the interleukin (IL)-1 and tumor necrosis factor (TNF) pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB3 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479 -553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574 -693 of TAB2 were shown to interact with TAK1. A green fluorescent protein fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1, and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), the crucial adaptor molecule of receptor activator of NF-κB (RANK), plays an essential role in governing the formation of multi-nucleated osteoclasts. TRAF6 is a RING-dependent ubiquitin (Ub) ligase that in conjunction with Ubc13/ Uev1A catalyzes its own auto-ubiquitination via Lys63-linked poly-Ub chains. While the receptoradaptor function of TRAF6 in RANK signaling is well understood, the significance of its Ub ligase activity in this process remains largely unknown. In this study, we show that retroviral expression of TRAF6, but not a RING mutant of TRAF6 was able to rescue TRAF6-deficient monocytes for the activation of IKK and osteoclast differentiation by RANKL. Furthermore, a catalytically inactive Ubc13 or stable knockdown of Ubc13 significantly prevents RANK-mediated TRAF6 ubiquitination and NF-κB and JNK activation. These data establish a signaling cascade in which regulated Lys63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of osteoclast differentiation.

TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.TRAF6, a member of the tumor necrosis factor receptorassociated factor (TRAF) 2 family, is a crucial docking molecule that mediates signaling events initiated by cytokines of the tumor necrosis factor superfamily, interleukin-1 (IL-1) family, and pathogen-associated microbial patterns that are recognized by the Toll-like receptor family (1, 2). Following binding to their respective receptors, these ligands induce a cascade of signaling events leading to the activation of transcription factors, such as the nuclear factor-B (NF-B) and AP1 (activator protein-1) family, through activation of upstream kinases, including inhibitor of -B kinase (IKK) and the mitogen-activated protein kinase (MAPK) family (i.e. p38, JNK, and extracellular signal-regulated kinase) (1, 2). As a result, these ligands induce numerous genes involved in the innate immune and inflammatory responses. Generation of TRAF6-deficient mice revealed that in addition to playing a critical role in the innate and adaptive immunity, TRAF6 has a crucial role in a wide range of biological functions, such as lymph node organogenesis, formation of skin appendices, nervous system development, and bone metabolism (3-9). TRAF6 contains three major domains: 1) the C-terminal domain, which facilitates oligomerization and interaction with receptors and other adaptor proteins in a sequence-specific manner; 2) t...

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