Scott J. Justus Adrian T. Ting

Cloaked in ubiquitin, a killer hides in plain sight: the molecular regulation of RIPK1

Authors’ addresses Scott J. Justus1,2, Adrian T. Ting1 1 Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute and Tisch Cancer Institute, New York, NY, USA. 2 Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Summary: In the past decade, studies have shown how instrumental programmed cell death (PCD) can be in innate and adaptive immune responses. PCD can be a means to maintain homeostasis, prevent or promote microbial pathogenesis, and drive autoimmune disease and inflammation. The molecular machinery regulating these cell death programs has been examined in detail, although there is still much to be explored. A master regulator of programmed cell death and innate immunity is receptor-interacting protein kinase 1 (RIPK1), which has been implicated in orchestrating various pathologies via the induction of apoptosis, necroptosis, and nuclear factor-jB-driven inflammation. These and other roles for RIPK1 have been reviewed elsewhere. In a reflection of the ability of tumor necrosis factor (TNF) to induce either survival or death response, this molecule in the TNF pathway can transduce either a survival or a death signal. The intrinsic killing capacity of RIPK1 is usually kept in check by the chains of ubiquitin, enabling it to serve in a prosurvival capacity. In this review, the intricate regulatory mechanisms responsible for restraining RIPK1 from killing are discussed primarily in the context of the TNF signaling pathway and how, when these mechanisms are disrupted, RIPK1 is free to unveil its program of cellular demise.

Correspondence to: Adrian T. Ting Immunology Institute, Icahn School of Medicine at Mount Sinai One Gustave L. Levy Place, Box 1630 New York, NY 10029, USA Tel.: +1 212 659 9410 e-mail: [email protected] Acknowledgements We thank Diana Legarda for critically reading the manuscript and Courtney McKenna for the illustrations. This study was supported by National Institutes of Health (NIH) grants AI052417, AI104521 and DK072201 (A. T. T.). A. T. T is a recipient of a Senior Research Award from the Crohn’s and Colitis Foundation. S. J. J. is supported in part by a Public Health Service Institutional Research Training Award (AI07647) and a Helmsley Trust fellowship. The authors have no conflicts of interest to declare.

This article is part of a series of reviews covering Ubiquitination in the Immune System appearing in Volume 266 of Immunological Reviews.

Immunological Reviews 2015 Vol. 266: 145–160

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Keywords: RIPK1, RIPK3, Caspase-8, CYLD, apoptosis, necroptosis, ubiquitin

The humble beginnings of RIPK1: initial insight into a killer’s deadly nature Prior to the discovery of receptor-interacting protein kinase 1 (RIPK1), studies of extrinsic programmed cell death (PCD) pathways had been focused on secondary signaling mediators of two primary death-inducing cytokines, tumor necrosis factor (TNF) and Fas ligand (FasL) (1, 2). However, little had been known in regard to how TNF, signaling through TNF receptor 1 (TNFR1)/TNFR2 (3, 4), or FasL, signaling through Fas (5–7), were able to transduce these death signals to initiate PCD. Due to the similar apoptotic morphology observed in both cell death programs, it was thought that these death receptors shared a similar signaling pathway to initiate apoptosis. In 1995, multiple groups identified three-related molecules that share homology with

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

145

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

the intracellular domains of TNFR1 and FAS: TNFR-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), and RIPK1 (8–11). This discovery indicated that while the distal biochemical mechanism utilized by the two pathways to execute death may be similar, the proximal signaling machinery of the two pathways were unique, as has been previously suggested (12). RIPK1 was discovered by Stanger et al. (9) via a yeast two-hybrid screen for proteins that interacted with the intracellular domain of human Fas. This interaction was shown to be mediated by the death domain shared by Fas and RIPK1 (9), which was also present in TRADD, FADD, and FAS. Previous studies implicated the death domain as being required for FAS and TNFR1 to induce apoptosis (13). Similarly, overexpression of RIPK1 induced apoptosis that was completely dependent on its death domain (9, 14). Soon after the initial suggestion of RIPK1’s role in FASinduced apoptosis, evidence began emerging that it had a role in TNF signaling. RIPK1 was found to be recruited to TNFR1 via TRADD (10, 14), and overexpression of RIPK1 induced nuclear factor (NF)-jB in a manner dependent on its intermediate domain (14). Genetic evidence demonstrating the essential requirement of RIPK1 for activation of NFjB downstream of TNFR1 came initially from Ting et al. (15) with the isolation of a RIPK1-deficient Jurkat T-cell mutant and subsequently from Kelliher et al. (16) with the generation of Ripk1 / mice. Reconstitution of RIPK1-deficient cells with various mutants of RIPK1 as well as overexpression of different RIPK1 mutants in 293 cells led to the surprisingly finding that the kinase activity of RIPK1 was dispensable for inducing NF-jB (14, 15). This was later confirmed by Lee et al. (17) who showed that TNF stimulation of Rip1 / mouse embryo fibroblasts (MEFs) overexpressing the kinase-dead point mutant of RIPK1 was still able induce IKK activity. The studies by Ting et al. (15) and Kelliher et al. (16) both demonstrated that RIPK1 was dispensable for Fas-induced apoptosis, consistent with the idea that the proximal signaling mechanisms of TNFR1 and FAS are different. Around the same time, different laboratories demonstrated that NF-jB was required to prevent TNF from inducing apoptosis via the induction of prosurvival genes (18–21). As the Ripk1 / cells were more susceptible to TNF-induced apoptosis, which correlated with a loss of NF-jB signaling (16, 22), this led to the conclusion that RIPK1 was required for TNF-mediated NF-jB and cell survival but not for TNFinduced apoptosis. However, this conclusion did not fit with a subsequent study showing that Jurkat T cells expressing

146

both TNFR1 and TNFR2, but not cells expressing only TNFR1, died by apoptosis in a RIPK1-dependent manner when treated with TNF (23). These disparate findings led to an air of contradiction around RIPK1: it can prevent cell death by activating NF-jB, yet it can also induce cell death in some contexts. Since sufficient evidence did not exist to implicate RIPK1 as a killer, focus shifted to RIPK1’s role in cell survival. RIPK1 as an inducer of NF-jB: a killer in disguise FADD was discovered as another FAS-interacting molecule (8, 11). Unlike the contradictory nature of RIPK1, FADD, and soon thereafter its enzymatic partner Caspase-8, was shown to be required for FAS and TNFR1 to induce apoptosis (24–29). In cells undergoing apoptosis, RIPK1 is proteolytically cleaved by Caspase-8 (30, 31). This cleavage of RIPK1 was shown to inhibit its ability to induce NF-jBdependent prosurvival genes, contributing further to the demise of the cell. These latest reports positioned RIPK1 as being a downstream substrate of Caspase-8, further strengthening the argument that RIPK1 has a prosurvival rather than a pro-apoptotic role. Along with RIPK1, other signaling molecules had been implicated as being recruited to TNFR1 to mediate NFjB, including TRADD (10, 14, 24), TNF receptor-associated factor 2 (TRAF2), and the cellular inhibitor of apoptosis 1 (cIAP1) (32). A major advancement in the understanding of the TNFR1 pathway came from Micheau and Tschopp (33), who described two sequential signaling complexes that regulated TNF-induced NF-jB and apoptosis. By utilizing gel filtration experiments, the authors found that stimulation of cells with TNF led to an initial complex (Complex I) being formed at TNFR1 that included TRADD, RIPK1, TRAF2, and cIAP1, but not the death effector molecules Caspase-8 and FADD. Complex I was shown to be responsible for the activation of NF-jB and induction of cellular FLICE-like inhibitory protein (cFLIP), an essential NF-jB-induced survival factor that functions by inhibiting the interaction of FADD and Caspase-8 (33–36). Upon blockade of NF-jB, TRADD, and RIPK1 translocate from TNFR1 to interact with FADD and Caspase-8 to form an apoptosis-inducing complex (Complex II) (33). Micheau and Tschopp’s findings (33) laid the foundation for future studies as efforts could now be focused to see how Complex I and Complex II were individually regulated. In particular, this would allow researchers to observe what was keeping a killer at bay: ubiquitin chains. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

The ubiquitin cloak distracted the revelation of RIPK1 as a killer As various groups continued to focus on elucidating the mechanism of formation and regulation of Complex I and its ability to activate NF-jB, it became apparent that this TNFinduced signaling pathway was intricate and involved multiple signaling mediators. Upon stimulation of the cells with TNF, it was shown that TRADD and RIPK1 were recruited to the receptor along with the E3 ligases TRAF2 and cIAP1 (14, 32, 33). In addition, multiple groups demonstrated that TNF stimulation induced the ubiquitination of RIPK1 with nondegradative, K63-linked polyubiquitin chains at the receptor, and this was shown to involve TRAF2 (17, 37–41). However, it would be shown later that polyubiquitination of RIPK1 was not restricted to K63-linked ubiquitin (42, 43). Ubiquitination of RIPK1 facilitated the recruitment of transforming growth factor-b activated kinase 1 (TAK1) and TAK1/ MAP3K7 binding protein 2/3 (TAB2/TAB3) through their conserved zinc finger domains as well as the recruitment of NF-jB essential modulator (NEMO) and the inhibitor of NFjB (IjB) kinase (IKK) complex (17, 37, 44, 45). These studies, among others, implicated the importance of ubiquitination in NF-jB signaling (46). However, definitive evidence that polyubiquitination of RIPK1 was required for NF-jB signaling did not come along until two groups discovered the lysine where RIPK1 is ubiquitinated, K377 (47, 48). Both Ea et al. (47) and Li et al. (48) showed that the point mutant RIPK1-K377R failed to undergo ubiquitination when cells were stimulated with TNF, and RIPK1-deficient cells reconstituted with the point mutant had impaired NF-jB signaling. The increased susceptibility of RIPK1-K377R cells to TNFinduced apoptosis was initially explained by the fact that loss of the polyubiquitin chains of RIPK1 prevented the recruitment of TAK1/TAB2/3 and the NEMO/IKKb kinase complexes to the receptor, an interaction thought to be necessary for I-jBa phosphorylation and NF-jB activation (47, 48). These studies solidified the concept that the ubiquitin chains of RIPK1 were important for NF-jB signaling and thus cell survival. Whether or not these chains had a function independent of NF-jB remained to be explored and the revelation of RIPK1 as a killer was yet to come. Caught red-handed with the smoking gun, RIPK1 was shown to be a killer when the ubiquitin cloak was removed The protective effects of NF-jB signaling had been shown to be dependent on transcription and translation of important cell survival factors such as cFLIP, as pretreatment of © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

cells with actinomycin D or cycloheximide sensitized cells to TNF-induced apoptosis (35, 36, 49). However, these findings also implied that the cell death machinery was already in place and did not require further protein synthesis. This raised an important question: how do TNF-treated cells keep the death machinery at bay during early time points, before NF-jB–induced cell survival genes are upregulated? Early insight into a possible mechanism came from Lee et al. (50) and Yeh et al. (51), who showed that TRAF2 played an important anti-apoptotic role independently of NF-jB signaling. Yeh et al. showed that Traf2 / mice exhibited postnatal lethality (51). They found that different TRAF2-deficient cell types were susceptible to TNF-induced cell death, and this prosurvival role of TRAF2 was independent of NF-jB as Traf2 / embryonic fibroblasts were more sensitive to TNF and cycloheximide treatment compared with Traf2+/+ cells (51). To investigate the role of TRAF2 in lymphocytes, Lee et al. generated mice expressing a TRAF2 dominant negative (lacking RING domain/E3 ligase activity) in B cells and T cells (50). They found that thymocytes expressing the TRAF2-DN were more susceptible to TNF and cycloheximide, consistent with the findings of Yeh et al. (51), and further indicating that TRAF2’s protective role was independent of NF-jB-induced gene expression (50). These observations on TRAF2 would remain unexplained for a decade until O’Donnell et al. (52) observed a surprising result when they examined the sensitivity of cells expressing RIPK1-K377R, the ubiquitin acceptor site mutant, to TNF-induced apoptosis. RIPK1-deficient Jurkat T cells that had been complemented with RIPK1-K377R, which had some residual NF-jB activity, were significantly more sensitive to apoptosis than those complemented with a control vector, which had almost no NF-jB activity. The fact that the RIPK1-K377R-complemented cells were dying more, despite their higher level of NF-jB, argued against the NF-jB defect as the reason for their enhanced apoptotic phenotype. To confirm this, RIPK1-deficient Jurkat cells reconstituted with RIPK1-WT or RIPK1-K377R were both then stably transfected with the IjBa super-repressor (IjBaSR) to negate the role of NF-jB. Under this NF-jBnull condition, the RIPK1-K377R complemented cells, where RIPK1 could not be ubiquitinated, were more sensitive to TNF-induced apoptosis. Loss of RIPK1’s protective polyubiquitin chains caused RIPK1 to associate with Caspase-8 to trigger apoptosis (52). This observation provided the smoking gun that revealed how RIPK1 came to be an inducer of apoptosis.

147

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

These and subsequent findings led to us to propose that there are two sequential cell death checkpoints upon activation of the TNFR1 pathway (53). The early transcription-independent checkpoint occurs when RIPK1 is rapidly modified by K63-linked polyubiquitin chains, enabling its association with NEMO and other partners in Complex I (Fig. 1). The initial function of this association is to prevent RIPK1 from interacting with Caspase-8 to initiate apoptosis. The second checkpoint occurs with the induction of NF-jBdependent prosurvival genes, which genetically programs the cell for long-lasting survival (Fig. 1). However, when the first checkpoint (i.e., ubiquitination) is disrupted, RIPK1 actively engages the apoptotic machinery (Fig. 2A). Disruption of the second checkpoint also leads to apoptosis but in this instance, RIPK1 is not required as it is likely held in check by ubiquitin and cleaved by Caspase-8 (30, 31, 33) (Fig. 2B). In this context, TRADD likely transduces the apoptotic signal from the receptor to the apoptotic FADD/Caspase-8 complex (54–56). Since most experimental systems up to this point in time sensitized cells to TNF-induced RIPK1-Dependent Survival

TNF TNFR1

Plasma ne Membra

TRAF2 TRADD

RIPK3

cIAP1 cIAP2 RIPK1

cFLIP FADD Caspase-8

Checkpoint 1

CYLD

RIPK1 FADD

TAB2/3 TAK1

CYLD

CYLD

NEMO

Caspase-8

RIPK1 Checkpoint 2

Necroptosis

NF-KB

Apoptosis

Fig. 1. Regulation of tumor necrosis factor receptor 1 (TNFR1) cell death checkpoints. Upon TNF ligand binding to TNFR1, most cells survive as at least two cell death checkpoints are in place. Checkpoint 1 occurs with the recruitment of multiple signaling molecules to the receptor resulting in the conjugation of non-degradative ubiquitin chains to receptor-interacting protein kinase 1 (RIPK1). This early checkpoint does not require de novo protein synthesis, but is dependent on TAB2/3/TAK1 and NEMO binding to the ubiquitin chains on RIPK1. Maintenance of Checkpoint 1 requires the proteolytic cleavage of the CYLD deubiquitinase by a prosurvival Caspase-8/cFLIP/FADD complex. Loss of CYLD sustains ubiquitination of RIPK1, further restricting RIPK1’s ability to initiate cell death. Checkpoint 2 occurs downstream when the initial ubiquitin-dependent complex leads to the induction of IKK and phosphorylation of I-jBa to initiate NF-jB signaling. Subsequent NF-jB-dependent expression of cFLIP and other prosurvival genes generates a long-lasting survival program. Disruption of either checkpoint leads to cell death.

148

apoptosis by using cycloheximide or NF-jB blockade to disrupt Checkpoint 2, the role of RIPK1 as an apoptosissignaling molecule in the TNFR1 pathway had been overlooked. These findings provided a much needed explanation for the contradictory nature of RIPK1 that has been previously described: depending on its ubiquitination status, RIPK1 can be prosurvival or prodeath. Since TRAF2 is required for the ubiquitination of RIPK1 (17, 57), these findings also provided a mechanistic explanation for the earlier observations by Lee et al. and Yeh et al. (50, 51). Indeed, apoptosis conferred by expression of TRAF2-DN is dependent on RIPK1 (52). SMAC-king a killer around can be deadly and when unchained, RIPK1 is armed and dangerous Further evidence of the role of the ubiquitination of RIPK1 preventing cell death came from studies of chemotherapeutic agents termed SMAC mimetics (SM). SM were originally designed to mimic endogenous SMAC/DIABLO by targeting XIAP and the cIAPs, relieving them of their caspase-suppressing duty to induce or potentiate apoptosis in certain cancer cells (58–63). As SMAC is released from the mitochondria upon initiation of apoptosis, SM-induced death was thought to be dependent on Caspase-3 and -9 (59). However, it was found by three different groups that SMinduced death was caused by the autoubiquitination and degradation of the cIAPs, autocrine TNF production, and the activation of Caspase-8 (64–66). In addition, it was shown that this death was dependent on RIPK1 (64). This led to the question of whether or not the ubiquitination of RIPK1 repressed cell death in cancer cells, and if treating these cells with SM led to deubiquination of RIPK1 and cell death. As reports emerged that cIAPs were not able to directly inhibit caspase activity (67), it was possible that the prosurvival role of the cIAPs was to function as E3 ligases for RIPK1. This was indeed found to be the case as cIAP1 and cIAP2, in combination with TRAF2, were shown to act as E3 ligases for RIPK1 (17, 57, 68–70). Furthermore, cells doubly deficient in cIAP1 and cIAP2, or treated with SM, exhibited loss of RIPK1 ubiquitination and sensitivity to apoptosis induced by autocrine TNF production (68, 69, 71). These findings provided damning evidence of the true nature of RIPK1: that despite having a prosurvival role in TNF signaling, underneath the cloak of RIPK1’s ubiquitin chains is the heart of a killer. In contrast to the observation that the kinase domain is not required for the role of RIPK1 in NF-jB signaling (14, 15, 17), the early studies implicating RIPK1 as an apoptosis-sig© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

A RIPK1-Dependent Apoptosis

TNF Plasmarane Memb

TNFR1

Plasmarane Memb

TRAF2 TRADD

cIAP1 cIAP2

RIPK1

B RIPK1-Independent Apoptosis

TNF

TRAF2

CYLD RIPK1

TNFR1

TRADD FADD

cIAP1 cIAP2 RIPK1

SMAC Mimetics

TRADD

FADD

RIPK1

TAB2/3

TAB2/3 Caspase-8

TAK1 NEMO

TAK1 NEMO

Apoptosis

NF-KB

e id im x e R oh S cl IKBα y C

Caspase-8

Apoptosis

Fig. 2. Depending on which cell death checkpoint is disrupted, it can lead to receptor-interacting protein kinase 1 (RIPK1)-dependent or -independent apoptosis. (A) Disruption of Checkpoint 1 leads to RIPK1-dependent apoptosis. For instance, this can occur if ubiquitination of RIPK1 is blocked by treatment with SMAC mimetics to degrade the E3 ligases cIAP1/2. Loss of ubiquitin on RIPK1 switches RIPK1 to become a death-signaling molecule. Additional removal of ubiquitin chains from RIPK1 by CYLD is also required and ubiquitin-free RIPK1 is then able to interact with FADD/Caspase-8 to initiate apoptosis. This form of apoptosis is due to an active switching on of the apoptotic machinery by RIPK1 and it occurs rapidly. (B) Disruption of Checkpoint 2 also leads to apoptosis but via a different mechanism. This can occur with blockade of NFjB-dependent gene expression, which can be achieved by the use of cycloheximide or by expressing the IjBa super-repressor (IjBaSR). As Checkpoint 1 is maintained under these conditions, RIPK1 is not able to activate Caspase-8 to induce apoptosis. Instead, the turnover of cFLIP and the failure to replenish it due to NF-jB blockade will cause Caspase-8 to undergo autoprocessing and initiation of apoptosis. This form of apoptosis results from the loss of an apoptosis-inhibitory molecule and therefore happens more slowly. It does not require RIPK1, but instead there is evidence to suggest that RIPK1 may dampen this apoptotic response.

naling molecule had shown that its ability to induce apoptosis was at least partially dependent on its kinase domain (9, 23). The kinase activity of RIPK1 was also essential for apoptosis of Panc-1 and other tumor cell lines induced by TNF and SM (71). In this context, apoptosis required the deubiquitinase CYLINDROMATOSIS (CYLD) (71, 72), which deubiquitinates RIPK1 allowing it to function as an upstream activator of Caspase-8 (Fig. 2A). As a result RIPK1 is able to initiate apoptosis before Caspase-8 can cleave and inactivate RIPK1 and CYLD (30, 31, 71, 73, 74). This cell death complex that formed upon loss of the first cell death checkpoint, such as by treating cells with SM, was termed the ‘Ripoptosome’, and the Ripoptosome was defined as a complex consisting of RIPK1, FADD, and Caspase-8 that is regulated by the different cFLIP isoforms (75, 76). Sometimes a killer will not go quietly: the role for RIPK1 in programmed necrosis While many studies were being performed looking at RIPK1’s role in TNF-induced NF-jB and apoptosis, light was starting to be shed on another role of RIPK1: that of regulator of an alternative form of PCD known as programmed necrosis. The earliest studies of this pathway indicated that programmed necrosis was a cell death pathway © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

that was dependent on RIPK1 and reactive oxygen species (ROS) and negatively regulated by caspases (77–81). Holler et al. (77) initially reported the involvement of RIPK1 in programmed necrosis. They showed that Jurkat cells either pretreated with the pan-caspase inhibitor zVAD or genetically deficient in Caspase-8 were susceptible to FasL-induced death that was dependent on RIPK1’s kinase activity. Their data also implicated Caspase-8 as a prominent negative regulator of this caspase-independent death pathway (77). Preliminary evidence of the in vivo relevance of this pathway came from two studies. First, with the knowledge that TNF is able to induce caspase-dependent apoptosis, Cauwels et al. (82) set out to test whether or not caspase inhibition would protect mice from TNF-induced shock. Surprisingly, treatment with zVAD sensitized the mice to TNF-induced shock. It was shown that this sensitization was dependent on ROS, consistent with the involvement of programmed necrosis (82). A second study came from Chan et al. (73) who were studying the role of TNFR2 in the context of programmed necrosis. Previous studies by Chan and Lenardo (83–86) established that stable overexpression of TNFR2 could potentiate TNF-induced apoptosis in Jurkat cells, possibly through the recruitment and degradation of TRAF2, similar to that described by Pimentel-Mui~ nos and Seed (23).

