Death Receptor Activation Complexes: It Takes Two to Activate TNF Receptor 1

The extrinsic pathway of apoptosis originates at the membrane and engages membrane death receptors. Tumor necrosis factor receptor 1 (TNF-R1) is a death receptor that transduces both the death and survival signals but the molecular mechanisms via which TNF-R1 mediates these signals remain poorly understood. Recently, it has been reported that the TNF-R1 transduces these signals via two signaling complexes. The first complex (complex I) is formed at the membrane by TNF-R1, TRADD, RIP, TRAF2 and c-IAP1, while the second complex (complex II), formed in the cytosol, predominantly contains FADD and pro-caspases 8/10 but lacks TNF-R1. Complex I is responsible for activating NF-kB and thus, the transduction of survival signals. Complex II, on the other hand, is reported to transduce the apoptotic signals and it does so only if NF-kB is unable to promote upregulation of the anti-apoptotic FLIPL. These findings highlighting the complexities of TNF-R1-mediated signaling events are likely to further the progress in the constantly evolving area of death receptor-dependent signaling pathways.     

The ubiquitin-homology protein, DAP-1, associates with tumor necrosis factor receptor (p60) death domain and induces apoptosis

The tumor necrosis factor receptor, p60 (TNF-R1), transduces death signals via the association of its cytoplasmic domain with several intracellular proteins. By screening a mammalian cDNA library using the yeast two-hybrid cloning technique, we isolated a ubiquitin-homology protein, DAP-1, which specifically interacts with the cytoplasmic death domain of TNF-R1. Sequence analysis reveals that DAP-1 shares striking sequence homology with the yeast SMT3 protein that is essential for the maintenance of chromosome integrity during mitosis (Meluh, P. B., and Koshland, D. (1995) Mol. Biol. Cell 6, 793-807). DAP-1 is nearly identical to PIC1, a protein that interacts with the PML tumor suppressor implicated in acute promyelocytic leukemia (Boddy, M. N., Howe, K., Etkin, L. D., Solomon, E., and Freemont, P. S. (1996) Oncogene 13, 971-982), and the sentrin protein, which associates with the Fas death receptor (Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277-4281). The in vivo interaction between DAP-1 and TNF-R1 was further confirmed in mammalian cells. In transient transfection assays, overexpression of DAP-1 suppresses NF-kappaB/Rel activity in 293T cells, a human kidney embryonic carcinoma cell line. Overexpression of either DAP-1 or sentrin causes apoptosis of TNF-sensitive L929 fibroblast cell line, as well as TNF-resistant osteosarcoma cell line, U2OS. Furthermore, the dominant negative Fas-associated death domain protein (FADD) protein blocks the cell death induced by either DAP-1 or FADD. Collectively, these observations highly suggest a role for DAP-1 in mediating TNF-induced cell death signaling pathways, presumably through the recruitment of FADD death effector.     

LMP1, a viral relative of the TNF receptor family,signals principally from intracellular compartments

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.     

TRUSS, a novel tumor necrosis factor receptor 1 scaffolding protein that mediates activation of the transcription factor NF-kappaB

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

TNF signaling: early events and phosphorylation

Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.     

Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis

Death receptors are a subfamily of the tumor necrosis factor (TNF) receptor subfamily. They are characterized by a death domain (DD) motif within their intracellular domain, which is required for the induction of apoptosis. Fas-associated death domain protein (FADD) is reported to be the universal adaptor used by death receptors to recruit and activate the initiator caspase-8. CD95, TNF-related apoptosis-inducing ligand (TRAIL-R1), and TRAIL-R2 bind FADD directly, whereas recruitment to TNF-R1 is indirect through another adaptor TNF receptor-associated death domain protein (TRADD). TRADD also binds two other adaptors receptor-interacting protein (RIP) and TNF-receptor-associated factor 2 (TRAF2), which are required for TNF-induced NF-kappaB and c-Jun N-terminal kinase activation, respectively. Analysis of the native TNF signaling complex revealed the recruitment of RIP, TRADD, and TRAF2 but not FADD or caspase-8. TNF failed to induce apoptosis in FADD- and caspase-8-deficient Jurkat cells, indicating that these apoptotic mediators were required for TNF-induced apoptosis. In an in vitro binding assay, the intracellular domain of TNF-R1 bound TRADD, RIP, and TRAF2 but did not bind FADD or caspase-8. Under the same conditions, the intracellular domain of both CD95 and TRAIL-R2 bound both FADD and caspase-8. Taken together these results suggest that apoptosis signaling by TNF is distinct from that induced by CD95 and TRAIL. Although caspase-8 and FADD are obligatory for TNF-mediated apoptosis, they are not recruited to a TNF-induced membrane-bound receptor signaling complex as occurs during CD95 or TRAIL signaling, but instead must be activated elsewhere within the cell.     

TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.     

Differential regulation of TNF-R1 signaling: lipid raft dependency of p42mapk/erk2 activation, but not NF-kappaB activation

The TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42(mapk/erk2) and NF-kappaB. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42(mapk/erk2) and NF-kappaB in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-alpha. Disruption of lipid rafts by cholesterol depletion prevented the TNF-alpha-dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42(mapk/erk2), while the activation of NF-kappaB was unaffected. In addition, phosphorylated p42(mapk/erk2), but not receptor interacting protein, I-kappaB kinase-gamma, or I-kappaBalpha was detected in raft-containing fractions following TNF-alpha stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.     

Endogenous membrane tumor necrosis factor (TNF) is a potent amplifier of TNF receptor 1-mediated apoptosis

The heat shock protein 90 (Hsp-90) inhibitor, geldanamycin, and the proteasome inhibitor, MG-132, both inhibited tumor necrosis factor receptor 1 (TNF-R1)- but not TRAIL-induced apoptosis in Kym-1 cells, suggesting that TNF-R1-induced cell death is dependent on NF-kappaB activation in this model. Triggering of TNF-R1 by agonistic antibodies led to cell-type specific induction of endogenous TNF and apoptosis, the latter of which was abrogated by neutralizing TNF specific antibodies. TNF-R1-stimulated cells expressed TNF mainly in a cell-associated form, suggesting that the endogenously produced TNF act in its membrane-bound form. Geldanamycin failed to inhibit apoptosis induction by a combination of agonistic TNF-R1- and TNF-R2-specific antibodies, indicating that both TNF receptors co-operate in TNF-R1-triggered apoptosis in Kym-1 cells. Thus, TNF-R1 stimulation can elicit a strong and rapid apoptotic response via induction of membrane TNF and subsequent cooperation of TNF-R1 and TNF-R2. Moreover, we give evidence that this mechanism circumvents the need of the prolonged presence of exogenous soluble TNF for TNF-R1-mediated apoptosis induction.     

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.     

The TNF receptor 1: a split personality complex

The tumor necrosis factor receptor 1 (TNFR1), a prototypic member of the death receptor family signals both cell survival and apoptosis. In this issue of Cell, report that apoptotic TNFR1 signaling proceeds via the sequential formation of two distinct complexes. Since the first complex can activate survival signals and influence the activity of the second complex, this mechanism provides a checkpoint to control the execution of apoptosis.     

Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.     

Cell Fate Decisions Regulated by K63 Ubiquitination of Tumor Necrosis Factor Receptor 1

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding.