Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro

Receptor interacting protein (RIP) is recruited to tumor necrosis factor-alpha receptor 1 (TNFR1) complex upon stimulation and plays a crucial role in the receptor-mediated NF-kappaB activation. Among the components of the TNFR1 complex are proteins that possess ubiquitin-protein isopeptide ligase (E3) activities, such as TNFR1-associated factor 2 (TRAF2), cellular inhibitor of apoptosis proteins (c-IAPs) namely, c-IAP1 and c-IAP2. Here, we showed that ectopically expressed RIP is ubiquitinated, and either the intermediate or death domain of RIP is required for this modification. Expression of c-IAP1 and c-IAP2 decreased the steady-state level of RIP, which was blocked by inhibition of the 26S proteasome. RIP degradation requires intact c-IAP2 containing the RING domain. Our in vitro ubiquitination assay revealed that while TRAF2 had no effect, both c-IAP1 and c-IAP2-mediated RIP ubiquitination with similar efficiency, indicating that c-IAPs can function as E3 toward RIP.     

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.     

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.     

Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2)

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.     

Mass spectrometric analysis of tumor necrosis factor receptor-associated factor 1 ubiquitination mediated by cellular inhibitor of apoptosis 2

Signaling complexes formed on tumor necrosis factor receptor 2 (TNF-R2) contain adaptor proteins TNF-R-associated factors (TRAFs) 1 and 2, and cellular inhibitors of apoptosis (cIAPs) 1 and 2 which function as regulators of programmed cell death. TRAF2, cIAP1 and cIAP2 all have RING finger domains known to possess E3 ubiquitin ligase activity, implying that ubiquitination may play an important role in the TNF signaling pathway. In this report, we have shown that cIAP2 specifically mediated ubiquitination and proteasome-dependent degradation of TRAF1. To identify the sites for cIAP2-mediated ubiquitination of TRAF1, we used high pressure liquid chromatography coupled with tandem mass spectrometry. Lys185 and Lys193 of TRAF1 were found to be modified with ubiquitin chains. Mutation of Lys185 and Lys193 to Arg almost completely blocked cIAP2-mediated ubiquitination of TRAF1, indicating that they are the major, if not the only, sites of TRAF1 ubiquitination. Our data suggest that cIAP2 may regulate the turnover of TRAF1 by adding polyubiquitin chains on Lys185 or Lys193 following its recruitment to TNF-R signaling complexes.     

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1)

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.     

Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys⁶³-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.     

Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling

Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.     

Enhanced Cytoprotective Effects of the Inhibitor of Apoptosis Protein Cellular IAP1 through Stabilization with TRAF2

Inhibitor of apoptosis (IAP) proteins are key regulators of intracellular signaling that interact with tumor necrosis factor (TNF) receptor superfamily members as well as proapoptotic molecules such as Smac/DIABLO and caspases. Whereas the X-linked IAP is an established caspase inhibitor, the protective mechanisms utilized by the cellular IAP (c-IAP) proteins are less clear because c-IAPs bind to but do not inhibit the enzymatic activities of caspases. In this study, c-IAPs are shown to be highly unstable molecules that undergo autoubiquitination. The autoubiquitination of c-IAP1 is blocked upon coexpression with TNF receptor-associated factor (TRAF) 2, and this is achieved by inhibition of the E3 ubiquitin ligase activity intrinsic to the RING of c-IAP1. Consistent with these observations, loss of TRAF2 results in a decrease in c-IAP1 levels. Stabilized c-IAP1 was found to sequester and prevent Smac/DIABLO from antagonizing X-linked IAP and protect against cell death. Therefore, this study describes an intriguing cytoprotective mechanism utilized by c-IAP1 and provides critical insight into how IAP proteins function to alter the apoptotic threshold.The inhibitors of apoptosis (IAPs)² are an evolutionarily conserved gene family described originally as encoding cell death inhibitors. IAP proteins have subsequently been found to participate in a variety of additional intracellular signaling processes (1), and it has become evident that IAP proteins are versatile molecules playing numerous distinct roles within the cell. Although a more complete understanding of these additional functions for IAP proteins is emerging, the distinct mechanisms utilized by some IAP proteins to function in their originally defined roles as cell death inhibitors remain unclear.Members of the IAP family are characterized by the presence of 1–3 tandem repeats of an ∼70-residue baculovirus IAP repeat domain (2). The baculovirus IAP repeat domains of many IAP proteins have been shown to be the region within IAP proteins that associates with caspases and other proapoptotic molecules (3, 4). IAP proteins have remarkably different apoptotic inhibitory abilities. For example, X-linked IAP (XIAP) is a highly potent cell death inhibitor (5) and is thought to be the only mammalian IAP protein that directly inhibits the enzymatic activities of caspases (2–4, 6). Although cellular IAP1 and -2 (c-IAP1 and c-IAP2) are anti-apoptotic proteins that can bind to caspase-7 and -9, they do not inhibit the enzymatic activities of these caspases (2, 6).Many IAP proteins, including c-IAP1 and c-IAP2, contain a carboxyl-terminal RING domain that can function as an E3 ubiquitin ligase (7). The E3 ubiquitin ligase activity of the RING domain in c-IAP1 and c-IAP2 was previously shown to negatively regulate the apoptotic inhibitory properties of c-IAP proteins and to promote autoubiquitination and degradation of c-IAP1 (8, 9), thus hindering attempts to define the cellular properties of this protein.A specialized property of the c-IAP proteins is their involvement in tumor necrosis family (TNF) signaling (10–12). Both c-IAP1 and c-IAP2 were discovered in a biochemical screen for factors associated with the type 2 TNF receptor. This association was found to be indirect and bridged by interactions with TNF receptor-associated factors (TRAFs), most notably TRAF1 and TRAF2 (11). Though the consequences of the association between TRAF2 and c-IAP1 on TNF-mediated signaling have been investigated (12), less is known about the functional significance of the association between TRAF2 and c-IAP1 on cell death inhibition. Because both c-IAP1 and TRAF2 possess E3 ubiquitin ligase activity in their respective RING domains, it seemed that the association between these molecules might impact the protective properties of c-IAP1 and alter the apoptotic threshold.In this study, the role of TRAF2 in c-IAP1 stability and how the association of TRAF2 with c-IAP1 affects the apoptotic inhibitory properties of c-IAP1 were examined. The presence of TRAF2 greatly enhanced the stability of c-IAP1, and these data suggest that the interaction between TRAF2 and c-IAP1 inhibits the E3 ubiquitin ligase activity intrinsic to the RING domain of c-IAP1. Using stabilized c-IAP1, the anti-apoptotic activity of c-IAP1 was characterized, and it was found that c-IAP1 suppresses apoptosis to a degree comparable with XIAP. Furthermore, we show that c-IAP1 functions to prevent the IAP antagonist, Smac/DIABLO (13, 14), from interfering with XIAP inhibition of caspases. Together, this study demonstrates that although c-IAP1 does not directly inhibit caspase activity, stabilized c-IAP1 can sequester Smac/DIABLO, prevent Smac/DIABLO from antagonizing XIAP, and inhibit cell death.     

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6

TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKβ, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.     

Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin

The stress-activated protein kinases (SAPKs, also called c-Jun NH(2)-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.