Regulation of TNFR1 and CD95 Signalling by Receptor

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Death-receptor signalling o p i n i o n The TNF superfamily of cytokines comprises 19 members and the corresponding Regulation of TNFR1 and TNFR superfamily includes 23 related receptors. A subgroup of this family includes the ‘death receptors’, TNFR1, CD95 signalling by receptor CD95, the TNF-related apoptosis-inducing ligand (TRAIL) receptors (TRAIL R1, also compartmentalization known as DR4; and TRAIL R2, also known as DR5), DR3, DR6 and p75NTR. These Stefan Schütze, Vladimir Tchikov and Wulf Schneider-Brachert receptors share a ‘death domain’, a conserved 80-amino-acid sequence in the cytoplasmic Abstract | The death receptors tumour-necrosis factor receptor‑1 (TNFR1) and tail that is necessary for the direct activation CD95 (also known as FAS and APO‑1) transduce signals that promote cell death by of the apoptotic programme by some of apoptosis. However, these receptors are also capable of inducing anti-apoptotic these receptors (TNFR1, CD95, TRAIL R1 signals through the activation of the transcription factor nuclear factor-κB (NF-κB) and TRAIL R2). TNF-induced apoptosis is or through activation of the proliferative mitogen-activated protein kinase (MAPK) mediated by the recruitment of the adaptor proteins TNFR-associated death-domain cascade. Recent findings reveal a role for receptor internalization and endosomal (TRADD) protein, FAS-associated death- trafficking in selectively transmitting the signals that lead either to apoptosis or to domain protein (FADD) and caspase‑8 the survival of the cell. to the cytoplasmic death domain of the receptor7. CD95 and TRAIL receptors do not require TRADD for the recruitment of FADD Several biological responses, ranging from However, the regulation of receptor and caspase‑8 (see BOX 2 for details)8–10. proliferation and differentiation to cell death, endocytosis and signal transduction, TRADD also functions as an assembly are mediated by ligand-triggered receptor as well as the linkage of compartmen- platform to diverge TNFR1 signalling from activation at the plasma membrane. The talization to selective biological outcomes the death domain; interaction of TRADD classic model of signal transduction involves in vivo, is still poorly understood. Whether with receptor-interacting protein (RIP) and cell-surface receptors that are activated signalling from endosomal compartments TNFR-associated factor-2 (TRAF2) leads to after the binding of a ligand and transmit generates a different biological response the activation of the survival transcription intracellular signals to generate secondary to signalling from the plasma membrane factor nuclear factor-κB (NF-κB) and to the messengers. The activation of many receptors remains to be determined3. In this context, induction of the c‑Jun N‑terminal kinase also triggers the accelerated endocytosis of recent findings concerning death-recep- (JNK) cascade7,11–13. ligand-receptor complexes. This suggests tor signalling provide novel insights into Earlier reports suggested that the forma- that endocytic vesicles are important sites for the physiological role of both receptor tion of a TNF-mediated apoptosis signalling organizing the recruitment of specific com- internalization and endosomal trafficking complex follows a different mechanism than ponents in the activated signalling cascade. in selectively transmitting signals that lead the mechanism that is activated by the CD95 Endocytosis has long been regarded solely as either to apoptosis or to the survival of ligand or TRAIL14,15. Micheau and Tschopp15 a mechanism to terminate signalling through the cell. proposed a model in which TNFR1 receptor internalization and subsequent lyso- signalling involves the assembly of two somal degradation. However, it has become ...endocytosis orchestrates molecularly and spatially distinct signalling clear that certain signalling pathways require cell signalling by coupling and complexes that sequentially activate NF‑κB receptor internalization for full activation to integrating different cascades and caspases. Within a few minutes of TNF occur (for example, epidermal growth factor on the surface of endocytic binding, TNFR1 recruits TRADD, RIP1 and receptor, TRK, nerve growth factor receptor TRAF2 to form a signalling complex at the and insulin receptor). Emerging data suggest vesicles... cell surface (which is known as ‘complex I’) that endocytosis orchestrates cell signal- that activates NF‑κB through recruitment of ling by coupling and integrating different In the following sections, we discuss the the inhibitor of κB (IκB) kinase (IKK) ‘sig- cascades on the surface of endocytic vesicles dichotomy of pro-apoptotic and anti- nalosome’. The signalosome is a high-molec- (reviewed in REFS 1–4). apoptotic signalling properties of the death ular-weight complex that comprises three Following receptor activation, receptors receptors tumour-necrosis factor recep- main proteins: two IκB kinases (IKK1 and and ligands can internalize from the cell tor‑1 (TNFR1; also known as p55/60 and IKK2) and IKKγ (also known as NEMO). surface by various routes (summarized in CD120a) and CD95 (also known as FAS This model implies that, at later time points BOX 1). The best-characterized mechanism and APO‑1). We highlight the early events and after TNFR1 internalization, RIP1, of endocytosis is clathrin-mediated endo- in the formation of ligand-activated recep- TRAF2 and TRADD become modified and cytosis. In addition, clathrin-independent tor complexes and we address the role of dissociate from the receptor. TRADD and/or pathways that involve cholesterol and post-translational modifications of CD95 RIP then bind to FADD, which results in the sphingolipid-enriched membrane domains in apoptosis signalling. We also discuss the recruitment of caspase‑8 to a secondary sig- (lipid rafts) and special membrane invagi- role of internalization in determining the nalling complex within the cytosol (known nations (caveolae) also have important roles fate of the receptors and subsequently the as ‘complex II’). This complex subsequently in endocytosis (reviewed in REFS 5,6). specificity of signalling events. mediates apoptosis.