149

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

Similar to their studies of apoptosis, Chan et al. demonstrated that Caspase-8 or FADD-deficient Jurkat cells transfected with TNFR2 were sensitized to RIPK1-dependent programmed necrosis when the cells were prestimulated through TNFR2. They also showed that the in vivo pathogenesis of vaccinia virus, which encodes the caspase inhibitor B13R/SPI-2, was greatly restricted in Tnfr2 / mice (73). Taken together, these studies provided preliminary evidence that RIPK1-dependent programmed necrosis was relevant in vivo and that it was negatively regulated by FADD and Caspase-8. These findings would set the stage for subsequent studies to examine the role of ubiquitin in the regulation of programmed necrosis. As TRAF2 was later shown to function in the E3 ligase complex that ubiquitinates RIPK1 (57), TNFR2 was likely sensitizing cells to RIPK1-dependent death by degrading TRAF2 and unmasking RIPK1. In other words, TNFR2 may be an external cue that can disrupt Checkpoint 1 of TNFR1, leading to RIPK1-dependent cell death. Breaking bad: the ringleader CYLD directs RIPK1 and RIPK3 down the murderous path of necroptosis In 2008, two studies from Yuan and colleagues (87, 88) put programmed necrosis (now termed necroptosis) back in the spotlight. Degterev et al. (88, 89) had discovered that the necroptosis inhibitor necrostatin-1 was a specific, allosteric inhibitor of RIPK1’s kinase activity. This was a significant finding, A RIPK1-Dependent Necroptosis When Apoptotic Caspase-8 is Inhibited

TNF Plasmarane Memb

TNFR1

as it would allow future studies to investigate the role of the kinase activity of RIPK1 in PCD both in vitro and in vivo. Hitomi et al. (87) sought to determine the molecular signaling network that regulated necroptosis. By using a genome-wide siRNA screen, the authors found 432 genes that regulated necroptosis, with one of the essential genes being the RIPK1 deubiquitinase CYLD (72, 87). Together with the data from Wang et al. (71) implicating CYLD in RIPK1-dependent apoptosis, these findings fit the model that disruption of Checkpoint 1 via the deubiquitination of RIPK1 was critical for initiating either apoptosis (Fig. 2A) or necroptosis (Fig. 3). Although more information was now known about the downstream events of necroptosis, very little was known about how the necroptotic signal was transduced downstream of RIPK1 (90–92). However, in 2009 three different groups found the missing link: RIPK3 (93–95). RIPK3 was found to be expressed in select cell types including hematopoietic cells and fibroblasts, implicating RIPK3 expression as a determining factor for whether or not different cell types could undergo necroptosis (94). All three groups found that, like RIPK1, the kinase activity of RIPK3 was required for necroptosis. A series of biochemical experiments showed that RIPK1 and RIPK3 interacted through their RIP homotypic interaction motif (RHIM) to form an amyloid signaling complex that was stabilized by the kinase activity of RIPK1 (93, 96, 97). However, Cho et al (93) found that B RIPK1-Dependent Necroptosis When Survival Caspase-8 is Inhibited

TNF Plasmarane Memb

TRAF2

TRAF2 TRADD

cIAP1 cIAP2

Caspase-8

zVAD + SMAC Mimetics

RIPK1

RIPK3 NEMO

TRADD

CYLD RIPK1

Necroptosis

cFLIP FADD Caspase-8

cIAP1 cIAP2

RIPK1

TAB2/3 TAK1

TNFR1

zVAD

TAB2/3

RIPK3

TAK1 NEMO

CYLD RIPK1

Necroptosis

Fig. 3. Disruption of Checkpoint 1 can also lead to necroptosis. (A) The dominant response to TNF when receptor-interacting protein kinase 1’s (RIPK1’s) ubiquitination in Checkpoint 1 is disrupted by SMAC mimetics is apoptosis. Apoptotic Caspase-8 cleaves RIPK1, which further promotes apoptosis in a positive feedback manner. Cleavage of RIPK1 and CYLD also serves to suppress necroptosis. If the cells are also treated with a caspase inhibitor such as zVAD-fmk, this prevents Caspase-8-mediated cleavage of RIPK1 and CYLD. Retention of these molecules allows CYLD to further deubiquitinate RIPK1, enabling the formation of the RIPK1-RIPK3 necrosome to initiate necroptosis. (B) TNFR1 ligation also induces a prosurvival Caspase-8 that disables CYLD, thereby maintaining RIPK1 ubiquitination and Checkpoint 1. Disruption of Checkpoint 1 can also be achieved by blocking the prosurvival Caspase-8 from cleaving CYLD by using zVAD-fmk. Retention of CYLD then allows it to deubiquitinate RIPK1, thus switching on the death-signaling capability of RIPK1. Due to the presence of zVAD-fmk, the only death effector available to RIPK1 is RIPK3 and thus in this case, necroptosis is the only option.

