TRAF6 is one of the key adaptor molecules in the signal transduction of members of the TNF receptor superfamily and the IL-1 receptor/Toll-like receptor superfamily. To date, TRAF6 is the only member of the TRAF family that plays a critical role in osteoclastogenesis; its recruitment to the cytoplasmic domain of RANK initiates a signaling cascade that is crucial for the maturation of monocyte precursors to fully differentiated osteoclasts. However, the molecular events associated with TRAF6 in this process are not well understood. In this study, we clearly show that the RING domain of TRAF6 and the dimeric E2 enzyme Ubc13/Uev1A are critical for Lys-63-linked TRAF6 auto-ubiquitination and the ability of TRAF6 to activate IKK leading to the activation of NF-κB. In contrast to most studies of Ub-dependent TRAF6 signaling that only examined the activation of IKK, we further addressed this molecular mechanism on TRAF6 for its capacity to regulate osteoclast differentiation and maturation. Similar to previous reports (29
), we observed that retroviral infection of RAW cells or mouse BMM with TRAF6 caused RANKL-independent osteoclast differentiation. However, we further show that a RING domain mutant of TRAF6 could not induce expression of NFATc1 nor induce the differentiation of monocytes to mature functional osteoclasts. Collectively, our results demonstrate the biological relevance of the RING domain of TRAF6, and hence its auto-ubiquitination for its ability to induce NFATc1 and terminal differentiation of osteoclasts.
Without the knowledge of specific ubiquitination sites, the precise role of TRAF6 auto-ubiquitination in its ability to activate downstream signaling events cannot be rigorously addressed. Therefore, we took on the task of identifying the Ub acceptor Lys residues in TRAF6 that are responsible for its auto-ubiquitination. Because the RING domain of TRAF6 interacts with its E2 and the ubiquitination site(s) should be sufficiently close to the E2 for ubiquitination to occur, we mutated the seven Lys residues within and surrounding the RING domain of TRAF6. By using a molecular model of the TRAF6 RING structure, we predicted that all of the mutated lysine residues are solvent exposed and their mutation to Arg would not alter the RING structure. Consistent with our prediction, each of the TRAF6 Lys mutants retained their ability to synthesize free poly-Ub chains. Through mutagenesis, we demonstrated that K124 is the major Ub acceptor site in TRAF6, which is required for its auto-ubiquitination. Importantly, the transient expression of the K124R mutant results in both signaling and biological defects: decreased TRAF6 auto-ubiquitination, loss of NEMO ubiquitination, lack of TAK1 and IKK activation, deficiency in NFATc1 induction, and impairment in mature osteoclast formation. Significantly, IL-1-dependent TRAF6 ubiquitination and IKK activation was abolished in stable TRAF6-deficient MEFs reconstituted with either the RING domain or the K124R mutant of TRAF6. Thus, by identifying a specific Lys-63-linked ubiquitination site on TRAF6 and determining the importance of this site, we have established a link between site-specific TRAF6 auto-ubiquitination and its ability to activate the IKK complex.
The identification of a single Lys residue for TRAF6 auto-ubiquitination points to a model of poly-, rather than multi-ubiquitination in NF-κB signaling, a question raised in a recent review (31
), and reveals the potential specificity in this pathway of Lys-63-linked ubiquitination. In contrast, K48-linked ubiquitination is often not so site-specific. This is perhaps in tune with the different roles of K48- versus Lys-63-linked ubiquitination. The former is simply a “signal” for proteasomal destruction, which does not need to be site-specific. In contrast, Lys-63-linked ubiquitination may be important for assembly of signaling complexes, which might require the poly-Ub chains to be at a specific site of the target. Because Lys-63-linked ubiquitination uses the heterodimeric E2 enzyme Ubc13/Uev1A while K48-linked ubiquitination uses single component E2 enzymes, it is tempting to speculate that this difference may play a role in the apparent specificity.
Strikingly, the lack of TRAF6 auto-ubiquitination also resulted in the loss of NEMO ubiquitination, suggesting that TRAF6 may also be the E3 for NEMO. Up to now, the E3 for NEMO has been obscure. In the TNF pathway, cIAP1 was shown to be the E3 for NEMO ubiquitination (26
), while the E3 for NOD2- or Bcl10-induced NEMO ubiquitination has not been identified (25
). In this regard, because TRAF6 and TRAF2 appear to be the common elements in these pathways, it is possible that TRAFs are the unifying E3s for NEMO ubiquitination (32
). Indeed, we have clearly demonstrated that TRAF6 serves as an E3 for NEMO ubiquitination using recombinant proteins, which is further supported by the lack of NEMO ubiquitination in TRAF6-deficient MEFs reconstituted with either a TRAF6 RING or K124R mutant. The failure of NEMO ubiquitination with these two TRAF6 mutants, may reflect the loss of recruitment of NEMO to the TRAF6 complex, because neither of these TRAF6 mutants are ubiquitinated. Consistent with this hypothesis, recent data have emerged implicating proteins with binding-specificity towards Lys-63-linked poly-Ub chains. Two such molecules TAB2 (or TAB3) through its C-terminal zinc finger domain and NEMO through a novel Ub-binding domain have been shown to preferentially bind Lys-63-linked poly Ub chains (10
) (B. G. D., unpublished observations). These data may explain the necessity of Lys-63-linked auto-ubiquitination of TRAF6 to facilitate the recruitment and activation of the IKK complex. Nevertheless, the functional mechanism of ubiquitinated NEMO in assisting the activation of IKK remains to be investigated. One way to resolve these questions would be to identify the Ub acceptor sites on NEMO and assess the functional effects of eliminating them, which is currently under investigation.
In summary, the data presented here may provide molecular insight into the non-traditional role of Ub in TRAF6 signaling. In particular, we have shown that site-specific auto-ubiquitination of TRAF6 via the Lys-63 linkage is crucial for the activation of downstream kinases and transcription factors and further define a role of TRAF6 auto-ubiquitination in regulating osteoclast differentiation. Site-specific TRAF6 auto-ubiquitination appears to be a prerequisite that initiates a cascade of further downstream ubiquitination events, including the ubiquitination of components in the TAK1 and IKK complexes. Further studies are required to determine the specific role of each of the ubiquitination events in TRAF6-mediated IKK activation.