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Box 1 | Different mechanisms of receptor endocytosis known as p75/80 and CD120b). Both TNFRs contain four Cys-rich repeats in their extra- Lipid-raft-dependent internalization Clathrin-mediated cellular domains that mediate homophilic internalization Caveolin-mediated Clathrin- and caveolin- interactions in the absence of a ligand at Dynamin internalization independent internalization the pre-ligand assembly domain. These AP2 Cell Lipid raft homophilic interactions form homotrimers Clathrin membrane of each receptor type. Ligand binding to pre- Caveolin formed TNFR complexes induces intracellular signal transduction. In contrast to TNFR1, Caveolae CCP TNFR2 does not contain a death domain and cannot transmit apoptotic signals.

Role of internalization in TNFR1 signalling. Binding of TNF initiates the rapid clustering of TNFR1, followed by internalization of the Caveosome ligand-receptor complex via clathrin-coated Early pit formation18–22. In human endothelial CCV endosome cells, TNFR endocytosis has been linked to TNF-induced expression of NF‑κB-regulated Early genes19; however, earlier reports suggest that endosome TNF-receptor internalization in other cell types might have a role in mediating TNF cytotoxicity23–25. trans-Golgi TNF activates two types of sphingo vesicle myelinases — an endolysosomal acid sphingomyelinase (aSMase) and a membrane-bound neutral sphingomyelinase (nSMase)26. The lipid second messenger Nucleus Golgi Late endosome Lysosome ceramide, which is generated by sphingo Receptors and ligands can be internalized from the cell surface by various routes. The first step in myelinases, is a potent pro-apoptotic media- clathrin-mediated endocytosis involves the selective recruitment of transmembrane receptors REFS 27,28 Nature Reviews | Molecular Cell Biology tor (reviewed in ). Conflicting data and their bound ligands into specialized membrane microdomains (known as clathrin-coated pits on the role of ceramide and sphingomyelinase (CCP); see figure). Several adaptor-protein complexes participate in this process, which is initiated in signalling have also been published27,29,30. by the binding of adaptor protein complex‑2 (AP2) to the plasma membrane through its lipid- nSMase is activated via FAN (factor associ- binding domains. AP2 also binds to specific transport sequences within the intracellular sequence of the activated receptor. The best-characterized endocytosis motifs of cargo proteins are the ated with nSMase) and leads to the accumula- 31,32 Tyr‑based YXXΦ motif (Y represents tyrosine, X any amino acid, and Φ a bulky hydrophobic amino tion of ceramide at the plasma membrane . acid) and dileucine-based motifs. Interaction of AP2 with the GTPase dynamin results in a ring The role of nSMase in TNF signalling remains formation around the neck of budding vesicles that leads to membrane fission and generation of unclear33. Activation of aSMase is dependent free clathrin-coated vesicles (CCVs). After uncoating during intracellular trafficking, CCVs fuse on TNFR1 internalization20,21 and is mediated with early endosomes and with trans-Golgi vesicles, from which the ligand-receptor complexes via the death domain of TNFR1 by the are sorted to various intracellular compartments, such as late endosomes (also known as recruitment of the adaptor proteins TRADD multivesicular bodies) and lysosomes. Receptor complexes that are destined for degradation fuse and FADD34,35. A role for aSMase in transmit- with lysosomes and are degraded. ting apoptotic signals of death receptors has Clathrin-independent pathways also have important roles in endocytosis. One form of clathrin- been reported for TNF36–38, CD95 (Refs 39–42) independent endocytosis known as raft or caveolar endocytosis (RCE) relies on cholesterol- and 43,44 sphingolipid-enriched membrane domains (lipid rafts) and special membrane invaginations and TRAIL . (caveolae). Membrane receptors located in these microdomains are severely limited in their ability In various cell lines, TNFR1 death- to diffuse freely across the plasma membrane. RCE uses a different compartment before fusion with domain signalling pathways, including the early endosome, the caveosome. In addition, several dynamin-independent endocytosis the pathways that lead to apoptosis, mechanisms that are distinct from both clathrin-coated pits and caveolae exist, but their exact are dependent on TNF-receptor inter- roles in receptor internalization and signalling is only beginning to be elucidated. nalization, whereas other pathways remain unaffected20–22. Blocking the formation of clathrin-coated pits inhibits the activation TNFR1 and CD95 receptors are capable TNFR1 compartmentalization of the endolysosomal aSMase and JNK, as of inducing both pro-apoptotic and anti- TNF was detected almost 30 years ago and well as TNF-induced cell death. By contrast, apoptotic signals. Therefore, it is of funda- is now recognized as a highly pleiotropic the interaction of the adaptor molecules mental interest to understand the molecular cytokine that elicits diverse cellular responses, FAN and TRADD with TNFR1 at the cell mechanisms that govern the decision of a which range from proliferation and differen- surface, the activation of plasma-membrane- cell to respond to death-receptor ligands tiation to activation of apoptosis (reviewed associated nSMase and the stimulation of by undergoing either apoptosis or other in REFS 16,17). The biological activities of proline-directed protein kinases were not biological processes, such as the induction of TNF are mediated by two distinct cell-surface influenced by the inhibition of TNFR1 differentiation or proliferation. receptors: TNFR1 and TNFR2 (TNFR2 is also internalization20. Genetic interventions