150

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

RIPK1 could not directly phosphorylate RIPK3 and that RIPK3 only weakly phosphorylated RIPK1. They went on to show that RIPK3 was required for RIPK1 phosphorylation, although this was not blocked with the RIPK1 kinase inhibitor necrostatin-1 (93). These data implied that RIPK1 and RIPK3 likely phosphorylated other signaling intermediates to initiate necroptosis. Zhang et al. (95) showed that RIPK3 was required for downstream ROS production, and this was potentially dependent on the interaction of RIPK3 with different metabolic enzymes. In vivo, it was shown that Ripk3 / mice were protected from cerulein-induced acute pancreatitis as well as vaccinia virus-induced pathology (93, 94). These results firmly established the role of the RIPK1 and RIPK3 ‘necrosome’ in mediating necroptosis and that necroptosis played a role in vivo by mediating inflammation and functioning as an antiviral response (98). In 2012, MLKL and PGAM5 were implicated as necroptosis signaling molecules that functioned downstream of the necrosome (99–101). RIPK3 was shown to phosphorylate MLKL, allowing MLKL to oligomerize and localize to the plasma membrane. At the plasma membrane, oligomeric MLKL either interacts with ion channels to induce an influx of sodium and/or calcium or they can directly form pores, leading to the eventual rupture of the plasma membrane (102–105). Although MLKL has been found to be essential for necroptosis triggered by many different stimuli (106, 107), the involvement of PGAM5 in necroptosis appears to be more context dependent (99, 107, 108). Caspase-8, cFLIP, and FADD intercede to suppress necroptosis in vivo Early studies demonstrated the important role of apoptosis in cell turnover and homeostasis as mice lacking Fas or FasL develop a lymphoproliferative disorder and autoimmunity (2). Fas/FasL-induced apoptosis is dependent on FADD and Caspase-8, however, neither Caspase-8 / nor Fadd / mice phenocopy the Fas/FasL-deficient mice (27–29). Instead, the mice die prenatally at day E10.5 due to excessive cell death in the yolk sac (109). This established that although FADD and Caspase-8 have essential roles in apoptosis, both proteins were required to perform a non-apoptotic role during development. Kang et al. (110) further investigated this non-apoptotic role of Caspase-8 by conditionally knocking out Caspase-8 in different tissues. They found that deletion of Caspase-8 in the endothelial cells resulted in embryonic lethality, similar to the Caspase-8 / or Fadd / mice, and deletion of Caspase-8 in bone mar© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

row cells and MCSF-derived macrophages caused a decrease in these cell types via a non-apoptotic form of cell death (110). For years, this non-apoptotic, developmental role of Caspase-8 remained elusive. Reports indicated that this nonapoptotic role of Caspase-8 was important for T-cell maintenance (111, 112) and skin homeostasis (113, 114), and that this non-apoptotic role of Caspase-8 did not depend on its ability to self-process, an event that is essential for apoptosis (115). However, it was not until 2011 that the mystery was solved and Caspase-8 and FADD were confirmed as functioning as in vivo repressors of necroptosis. Three reports were published implicating Caspase-8 and FADD in repressing RIPK1/RIPK3-dependent necroptosis (116–118). Kaiser et al. (118) and Oberst et al. (116) showed that crossing Caspase-8 / mice with Ripk3 / mice rescued the prenatal lethality of the Caspase-8 / mice. These mice exhibited normal development and Caspase-8 / Rip3 / cells were shown to be resistant to both apoptosis and necroptosis. However, these mice eventually exhibited a lymphoproliferative disorder due to an increase in B220+ CD4 CD8 T cells, similar to the Fas-deficient mice (116, 118, 119). Since cFLIP-deficient animals phenocopy the Fadd / and Caspase-8 / mice and die at day E10.5, this indicates that cFLIP also participates in the prosurvival role of Caspase-8 (120). Further work by Green and colleagues showed that cFLIP was indeed required for the prosurvival role of FADD and Caspase-8 in suppressing necroptosis (116, 121), possibly by altering substrate specificity of Caspase-8 (122). They also showed that the prosurvival roles of cFLIP and Caspase-8 were required both in development as well as adult homeostasis (123). Finally, it had been shown by Zhang et al. (117) that lethality observed in Fadd / mice could be rescued by crossing the mice to Ripk1 / mice; however, these mice died soon after birth. An important question still remained, however: what substrate of Caspase-8 is cleaved to prevent necroptosis from occurring? Caspase-8 curtails the murderous rampage of RIPK1 by proteolytic inactivation of the ringleader CYLD Necroptosis was originally thought to function as a backup form of cell death in the context of apoptosis when caspases were compromised (77, 78). This can be demonstrated by treating RIPK3-expressing cells with TNF and cycloheximide to induce apoptosis, and treating the same cells with TNF, cycloheximide, and zVAD to induce necroptosis (94, 124). However, it has become clear that necroptosis can occur independently of apoptosis in some cell types as cells stimu-

