TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation

Lamothe, Betty; Webster, William K.; Gopinathan, Ambily; Besse, Arnaud; Campos, Alejandro D.; Darnay, Bryant G.

doi:10.1016/j.bbrc.2007.06.017

Cited by 114 publications

(117 citation statements)

References 20 publications

(37 reference statements)

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“…Other laboratories have reported that RANKL signaling cannot be restored to TRAF6 KO monocytes by the reexpression of TRAF6[C70A] (17,39). This could be related to the problem discussed in the preceding section in refolding this E3 ligase-inactive mutant in MEFs to a conformation that can support signaling.…”

Section: Il-1 Signaling In Mefs Frommentioning

confidence: 99%

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Strickson

Emmerich

Goh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

100

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It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligaseinactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligaseinactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKLinduced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.T NF receptor-associated factor 6 (TRAF6) is essential for many biological processes (1). These include the myeloid differentiation primary response gene 88 (MyD88) signaling network of the innate immune system, RANK ligand (RANKL)-dependent signaling and osteoclast formation, lymph node organogenesis (2), and the development of hair follicles, sweat glands, and sebaceous glands (3). TRAF6 expression is also needed for CD40 signaling in B cells (4), the maturation and development of dendritic cells (5), and the regulation of T-cell function (6, 7).In innate immunity, nearly all Toll-like receptors (TLRs), as well as the receptors of the interleukin 1 (IL-1) family of cytokines, initiate signaling by recruiting the adaptor protein MyD88. This is followed by the interaction of IL-1-receptor (IL-R)-associated kinase 4 (IRAK4) with MyD88 and then the interaction of other IRAK family members with IRAK4, to form an oligomeric complex, termed the Myddosome (8, 9). IRAK1 and IRAK2 can then interact with TRAF6 (10, 11) and induce TRAF6 dimerization (12), which triggers the activation of its E3 ligase activity (13).TRAF6 catalyzes the formation of Lys63-linked ubiquitin (K63-Ub) chains in vitro in the presence of Ubc13-Uev1a (also called UBE2N-UBE2V1), an E2 conjugating enzyme that directs the formation of this type of ubiquitin linkage (14, 15). Although truncated forms of TRAF6 lacking the really...

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Section: Il-1 Signaling In Mefs Frommentioning

confidence: 99%

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Strickson

Emmerich

Goh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

100

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“…Mutational analysis performed in authentic OC precursors using a chimeric receptor strategy showed that although a proximal TRAF6-binding site mediated NF-κB activation at the level of IκBα phosphorylation, two more distal sites, which also bind to TRAF2 and 5, could activate classical NF-κB as well [7]. Autoubiquitination of TRAF6 and interaction between aPKC and p62 are required for sustained NF-κB activation and OC differentiation [8,9]. CYLD is a negative regulator of TRAF6 ubiquitination and NF-κB activation, and its deletion enhances RANKL-mediated osteoclastogenesis [10].…”

Section: Rankl-induced Nf-κbmentioning

confidence: 99%

Role of NF-κB in the skeleton

2010

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Since the discovery that deletion of the NF-κB subunits p50 and p52 causes osteopetrosis in mice, there has been considerable interest in the role of NF-κB signaling in bone. NF-κB controls the differentiation or activity of the major skeletal cell types -osteoclasts, osteoblasts, osteocytes and chondrocytes. However, with five NF-κB subunits and two distinct activation pathways, not all NF-κB signals lead to the same physiologic responses. In this review, we will describe the roles of various NF-κB proteins in basal bone homeostasis and disease states, and explore how NF-κB inhibition might be utilized therapeutically.

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“…Protein expression and purification from Escherichia coli were performed as described previously for His-tagged proteins (17) or for glutathione S-transferase (GST)-tagged proteins (34).…”

Section: Cell Lines and Antibodiesmentioning

confidence: 99%

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1

Ning¹,

Campos

Darnay³

et al. 2008

Molecular and Cellular Biology

Self Cite

100

136

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We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6 ؊/؊ mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IB kinase . In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor. Intracellular signaling initiated by latent membrane protein 1 (LMP1), the principal oncoprotein of the human gammaherpesvirus Epstein-Barr virus (EBV), is of interest not only for viral oncogenesis but also for the reason that LMP1 shares early steps in pathways used by CD40, interleukin-1 (IL-1) receptor-Toll-like receptor (TLR), and tumor necrosis factor receptor (TNFR) for activation of NFB (reviewed in references 16 and 35). As a member of the TNFR superfamily, LMP1 recruits TNFR-associated death domain protein, TNFRinteracting protein (RIP), and several TNFR-associated factors (TRAFs). Unlike CD40 and receptor activator of NF-B (RANK), which contain TRAF6-binding sites, LMP1 does not have a consensus TRAF6-binding sequence but associates with TRAF6 indirectly (reviewed in references 8, 16, 35, 51, and 61).Ubiquitination through K48-polyubiquitin linkage is well known as a process whereby proteins are targeted for proteasomal degradation. Recently, proteasome-independent functions for ubiquitination through K63 polyubiquitin or monoubiquitin linkages have been identified, and the importance of these ubiquitin...

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TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation

Cited by 114 publications

References 20 publications

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Role of NF-κB in the skeleton

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1

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