656 | August 2008 | volume 9 www.nature.com/reviews/molcellbio © 2008 Macmillan Publishers Limited. All rights reserved. required for the activation of NF in a cascade-like manner. Once released released manner. Once cascade-like a in activated be can CTSD as such proteases activa to the BID.cleaves CTSD leads This membrane, endosomal the through cation translo ceramide-mediated TNF-induced, After site activation. of CTSD subcellular at the located is that BID indicates This endosomes. early RAB5-positive in located areand BID CTSD both stimulation, TNF and, following vesicles CTSD-positive with pro-apoptotic The protein compartment endolysosomal same the within of target ceramide downstream D ( cathepsin protease Asp-The ceramide. generates which aSMase enzyme endolysosomal the activates TNFR1 above, triggering cussed in death-signalling vesicles. TNFR1 this intr apoptotic signals further transmitted from death signals from TNFR1. So, how are that is involved in selectively transmitting recognized as a novel signalling organelle that the endosomal compartment should be matory) signalling pathways. We believe mediated anti-apoptotic and pro-inflam and internalization-independent (NF- ization-dependent (cytotoxic, pro-apoptotic) production were not investigated. apoptotic consequences of blocking ROS TNF-induced cell death, but the functional production is also known to have a role in and mitogen-activated protein kinases (MAPKs) species (ROS) and for the activation of both TNF-induced production of reactive oxygen cells, TNFR internalization is required for compartments. In HEK293 and HeLa ting pro-apoptotic signals from intracellular a role of TNFR1 internalization in transmit NF tosis, but does not affect TNF-mediated formation and prevents TNF-induced apop death-inducing signalling complex (DISC) 14.7K also inhibits TNFR1 internalization, induced apoptosis complex; and significantly inhibited TNF- and caspase internalization; abolished TRADD, FADD — resulted in the elimination of TNFR1 G thatpoint carry mutations within the Tyr- internalization domain (TRID) or mutants — such as TNFR1 mutants that lack the nat might proteases these cytoplasm, the to tion of tion caspase have an important role for selective internal ln-Arg-Trp TNFR1 internalization motif

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of activated caspase caspase procaspase (TRAIL) receptors directly bind to FADD and procaspase Box 2 | TNFR internalization domain. apoptotic stimuli to guarantee caspase activation. NSD, neutral sphingomyelinase domain; TRID, counteracted by mitochondrial proteins such as Smac/Diablo, which are released in responseprotein to XIAP which prevents caspase counterparts (such as BAX and BAD) to block tBID-mediated apoptosis, or by the inhibitory apoptotic BCL2 proteins such as BCL2 or BCL-x of caspase recruitment of procaspase tightly regulated; at the level of the DISC, the inhibitory proteins (cFLIP, cIAP) can prevent the activating factor-1 the BCL2 proteins BAX and BAD results in the release of cytochrome resulting in the translocation of truncated (t)BID to the mitochondria. The interaction of cells),tBID withwhich involves cleavage of the B-cell lymphoma protein-2 (BCL2) family protein BID, adaptor proteins to TNFR1 and CD95, see procaspase (TRADD) protein is required for the recruitment of FAS-associated death-domain (FADD)For and tumour-necrosis factor receptor to form the death-inducing signalling complex (DISC) via their intracellular death domains (DD). After ligand binding, pre-assembled (trimeric) death receptors recruit specific adaptor proteins subsequently leads to the activation of caspase procaspase caspase

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execute caspase-independent apoptosis or Although it is generally accepted that CD95 compartmentalization might individually participate in different TRADD is required for RIP recruitment CD95 is another important member of the apoptotic or cell-death-signalling cascades to TNFR1, this observation is in line with TNFR superfamily (reviewed in REF. 57). by connecting the endosomal compart- previous reports that support a direct This receptor is a key mediator of apoptosis, ment to the classic apoptotic signalling interaction of RIP with TNFR1 and CD95 and has a pivotal role in the regulation of pathways. (Refs 50,51). In addition, it has been shown peripheral tolerance and lymphoid home- that TRADD and RIP1 can independently ostasis. Accumulating evidence suggests DISC assembly at TNF receptosomes. and competitively associate with TNFR1 that the ligation of CD95 can also mediate The key elements of the signalling pathways (Refs 52,53). RIP is essential for TNFR1- several non-apoptotic activities (recently of apoptosis (the TNFR1 adaptor proteins mediated NF‑κB activation, as RIP-deficient discussed in REF. 58). Thus, under certain TRADD, FADD and caspase‑8) and of cells showed abolished NF‑κB activation conditions and in some cell types, CD95 NF‑κB (TRADD, RIP1 and the TRAFs) and were highly sensitive to the induction of ligation might also protect cells and regulate are well defined (reviewed in REFS 17,47). apoptosis54. RIP is recruited to TRADD via tissue regeneration and proliferation. A clue However, the molecular mechanisms that its own death domain51, whereas TRAF2 can to the understanding of these contradicting regulate the formation of the initial signal- be recruited to the N terminus of TRADD biological activities of CD95 might lie in the ling complexes at the activated TNFR1, in via its TRAF2 domain. The interaction of dynamics of CD95 