151

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

lated with TNF can switch from survival (TNF stimulation alone) to death (TNF + zVAD) by only introducing caspase inhibitors (95, 116) (Fig. 3B). As Pop et al. (122) demonstrated, cFLIP likely functions by altering the substrate specificity of Caspase-8 during this non-apoptotic, prosurvival cleavage event. From this, it can be assumed that Caspase-8 may have both distinct and overlapping substrates during its roles in apoptosis and cell survival. During apoptosis, it has been shown that Caspase-8 is able to cleave RIPK1, RIPK3, and CYLD (30, 31, 73, 74, 125, 126); however, endogenous RIPK3 cleavage has not been observed to date. These studies demonstrated that the apoptosis-inducing Caspase-8 may suppress necroptosis by cleaving multiple substrates. However, this does not hold true under survival signaling conditions when Caspase-8 is under the regulation of cFLIP. O’Donnell et al. (74) showed that stimulation of MEFs with only TNF induced Caspase-8dependent CYLD cleavage when the cells were surviving and there was no detectable sign of apoptosis. In this context, no cleavage or change in the protein expression level of RIPK1 or RIPK3 was detected (74). Cleavage of CYLD led to the rapid degradation of the C-terminal fragment, which contains the enzymatic domain, resulting in loss of CYLD’s deubiquitinase activity. To test whether or not CYLD proteolysis by Caspase-8 prevented necroptosis, O’Donnell et al. reconstituted Cyld / MEFs with CYLD-D215A, a CYLD mutant that cannot be cleaved by Caspase-8. The CYLDD215A expressing cells were able to undergo necroptosis with TNF treatment alone to a similar extent as cells reconstituted with wildtype CYLD when treated with both TNF and zVAD (74). Thus, cleavage of CYLD by a prosurvival Caspase-8 was necessary and sufficient to suppress necroptosis. Removal of CYLD led to sustained ubiquitination of RIPK1, whereas when CYLD was stabilized it was able to deubiquitinate RIPK1 and initiate necroptosis (74). Thus during cell survival, unlike during apoptosis when Caspase-8 cleaves multiple substrates to suppress necroptosis, Caspase8 appears to cleave only CYLD. The removal of CYLD by Caspase-8 returns us to a familiar theme: this event maintains RIPK1 ubiquitination, i.e. the first cell death checkpoint, thereby preventing RIPK1 from becoming a killer. The chains that bind: whether RIPK1 goes rogue depends on who its friends are How does ubiquitination of RIPK1 function to inhibit its proclivity to be a killer? A simple model is that ubiquitination of RIPK1 causes it to associate with certain partners while precluding its interaction with other partners. During

152

NF-jB signaling, TNFR1 recruits RIPK1 and the E3 ligase machinery to ubiquitinate the former. This modification on RIPK1 is then thought to recruit two kinase complexes, TAB2/TAK1 and NEMO/IKKb, bringing TAK1 into close proximity to phosphorylate IKKb and activate downstream NF-jB signaling (47, 48). The recruitment of these factors to the ubiquitin chains of RIPK1 prevents RIPK1 from interacting with death-signaling molecules, and this may be how the early NF-jB-independent Checkpoint 1 functions. Consistent with this idea, NEMO has been shown to have an NF-jB-independent prosurvival function that opposes both apoptosis and necroptosis. NEMO-deficient Jurkat cells were found to be more sensitive to TNF-induced apoptosis than their NEMO-sufficient counterparts, with the IjBaSR being present in both lines to block NF-jB-mediated gene transcription (127). This prosurvival function of NEMO was shown to be dependent on the ubiquitin-binding UBAN domain, which is also needed for binding ubiquitinated RIPK1 (47, 127, 128). The failure of NEMO to bind ubiquitinated RIPK1 due to mutations in the UBAN domain, or the loss of NEMO entirely, allowed RIPK1 to interact with Caspase-8 to induce apoptosis, similar to that in RIPK1-K377R-expressing cells (52, 127). Similarly, NEMOdeficient Jurkat cells stably expressing IjBaSR were more sensitive to zVAD and TNF-induced necroptosis than NEMO-sufficient cells stably expressing IjBaSR (129). Furthermore, this survival function of NEMO was dependent on the ubiquitination of RIPK1 as NEMO was no longer able to protect cells from death if they were also treated with SM (129). Thus, the interaction of ubiquitinated RIPK1 with prosurvival binding partners could sequester it from deathsignaling molecules or sterically hinder RIPK1 from interacting with them. Another possibility, which is not mutually exclusive, is that the catalytic enzymes present in this first cell death checkpoint may be suppressing RIPK1. For instance, TAK1 has been shown to suppress TNF- and TRAIL-induced apoptosis independently of its role in NF-jB signaling (130, 131). Subsequent studies showed that TAK1 was important not only for suppressing apoptosis but also necroptosis (132, 133). Furthermore, NEMO forms a complex with IKKb, which may phosphorylate substrates that reinforce RIPK1 sequestration from death-signaling molecules or prevent RIPK1 from activating them. IKKb has been reported to phosphorylate the Bcl-2-associated death promoter (BAD) as a way to inhibit apoptosis, so there is a good possibility that other IKKb substrates exist (134). These observations together indicate that multiple regulatory mechanisms are © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

