The RING Domain and First Zinc Finger of TRAF6 Coordinate Signaling by Interleukin-1, Lipopolysaccharide, and RANKL

Abstract

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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“…Our observations are consistent with earlier reports describing the role of TRAF6 as a key ubiquitin ligase and TAK1 and NEMO as major ubiquitinated pilars that facilitate recruitment of signaling molecules (21,25). In support of this notion, recent evidence points to the paradigm that TRAF6-mediated ubiquitination is an important step for the formation and activation of a signaling complex that includes UBC13, TAK1, and its adaptors TAB1 and TAB2 (40,(45)(46)(47)(48). In a more recent study, Walsh et al, (42) showed that the RING finger of TRAF6 is required for the activation of TAK1, and TRAF6 was found to interact with TAK1.…”

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

“…MEFs derived from WT, Irgm1/m3 −/− , TRAF6 −/− , p62 −/− , and GBP chr3−/− mice were previously described (6,(59)(60)(61). Primary murine BMMs were isolated from the tibia and femurs of 2-4-mo-old mice as described before (10).…”

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

“…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.…”

“…Although truncated forms of TRAF6 lacking the really interesting new gene (RING) domain were reported to restore IL-1-signaling to TRAF6 knockout (KO) mouse embryonic fibroblasts (MEFs) many years ago (16), other laboratories reported subsequently that the E3 ligase-inactive TRAF6[C70A] mutant could not (12,17,18). These reports led to widespread acceptance of the notion that the E3 ligase activity of TRAF6 is essential for IL-1 signaling.…”