membrane localization order to selectively transmit specific signal TRAF2 with the IKK complex is supported as well as internalization-dependent and transduction events to various intracellular by RIP and leads to NF‑κB activation55. internalization-independent pathways that compartments, are poorly understood. How are signalling events during signal for apoptosis and other functions. Three recent reports from our the endocytosis of TNFR complexes group21,22,48 confirmed the existence of regulated? Because TRADD, RIP1 and Early events in CD95 activation. The initial spatially distinct signalling complexes at the TRAF2 co-internalize with TNFR1, NF‑κB events in CD95 activation are just begin- cell surface and in the cytoplasm, as pro- signalling has to be downregulated during ning to be understood (FIG. 2). Earlier work posed by Micheau and Tschopp15. By using TNFR endocytosis. This downregulation on the discovery of the CD95 DISC8–10 a novel experimental approach — in which allows for the full propagation of DISC- showed that the recruitment of FADD and TNFRs were labelled with biotin–TNF mediated pro-apoptotic signalling. The caspase‑8 occurs in an initial rapid phase coupled to streptavidin-coated magnetic ubiquitin ligase caspase-associated ring within seconds of CD95 activation. A nanobeads and intact TNF–TNFR com- protein‑2 (CARP2) was recently identi- further significant enhancement of p26/p28 plexes were isolated within their native fied as a constitutive negative regulator and p18/p10 active caspase‑8 was detected membrane environment using a special- of TNF-induced NF‑κB activation56. between 5 and 10 minutes after CD95 ized magnetic device49 — we showed that CARP2 is localized to endocytic vesicles, ligation, which indicates that there are two recruitment of RIP1 and TRAF2 to cell-sur- in which it interacts with internalized phases of caspase‑8 activation. The forma- face TNFR1 is sufficient to activate NF‑κB TNF receptosomes and, together with tion of SDS-stable and mercaptoethanol- (which corresponds to complex I). We also the zinc-finger protein A20, mediates the stable CD95 aggregates was also observed demonstrated that the DISC was associated ubiquitylation and degradation of RIP1 within seconds of ligand binding8,59,60. These with the internalized TNFR1 in endosomal and the subsequent downregulation of highly aggregated CD95 forms of approxi- compartments — termed TNFR1 recep- NF‑κB signalling. mately 180 kDa in SDS–PAGE were termed tosomes (which do not correspond to the In summary, two temporary and spa- CD95hi (Ref. 60). CD95hi forms higher order cytosolic complex II) — or in immunopre- tially distinct TNFR1 signalling complexes aggregates through interactions with actin cipitates of TNFR1 from TNF-treated U937 are formed with the capacity to signal filaments within 15 minutes of ligand cells21,22. either for NF‑κB activation from the cell binding59. These aggregates can be detected In our reports, recruitment of the adap- surface or for apoptosis from internalized by fluorescence microscopy as ‘signalling tor proteins TRADD, FADD and caspase‑8 receptosomes (FIG. 1), indicating that protein oligomerization transduction struc- to form the DISC complex occurred within TNFR1 compartmentalization has an tures’ (SPOTS) in type I cells61. However, 3 minutes of TNF stimulation. The DISC important role in the diversification of the early formation of CD95hi is not suffi- was still associated with TNFR1 after 60 TNF-mediated biological responses. The cient to induce apoptosis. Only one form of minutes. Inhibition of TNFR1 internaliza- reason for the discrepancy between our CD95hi shifted to a high-molecular-weight tion, either by deletion of the TNFR1 inter- findings of TNFR1-associated DISC and structure of many megadaltons; this was nalization domain21 or by infection with an the results of Harper et al.14 and Micheau termed hiDISC60. In these hiDISC struc- adenovirus22, blocked DISC recruitment and Tschopp15 might be due to the different tures, caspase‑8 is activated, followed by the and apoptosis but still allowed the recruit- detergents used for the solubilization of initiation of apoptosis. ment of RIP1 and TRAF2 to signal for TNFR1 from clathrin-coated endosomes NF‑κB activation. Immunomagnetic isola- and the fact that different labelling and Receptor internalization in CD95 signal- tion of the morphologically intact vesicles precipitation protocols were used. ling. Between 15 minutes and 1 hour showed that trafficking and maturation of Furthermore, early DISC formation at after initial caspase‑8 activation, lateral TNF receptosomes occurs along the endo- TNF-receptosomes occurred rapidly and segregation of CD95 complexes forms cytic pathway, and also showed the fusion transiently and might only have been large clusters on one pole of the cells; these of TNF receptosomes with trans-Golgi detectable when the internalization was large clusters, which are known as ‘caps’, membranes, resulting in the formation of synchronized by pre-labelling the recep- correspond to internalized receptors that multivesicular endosomes21, thus allowing tors with TNF at 4 °C, followed by a rapid are derived from hiDISC structures59,60,62. for the activation of aSMase and CTSD46. increase in the temperature to 37 °C. Electron microscopy has revealed that after

