Site-specific Lys-63-linked Tumor Necrosis Factor Receptor-associated Factor 6 Auto-ubiquitination Is a Critical Determinant of IκB Kinase Activation

Abstract

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...

TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme

“…In the cytoplasm, TRAF6 interacts with the E2 ubiquitin-conjugating enzyme complex Ubc13/ Uev1A, thus forming complex III. Ubc13/Uev1A cooperates with the RING finger domain of TRAF6, which acts as an E3-ubiquitin ligase, and results in K63-linked poly-ubiquitination of TRAF6, which is necessary for IKK and c-jun N-terminal kinase (JNK) activation [22,38,39]. The RING finger domain of TRAF6 is necessary for Ubc13 interaction and selfassociation [40].…”

“…In addition, overexpression of TIFA induces NF-kB and JNK activation in HEK293 cells [52]. Lysine 124 in TRAF6 was identified as the main ubiquitin acceptor site for auto-ubiquitination, and mutation of this lysine leads to impaired TAK1, IKK and JNK activation [22]. Moreover, complementation of TRAF6-deficient cells with this mutant or a RING finger mutant does not restore TRAF6 auto-ubiquitination, IKKg polyubiquitination, and subsequent IKK activation upon IL-1 stimulation [22], demonstrating an essential role for TRAF6 E3 ligase activity in signaling.…”

“…Lysine 124 in TRAF6 was identified as the main ubiquitin acceptor site for auto-ubiquitination, and mutation of this lysine leads to impaired TAK1, IKK and JNK activation [22]. Moreover, complementation of TRAF6-deficient cells with this mutant or a RING finger mutant does not restore TRAF6 auto-ubiquitination, IKKg polyubiquitination, and subsequent IKK activation upon IL-1 stimulation [22], demonstrating an essential role for TRAF6 E3 ligase activity in signaling. To conclude, oligomerization of TRAF6 might lead to auto-polyubiquitination of TRAF6, which is necessary for IL-1-and LPS-induced NF-kB activation, whereas TRAF6-induced poly-ubiquitination of NEMO might rather play a role in IL-1-induced JNK activation.…”

Identification of a consensus site for TRAF6/p62 polyubiquitination

“…There are only a few other reports on TRAF6-mediated polyubiquitination that include TRAF6 auto-ubiquitination [13], NEMO [13], TAB2 and TAB3 [14]. High substrate specificity of the E3-ubiquitin ligase ensures correct transmission of signals.…”

The role of K63‐linked polyubiquitination in cardiac hypertrophy

“…It is well defined that the expression of TRAF6 and the activity of TAK1‐JNK1/2/p38 cascade are increased in failing human heart. Thus, NADPH oxidase‐dependent accumulation of ROS triggers TRAF6 auto‐ubiquitination and subsequent K63‐linked ubiquitination and phosphorylation of TAK1 triggering downstream cascade of pathological hypertrophy 65, 66 (Figure 2). Together, these reports indicate that ubiquitination machineries have diverse role in cellular function and disease development, in particular, in cardiovascular system.…”