Bryant G. Darnay

Distinct molecular mechanism for initiating TRAF6 signalling.

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40° difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide–TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.

Characterization of the Intracellular Domain of Receptor Activator of NF-κB (RANK) INTERACTION WITH TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTORS AND ACTIVATION OF NF-κB AND c-JUN N-TERMINAL KINASE

Various members of the tumor necrosis factor (TNF) receptor superfamily interact directly with signaling molecules of the TNF receptor-associated factor (TRAF) family to activate nuclear factor κB (NF-κB) and the c-Jun N-terminal kinase (JNK) pathway. The receptor activator of NF-κB (RANK), a recently described TNF receptor family member, and its ligand, RANKL, promote survival of dendritic cells and differentiation of osteoclasts. RANK contains 383 amino acids in its intracellular domain (residues 234–616), which contain three putative TRAF-binding domains (termed I, II, and III). In this study, we set out to identify the region of RANK needed for interaction with TRAF molecules and for stimulation of NF-κB and JNK activity. We constructed epitope-tagged RANK (F-RANK616) and three C-terminal truncations, F-RANK330, F-RANK427, and F-RANK530, lacking 85, 188, and 285 amino acids, respectively. From this deletion analysis, we determined that TRAF2, TRAF5, and TRAF6 interact with RANK at its C-terminal 85-amino acid tail; the binding affinity appeared to be in the order of TRAF2 > TRAF5 > TRAF6. Furthermore, overexpression of RANK stimulated JNK and NF-κB activation. When the C-terminal tail, which is necessary for TRAF binding, was deleted, the truncated RANK receptor was still capable of stimulating JNK activity but not NF-κB, suggesting that interaction with TRAFs is necessary for NF-κB activation but not necessary for activation of the JNK pathway.

Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaB-inducing kinase. Identification of a novel TRAF6 interaction motif.

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor κB (NF-κB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-κB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-κB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-κB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565–568 and 606–611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340–358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-κB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-κB activation. However, RANK-mediated JNK activation required a distal portion (427–603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-κB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-κB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.

The E3 Ligase TRAF6 Regulates Akt Ubiquitination and Activation

Regulating Akt The protein kinase Akt is activated in response to receptor-activated generation of the signaling second messenger phosphatidylinositol 3,4,5-trisphosphate and has roles in regulation of diverse processes from metabolism and cell survival to transcription and tumorigenesis. Yang et al. (p. 1134; see the Perspective by Restuccia and Hemmings) report a previously unrecognized mode of regulation of Akt: covalent modification of Akt by linkage to lysine 63 of ubiquitin molecules. Such ubiquitination of Akt promotes localization to the cell membrane and consequent activation in cells stimulated with growth factors. TRAF6 (TNF receptor–associated factor 6) was implicated as the E3 ubiquitin ligase that mediates ubiquitination of Akt. Ubiquitination of Akt may influence its role in cancer cells: A mutant form of Akt associated with human cancer showed increased ubiquitination, and depletion of TRAF6 decreased tumorigenicity of a prostate cancer cell line in a mouse cancer model. Localization and activation of signaling proteins in cancer cells are controlled by ubiquitin labeling. Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. We found that the protein kinase Akt undergoes lysine-63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.

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

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-κB. 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-κB 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-κB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-κB-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-β-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.

Sanguinarine (Pseudochelerythrine) Is a Potent Inhibitor of NF-κB Activation, IκBα Phosphorylation, and Degradation

The nuclear factor NF-κB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-κB is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-κB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-κB, this activation was completely suppressed by sanguinarine in a dose- and time-dependent manner. Sanguinarine did not inhibit the binding of NF-κB protein to the DNA but rather inhibited the pathway leading to NF-κB activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-κB activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IκBα, an inhibitory subunit of NF-κB, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-κB activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-κB activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-κB activation and it acts at a step prior to IκBα phosphorylation.

E2 interaction and dimerization in the crystal structure of TRAF6

Tumor necrosis factor (TNF) receptor–associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-κB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.

Early events in TNF signaling: a story of associations and dissociations

At the cellular level, the multifunctional cytokine tumor necrosis factor (TNF) modulates growth and activates genes through various intermediates, including protein kinases, protein phosphatases, reactive oxygen intermediates, phospholipases, proteases, sphingomyelinases, and transcription factors. Unlike many cytokine receptors, however, the cytoplasmic domain (CD) of the TNF receptors lacks an intrinsic protein kinase activity and yet on interaction with ligand it phosphorylates various proteins. Although the kinetics of most of these activities differ, their interactions are coordinated through the selective interplay between the CD of the receptors and the associated proteins. A unique pathway has been identified by the ability of the TNF receptors to associate with a novel family of proteins. Two distinct families of proteins have emerged, the TNF receptor‐associated factors (TRAFs) and the death domain homologues. The cloning of members of these gene families and the identification of the protein‐interaction motifs found within their gene products has initiated the molecular identity of factors (TRADD, FADD/MORT, RIP, FLICE/ MACH, and TRAFs) associated with both of the p60 and p80 forms of the TNF receptor and with other members of the TNF receptor superfamily. In this review, we summarize these and other TNF receptor‐associated proteins and their potential roles in regulating the activation of nuclear factor‐κB and apoptosis, two major responses activated by engagement of TNF receptors by the ligand. J. Leukoc. Biol. 61: 559–566; 1997.

Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice

The transcription factor Osterix (Osx) is required for osteoblast differentiation and bone formation during embryonic development, but it is not known whether Osx has an essential function in postnatal bone growth and in bone homeostasis. Conditional deletion of Osx at several time points postnatally revealed that Osx was essential for osteoblast differentiation and new bone formation in growing and adult bones. Additionally, inactivation of Osx in bones severely disrupted the maturation, morphology, and function of osteocytes. These findings identify Osx as having an essential role in the cell-specific genetic program of osteocytes. Interestingly, Osx inactivation also led to the massive accumulation of unresorbed calcified cartilage in a large area below the growth plate of endochondral bones. This specific area was also marked by an unanticipated almost complete lack of bone marrow cells and a marked decrease in the density and size of osteoclasts. This diminished density of osteoclasts could contribute to the lack of resorption of mineralized cartilage. In addition, we speculate that the abnormally accumulated, mainly naked cartilage represents an unfavorable substrate for osteoclasts. Our study identifies Osx as an essential multifunctional player in postnatal bone growth and homeostasis.

A20 Negatively Regulates T Cell Receptor Signaling to NF-κB by Cleaving Malt1 Ubiquitin Chains

The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IκB kinase (IKK)/NF-κB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-κB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1−/− T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-κB signaling in CD4+ T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-κB signaling.