658 | August 2008 | volume 9 www.nature.com/reviews/molcellbio © 2008 Macmillan Publishers Limited. All rights reserved. tumour-necrosis factor receptor tein of cell surface TNFR1 is sufficient for NF cles caspase cles within 3 minutes of ligand binding, high 3 minutes binding, of ligand within family. death-receptor TNF the of formember another valid be also could but to TNFR1, restricted not be might tosis role of that the recep suggest reports These vesi these and within vesicles, endosomal in large clusters in that are internalized aggregate lipid rafts stimulation, CD95 and NF ligand binding, TNFR1 is internalized via clathrin-dependent endocytosis (DD) domain death cytoplasmic the to TRAF2 and RIP1 of recruitment the 14.7K), protein adenovirus the by or (TRID) domain internalization When receptor internalization is blocked (by mutations within the TNFR pro receptor-interacting protein, (TRADD) domain death associated factor- nuclear factor transcription Figure 1 | nat of endocytosis byrecruitment the lighted tor internalization for signalling in apopin for signalling tor internalization ligases caspase-associated ring protein ring caspase-associated ligases

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CA EEA A20 EE endocytic pathway, TNF-receptosomes fuse with syntaxin with fuse TNF-receptosomes pathway, endocytic pase induce caspase Within the receptosome-bound DISC, caspase DISC, receptosome-bound the Within domain. caspase of activation of mediating apoptosis through cleavage of the apoptotic protein BID and cas activated MVB, the Within (MVBs). bodies multivesicular form to positive VTI-1b and p47a protein (FADD) and caspase degradation of RIP1. TRADD then recruits FAS-associated death domain sphingomyelinase (pro RP TNF-r A1 2 .7 1 ‑ K ) and cathepsin ) and cathepsin 8 was also also 8 was ‑ 8 stimulates the aSMase–ceramide–CTSD cascade, which is capable Apoptosis RIP1 TRADD ec eptosome DIS TRAF2 RAB4 60,62 C TRADD

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CD95L SDS-stable microaggregates, Actin/palmitoylation-dependent CD95 palmitoylation, recruitment of CD95hi into low level DISC lipid rafts, SPOTS CD95hi clustering, capping, large lipid-raft platforms, Endosomal trafficking of CD95 receptosomes, clathrin-mediated recruitment of large amounts of DISC (hiDISC), Lipid internalization massive caspase-8 activation PA PA PA PA raft Ezrin Ezrin CCP Clathrin CD95-receptosome CCV Actin PA PA DD DD DD DD

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Non-apoptotic pathways Apoptosis Figure 2 | CD95 compartmentalization. Ligation of CD95 leads to the activation of the mitogen-activated protein kinase (MAPK) and the tran- rapid formation of SDS-stable microaggregates (CD95hi), which trans scription factor nuclear factor-κB (NF-NaturκeB), Re leadingviews | Mol to ecularcell proliferation Cell Biology locate to lipid-raft plasma-membrane microdomains, a process that is and migration, but is unable to kill type I cells. Between 5 to 15 minutes regulated by palmitoylation (PA) and ezrin-mediated association of the after CD95 triggering, CD95 is internalized in a clathrin-dependent man- receptor with the actin cytoskeleton. Low amounts of FAS-associated ner into endosomal compartments via ezrin-mediated actin-filament death-domain protein (FADD) and caspase‑8 are recruited within this association. During endosomal trafficking, high levels of death-inducing early time frame and ‘signalling protein oligomerization transduction signalling complex (DISC) proteins (FADD and caspase‑8) are recruited, structures’ (SPOTS) are then formed, resulting in the clustering of CD95 resulting in strong caspase‑8 activation in high-molecular-weight struc- (a process known as ‘capping’) which depends on active caspase‑8. This tures of many megadaltons (hiDISC complexes) resulting in the propaga- leads to the formation of large lipid-raft platforms. At this stage, CD95 tion of apoptosis signalling. CCP, clathrin-coated pit; CCV, clathrin‑coated has the potential to activate non-apoptotic pathways by inducing the vesicle. in both lymphoblasts and T cells. This sug- of caspase‑8 activation as compared to CD95-dependent proliferative signalling gests a regulatory role for caspase‑8 in CD95 stimulation with crosslinked sCD95L62. could also be mediated through the caspase‑8 internalization. CD95 internalization was not Inhibition of CD95 internalization enabled inhibitor cFLIP to promote the activation observed in type II cells and disruption of the activation of NF‑κB and also of extra of the NF‑κB and ERK signalling pathways, the actin filaments by latrunculin A did not cellular signal-regulated kinase (ERK)1/2 either by the inhibition of caspase‑8, by affect apoptosis sensitivity, suggesting that following CD95 engagement. Treatment of recruiting TRAF2 and RIP into the DISC or CD95 signalling in type II cells is different CD95L-resistant MCF7 cells (a human breast by interacting with IKKγ in the IKK com- from that in type I cells59. adenocarcinoma cell line) with anti-APO‑1 plex69,70. Whether these events are initiated As the physiological stimulus of CD95 is antibody or with sCD95L (which does not from cell-surface or internalized CD95 is more likely to be a membrane-bound ligand induce CD95 internalization) increased unknown. (mCD95L) than a soluble ligand (sCD95L), tumour-cell motility and invasiveness62. Thus, it appears that the dynamics of the intriguing question is whether CD95 These observations provided the molecular CD95 membrane localization and internali- internalization also takes place after basis for the assumption that the activation of zation also have a critical role in balancing stimulation of CD95 with mCD95L. In co- non-apoptotic signalling pathways by CD95L, internalization-dependent apoptotic and culture experiments using cells that express including the MAPK and NF‑κB signalling internalization-independent non-apoptotic non-cleavable mCD95L, the ligand induces pathways, have a role in the tumorigenesis of pathways, which drive cell death and other internalization of CD95 and similar levels CD95-resistant tumours67,68. functions, respectively (FIG. 2).

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CD95 post-translational modifications. How To further address the role of TNFR1 and 10. Medema, J. P. et al. FLICE is activated by association with the CD95 death-inducing signaling complex are the different steps in CD95 activation reg- CD95 internalization in vivo, a knock-in (DISC). EMBO J. 16, 2794–2804 (1997). ulated at the molecular level? It is well known approach is currently in progress to study the 11. Yeh, W. C. et al. Early lethality, functional NF‑κB activation, and increased sensitivity to TNF-induced that membrane proteins that are post-transla- effect of TNFR1-internalization-deficient cell death in TRAF2-deficient mice. Immunity 7, tionally modified by N‑myristoylation and/or and CD95-internalization-deficient mutants 715–725 (1997). 12. Lee, S. Y. et al. TRAF2 is essential for JNK but not S‑palmitoylation can be found in lipid rafts, in different mouse models of inflammation, NF‑κB activation and regulates lymphocyte whereas proteins that have been modified infection and tumour development. Another proliferation and survival. Immunity 7, 703–713 (1997). by unsaturated fatty acids or prenyl groups challenge for the future will be to define the 13. Grech, A. P. et al. TRAF2 differentially regulates the are excluded from lipid rafts (reviewed in receptor-specific molecular composition and canonical and noncanonical pathways of NF‑κB activation in mature B cells. Immunity 21, 629–642 REF. 71). Indeed, human and murine CD95 regulation of the TNFR1 and CD95 internal- (2004). proteins have been found to be palmitoylated ization machinery and to define the mecha- 14. Harper, N., Hughes, M., MacFarlane, M. & Cohen, G. M. Fas-associated death domain protein and at the membrane proximal Cys199 or nisms that decide how to compartmentalize caspase‑8 are not recruited to the tumor necrosis Cys194, respectively60,65. Mutations in these the receptors. Elucidation of the pathways factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. J. Biol. Chem. 278, sites or competition for CD95 palmitoylation involved in TRAIL-receptor internalization 25534–25541 (2003). prevented CD95hi complex formation and remains another challenge. TRAIL-stimu- 15. Micheau, O. & Tschopp, J. Induction of TNF receptor I‑mediated apoptosis via two sequential signaling resulted in a marked reduction of CD95 lated internalization of its cognate receptors complexes. Cell 114, 181–190 (2003). translocation to lipid rafts, CD95L-induced proceeds along both clathrin-dependent 16. Locksley, R. M., Killeen, N. & Lenardo, M. J. The TNF and TNF receptor superfamilies: integrating 72,73 receptor internalization, DISC-formation and clathrin-independent pathways , but mammalian biology. Cell 104, 487–501 (2001). and apoptosis. Thus, CD95 palmitoylation TRAIL-induced DISC formation, caspase 17. Wajant, H., Pfizenmaier, K. & Scheurich, P. Tumor necrosis factor signaling. Cell Death. Differ. 10, 45–65 might have an essential role in the initiation activation and apoptosis signalling also (2003). of CD95 apoptotic signalling pathway. occurs in the absence of clathrin-medi- 18. Mosselmans, R., Hepburn, A., Dumont, J. E., Fiers, W. 72,73 & Galand, P. Endocytic pathway of recombinant ated TRAIL-receptor endocytosis . The murine tumor necrosis factor in L‑929 cells. Conclusions and future perspectives molecular basis for the apparent differences J. Immunol. 141, 3096–3100 (1988). 19. Bradley, J. R., Johnson, D. R. & Pober, J. S. Four The current data suggest that the compart- between TRAIL receptors and TNFR1 different classes of inhibitors of receptor-mediated mentalization of TNFR1 and CD95 might and CD95 compartmentalization requires endocytosis decrease tumor necrosis factor-induced gene expression in human endothelial cells. be an important mechanism for the diver- further investigation, including genetic J. Immunol. 150, 5544–5555 (1993). sification of intracellular signalling, thus evidence from knock-in experiments using 20. Schütze, S. et al. Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor determining biological outcomes following internalization-defective receptor constructs. necrosis factor receptor death domain signaling. ligand binding. Clathrin-mediated internali- J. Biol. Chem. 274, 10203–10212 Stefan Schütze and Vladimir Tchikov are at the (1999). zation of both TNFR1 and CD95 seems to be Institute of Immunology, University Hospital of 21. Schneider-Brachert, W. et al. Compartmentalization of a prerequisite for the efficient recruitment of Schleswig-Holstein, Campus Kiel, Michaelisstr. 5, TNF receptor 1 signaling: internalized TNF D‑24105 Kiel, Germany. receptosomes as death signaling vesicles. Immunity DISC proteins and apoptosis signalling from 21, 415–428 (2004). endosomal compartments. By contrast, anti- Wulf Schneider-Brachert is at the Institute for Medical 22. Schneider-Brachert, W. et al. Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a Microbiology and Hygiene, University of Regensburg, apoptotic signalling through activation of novel immune escape mechanism. J. Clin. Invest. 116, NF‑κB and MAPKs occurs independently of Franz-Josef-Strauss-Allee 11, 2901–2913 (2006). D‑93053 Regensburg, Germany. 23. Kull, F. C. Jr. & Cuatrecasas, P. Possible requirement of receptor internalization through binding internalization in the mechanism of in vitro cytotoxicity of RIP and TRAF2 to TNFR1 or low-level Correspondence to S.S. in tumor necrosis serum. Cancer Res. 41, 4885–4890 e-mail: [email protected] (1981). recruitment of caspase‑8 to CD95, respec- 24. Watanabe, N. et al. Continuous internalization of doi:10.1038/nrm2430 tively. The physiological relevance of TNFR1 tumor necrosis factor receptors in a human Published online 11 June 2008 myosarcoma cell line. J. Biol. Chem. 263, compartmentalization becomes evident 10262–10266 (1988). when considering infection by pathogens; 1. McPherson, P. S., Kay, B. K. & Hussain, N. K. Signaling 25. Pastorino, J. G. et al. The cytotoxicity of tumor on the endocytic pathway. Traffic 2001. 2, 375–384 necrosis factor depends on induction of the for example, adenoviruses use the inhibi- (2001). mitochondrial permeability transition. J. Biol. Chem. tion of TNFR1 endocytosis to selectively 2. Sorkin, A. & Von Zastrow, M. Signal transduction and 271, 29792–29798 (1996). endocytosis: close encounters of many kinds. Nature 26. Wiegmann, K., Schütze, S., Machleidt, T., Witte, D. & prevent TNF-mediated apoptosis of infected Rev. Mol. Cell Biol. 3, 600–614 (2002). Krönke, M. Functional dichotomy of neutral and acidic cells, without affecting other signalling 3. Teis, D. & Huber, L. A. The odd couple: signal sphingomyelinases in tumor necrosis factor signaling. transduction and endocytosis. Cell Mol. Life Sci. 60, Cell 78, 1005–1015 (1994). events from the cell surface. Although our 2020–2033 (2003). 27. Lin, T. et al. Role of acidic sphingomyelinase in Fas/ proposed model concurs with the model of 4. Miaczynska, M., Pelkmans, L. & Zerial, M. Not just a CD95-mediated cell death. J. Biol. Chem. 275, sink: endosomes in control of signal transduction. 8657–8663 (2000). Micheau and Tschopp in that two distinct Curr. Opin. Cell Biol. 16, 400–406 (2004). 28. Morales, A., Lee, H., Goni, F. M., Kolesnick, R. & signalling complexes exist — one that 5. Le Roy, C. & Wrana, J. L. Clathrin- and non‑clathrin‑ Fernandez-Checa, J. C. Sphingolipids and cell death. mediated endocytic regulation of cell signalling. Apoptosis 12, 923–939 (2007). signals to NF‑κB from the cell surface and Nature Rev. Mol. Cell Biol. 6, 112–126 (2005). 29. Manthey, C. L. & Schuchman, E. H. Acid an intracellular one that signals after TNFR1 6. Mayor, S. & Pagano, R. E. Pathways of clathrin- sphingomyelinase-derived ceramide is not required for independent endocytosis. Nature Rev. Mol. Cell Biol. inflammatory cytokine signalling in murine internalization for cell death, there are 8, 603–612 (2007). macrophages. Cytokine 10, 654–661 (1998). some differences. We showed that NF‑κB 7. Hsu, H., Shu, H. B., Pan, M. G. & Goeddel, D. V. 30. Nix, M. & Stoffel, W. Perturbation of membrane TRADD–TRAF2 and TRADD–FADD interactions define microdomains reduces mitogenic signaling and signalling also occurs from the cell surface, two distinct TNF receptor 1 signal transduction increases susceptibility to apoptosis after T cell following recruitment of RIP and TRAF2 to pathways. Cell 84, 299–308 (1996). receptor stimulation. Cell Death. Differ. 7, 413–424 8. Kischkel, F. C. et al. Cytotoxicity-dependent APO‑1 (2000). internalization-deficient TNFR1 mutants (Fas/CD95)-associated proteins form a death-inducing 31. Adam, D., Wiegmann, K., Adam-Klages, S., Ruff, A. & or in adenovirus 14.7K protein-transduced signaling complex (DISC) with the receptor. EMBO J. Krönke, M. A novel cytoplasmic domain of the p55 14, 5579–5588 (1995). tumor necrosis factor receptor initiates the neutral cells. Furthermore, we detected recruitment 9. Scaffidi, C., Medema, J. P., Krammer, P. H. & Peter, sphingomyelinase pathway. J. Biol. Chem. 271, of DISC by TNFR1 at TNF receptosomes, M. E. FLICE is predominantly expressed as two 14617–14622 (1996). 15 functionally active isoforms, caspase‑8/a and 32. Adam-Klages, S. et al. FAN, a novel WD‑repeat whereas Micheau and Tschopp could not caspase‑8/b. J. Biol. Chem. 272, 26953–26958 protein, couples the p55 TNF-receptor to neutral detect any association of DISC with TNFR1. (1997). sphingomyelinase. Cell 86, 937–947 (1996).

P e r s p e c t i v e s

33. Boone, E., Vandevoorde, V., De Wilde, G. & 48. Neumeyer, J. et al. TNF-receptor I defective in 65. Parlato, S. et al. CD95 (APO‑1/Fas) linkage to the Haegeman, G. Activation of p42/p44 mitogen- internalization allows for cell death through activation actin cytoskeleton through ezrin in human T activated protein kinases (MAPK) and p38 MAPK by of neutral sphingomyelinase. Exp. Cell Res. 312, lymphocytes: a novel regulatory mechanism of the tumor necrosis factor (TNF) is mediated through the 2142–2153 (2006). CD95 apoptotic pathway. EMBO J. 19, 5123–5134 death domain of the 55-kDa TNF receptor. FEBS Lett. 49. Schütze, S. & Tchikov, V. in Programmed Cell Death (2000). 441, 275–280 (1998). Part A Volume 442 Ch. 5 (eds Khosravi-Far, R. et al.) 66. Koncz, G., Kerekes, K., Chakrabandhu, K. & Hueber, 34. Schwandner, R., Wiegmann, K., Bernardo, K., Kreder, D. (Academic Press, in the press). A. O. Regulating Vav1 phosphorylation by the SHP‑1 & Krönke, M. TNF receptor death domain-associated 50. Stanger, B. Z., Leder, P., Lee, T. H., Kim, E. & Seed, B. tyrosine phosphatase is a fine-tuning mechanism for proteins TRADD and FADD signal activation of acid RIP: a novel protein containing a death domain that the negative regulation of DISC formation and Fas- sphingomyelinase. J. Biol. Chem. 273, 5916–5922 interacts with Fas/APO‑1 (CD95) in yeast and causes mediated cell death signaling. Cell Death. Differ. 15, (1998). cell death. Cell 81, 513–523 (1995). 494–503 (2007). 35. Wiegmann, K. et al. Requirement of FADD for tumor 51. Hsu, H., Huang, J., Shu, H. B., Baichwal, V. & Goeddel, 67. Barnhart, B. C. et al. CD95 ligand induces motility necrosis factor-induced activation of acid D. V. TNF-dependent recruitment of the protein kinase and invasiveness of apoptosis-resistant tumor cells. sphingomyelinase. J. Biol. Chem. 274, 5267–5270 RIP to the TNF receptor‑1 signaling complex. EMBO J. 23, 3175–3185 (2004). (1999). Immunity 4, 387–396 (1996). 68. Ahn, J. H. et al. Non-apoptotic signaling pathways 36. Monney, L. et al. Role of an acidic compartment in 52. Zheng, L. et al. Competitive control of independent activated by soluble Fas ligand in serum-starved tumor‑necrosis‑factor‑α‑induced production of programs of tumor necrosis factor receptor-induced human fibroblasts. Mitogen-activated protein kinases ceramide, activation of caspase‑3 and apoptosis. Eur. cell death by TRADD and RIP1. Mol. Cell. Biol. 26, and NF‑κB‑dependent gene expression. J. Biol. Chem. J. Biochem. 251, 295–303 (1998). 3505–3513 (2006). 276, 47100–47106 (2001). 37. Garcia-Ruiz, C. et al. Defective TNF‑α‑mediated 53. Jin, Z. & El Deiry, W. S. Distinct signaling pathways in 69. Kataoka, T. et al. The caspase‑8 inhibitor FLIP hepatocellular apoptosis and liver damage in acidic TRAIL- versus tumor necrosis factor-induced promotes activation of NF‑κB and Erk signaling sphingomyelinase knockout mice. J. Clin. Invest. 111, apoptosis. Mol. Cell. Biol. 26, 8136–8148 (2006). pathways. Curr. Biol. 10, 640–648 (2000). 197–208 (2003). 54. Kelliher, M. A. et al. The death domain kinase RIP 70. Golks, A., Brenner, D., Krammer, P. H. & Lavrik, I. N. 38. Heinrich, M. et al. Cathepsin D links TNF-induced acid mediates the TNF-induced NF‑κB signal. Immunity 8, The c‑FLIP‑NH2 terminus (p22-FLIP) induces sphingomyelinase to Bid-mediated caspase‑9 and ‑3 297–303 (1998). NF‑κB activation. J. Exp. Med. 203, 1295–1305 activation. Cell Death. Differ. 11, 550–563 (2004). 55. Devin, A. et al. The distinct roles of TRAF2 and RIP in (2006). 39. Cifone, M. G. et al. Apoptotic signaling through CD95 IKK activation by TNF‑R1: TRAF2 recruits IKK to 71. Smotrys, J. E. & Linder, M. E. Palmitoylation of (Fas/Apo‑1) activates an acidic sphingomyelinase. TNF‑R1 while RIP mediates IKK activation. Immunity intracellular signaling proteins: regulation and J. Exp. Med. 180, 1547–1552 (1994). 12, 419–429 (2000). function. Annu. Rev. Biochem. 73, 559–587 40. Herr, I., Wilhelm, D., Bohler, T., Angel, P. & Debatin, 56. Liao, W. et al. CARP‑2 is an endosome-associated (2004). K. M. Activation of CD95 (APO‑1/Fas) signaling by ubiquitin protein ligase for RIP and regulates TNF- 72. Austin, C. D. et al. Death-receptor activation halts ceramide mediates cancer therapy-induced apoptosis. induced NF‑B activation. Current Biology 18, clathrin-dependent endocytosis. Proc. Natl Acad. Sci. EMBO J. 16, 6200–6208 (1997). 641–649 (2008). USA. 103, 10283–10288 (2006). 41. De Maria, R., Rippo, M. R., Schuchman, E. H. & Testi, R. 57. Li-Weber, M. & Krammer, P. H. Function and regulation 73. Kohlhaas, S. L., Craxton, A., Sun, X. M., Pinkoski, Acidic sphingomyelinase (ASM) is necessary for of the CD95 (APO‑1/Fas) ligand in the immune M. J. & Cohen, G. M. Receptor-mediated endocytosis Fas-induced GD3 ganglioside accumulation and system. Semin. Immunol. 15, 145–157 (2003). is not required for TRAIL-induced apoptosis. J. Biol. efficient apoptosis of lymphoid cells. J. Exp. Med. 58. Peter, M. E. et al. The CD95 receptor: apoptosis Chem. 282, 12831–12841 (2007). 187, 897–902 (1998). revisited. Cell 129, 447–450 (2007). 42. Brenner, B. et al. Fas/CD95/Apo‑I activates the acidic 59. Algeciras-Schimnich, A. et al. Molecular ordering of Acknowledgements sphingomyelinase via caspases. Cell Death. Differ. 5, the initial signaling events of CD95. Mol. Cell. Biol. Our work was supported by grants from the Deutsche 29–37 (1998). 22, 207–220 (2002). Forschungsgemeinschaft (DFG) SCHU 733/7‑1, SCHU733/8‑1, 43. Dumitru, C. A. & Gulbins, E. TRAIL activates acid 60. Feig, C., Tchikov, V., Schütze, S. & Peter, M. E. SCHU733/9-1and SFB 415, project A11 given to S.S. and sphingomyelinase via a redox mechanism and releases Palmitoylation of CD95 facilitates formation of SDS- FOR 876, TP1 given to W.S.‑B. ceramide to trigger apoptosis. Oncogene. 25, stable receptor aggregates that initiate apoptosis 5612–5625 (2006). signaling. EMBO J. 26, 221–231 (2007). 44. Thon, L., Mathieu, S., Kabelitz, D. & Adam, D. 61. Siegel, R. M. et al. SPOTS: signaling protein DATABASES The murine TRAIL receptor signals caspase- oligomeric transduction structures are early mediators Interpro: http://www.ebi.ac.uk/interpro independent cell death through ceramide. Exp. Cell of death receptor-induced apoptosis at the plasma SH2 Res. 312, 3808–3821 (2006). membrane. J. Cell Biol. 167, 735–744 (2004). UniProtKB: http://ca.expasy.org/sprot 45. Woo, C. H. et al. Inhibition of receptor internalization 62. Lee, K. H. et al. The role of receptor internalization AKT | BID | CARP2 | caspase‑8 | CD95 | CTSD | DR3 | DR6 | attenuates the TNFα-induced ROS generation in non- in CD95 signaling. EMBO J. 24, 1009–1023 (2006). EEA1 | ezrin | FADD | FAN | IKK1 | IKK2 | JNK | NEMO | p75NTR | phagocytic cells. Biochem. Biophys. Res. Commun. 63. Eramo, A. et al. CD95 death-inducing signaling RIP | TNFR1 | TNFR2 | TRADD | TRAF2 | TRAIL R1 | TRAIL R2 351, 972–978 (2006). complex formation and internalization occur in lipid 46. Heinrich, M. et al. Cathepsin D targeted by acid rafts of type I and type II cells. Eur. J. Immunol. 34, FURTHER INFORMATION sphingomyelinase-derived ceramide. EMBO J. 18, 1930–1940 (2004). Stefan Schütze’s homepage: 5252–5263 (1999). 64. Chakrabandhu, K. et al. Palmitoylation is required for http://www.immunologie-kiel.uk-sh.de 47. Chen, G. & Goeddel, D. V. TNF‑R1 signaling: a efficient Fas cell death signaling. EMBO J. 26, All links are active in the online pdf beautiful pathway. Science 296, 1634–1635 (2002). 209–220 (2007).