required to suppress cell death independently of NF-jB signaling, and that the underlying mechanism required for these events to unfold is the ubiquitination of RIPK1. What remains unclear is what substrates are phosphorylated by these kinases and how these phosphorylation events prevent RIPK1 from initiating death. Another form of ubiquitin chains known as linear or M1linked ubiquitin has been described, and this ubiquitination is mediated by the LUBAC E3 ligases consisting of HOIP, HOIL-1L, and SHARPIN, with mice deficient in SHARPIN displaying multi-organ inflammation that is primarily driven by TNF signaling (43, 135–138). Gerlach et al. (43) showed that both NEMO and RIPK1 were linearly ubiquitinated, and that SHARPIN-deficient MEFs stimulated with TNF were sensitive to FADD/Caspase-8-dependent apoptosis as well as necroptosis. This indicated that LUBAC/SHARPIN and linear ubiquitination functioned as an important prosurvival mechanism. This was confirmed in vivo when it was shown that the skin inflammation in the SHARPIN-deficient mice was reversed by the loss of FADD or Caspase-8, in combination with RIPK3, or the loss of RIPK1’s kinase activity (139–141). The results from these genetic crosses are consistent with SHARPIN/LUBAC functioning as part of the NFjB-independent Checkpoint 1 to suppress RIPK1-dependent death. Consistent with this hypothesis, the UBAN domain of NEMO that is necessary for its prosurvival function was subsequently shown to have a higher affinity for linear ubiquitin chains than for K63-linked chains (142, 143), suggesting that the recruitment of NEMO to linear ubiquitin chains may be essential for the NF-jB-independent cell death checkpoint. Finally, unanchored K63-linked ubiquitin chains have also been shown to activate TAK1 and IKKb by biochemical reconstitution in vitro (144). Whether these unanchored ubiquitin chains also participate in suppressing RIPK1-mediated cell death remains to be formally tested. Thus, multiple forms of ubiquitin chains are likely involved in this suppression and the challenge in the future is to decipher the role of each of these chains. RIPK1 is a killer who does not discriminate: the role of RIPK1 in TLR-induced, TRIF-dependent necroptosis While the majority of the studies on necroptosis were carried out in the TNF pathway, studies from Han and colleagues in the early 2000s (145, 146) showed that LPS, in the presence of caspase inhibitors, could also induce necroptosis in the RAW 264.7 macrophage cell line, similar to the death that was observed with TNF. In subsequent years, many reports were published trying to establish a mechanism for © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

LPS-induced necroptosis and how caspases were able to negatively regulate this process (147–151). However, it was not until 2011 that the underlying mechanism was made clear. He et al. (152) demonstrated that LPS, signaling through Toll-like receptor 4 (TLR4), and poly(I:C), signaling through TLR3, were able to induce TRIF-dependent necroptosis in murine bone marrow-derived macrophages (BMDMs) when caspases were inhibited. This death was found to be independent of TNF and was mediated by the RHIM domains of TRIF and RIPK3 and the kinase activity of RIPK1. However, as RIPK1 and RIPK3 are recruited to TRIF independently of necroptosis when macrophages are stimulated with LPS or poly(I:C), whether or not the necrosome was forming at TRIF or downstream remained unclear (152, 153). Further work by other groups has shown that in addition to macrophages, TLR-induced necroptosis occurs in fibroblasts, microglia, and intestinal epithelial cells (154–156). The requirement for caspase inhibitors to induce RIPK1dependent necroptosis in the TLR3/4 pathway suggests that the early NF-jB-independent cell death checkpoint that restrains RIPK1 in the TNF pathway may also be operational downstream of the TRIF-coupled TLRs. As Caspase-8 has been implicated in being a predominant negative regulator of TLR-induced necroptosis (155, 156), an important question needed to be addressed: what is the mechanism by which Caspase-8 is being activated downstream of the TLRs in this non-apoptotic, prosurvival context, and what is its substrate? An important observation was that TRIF is required for TLR-induced necroptosis and that necrosome formation only occurs in the context of Caspase-8 inhibition (152, 156), indicating that Caspase-8 must regulate TLRinduced necroptosis at the level of TRIF necrosome formation or further upstream. Since TRIF has previously been implicated in interacting with Caspase-8 (157, 158), our laboratory decided to investigate whether or not TRIF could activate Caspase-8 in this prosurvival context in macrophages. Furthermore, we wanted to determine if the cleavage of CYLD occurred in cells stimulated with LPS or poly(I:C), similar to what is seen in cells stimulated with TNF (74). In unpublished studies, we found that the stimulation of wildtype BMDMs with LPS or poly(I:C) induced cleavage of CYLD, but not RIPK1 or RIPK3, and this cleavage requires TRIF and Caspase-8. Furthermore, this cleavage of CYLD in BMDMs was not dependent on TNF. In addition to the TLRs, we have found that CYLD cleavage occurs in T cells following TCR stimulation, indicating that cleavage of CYLD by Caspase-8 may be an important regulatory event down-

153

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

stream of pattern recognition receptors and antigen receptors. In all these cases, blockade of CYLD cleavage by caspase inhibitors correlated with the induction of necroptosis (D. Legarda, S. J. Justus and A. T. Ting, unpublished data). Thus, our speculation is that CYLD cleavage and removal by a FADD/Caspase-8/cFLIP complex may be a more general survival mechanism utilized by multiple immune receptors to sustain the ubiquitination of RIPK1 to restrain its prodeath tendencies. The redemption of RIPK1: RIPK1 is both an activator and suppressor of death While the role of RIPK1 in necroptosis has now been established, various reports have also indicated that RIPK1 is not essential for necroptosis (152, 156, 159–163). Definitive evidence of the existence of RIPK1-independent necroptosis came from three groups that extensively characterized Ripk1 / mice by crossing them on to various genetic knockouts (164–166). As Ripk1 / mice die soon after birth, this lethality was thought to be due to the loss of RIPK1 in its prosurvival role in TNF-induced NF-jB, rendering cells susceptible to excessive TNF-driven apoptosis in vivo (16). However, all three groups found that Ripk1 / Caspase-8 / mice were not fully protected from lethality, although they were rescued to a similar extent as Ripk1 / Fadd / mice (117, 164–166). Interestingly, Ripk1 / mice crossed to Tnfr1 / to prevent both TNF-induced, Caspase-8-dependent apoptosis and necroptosis, or Ripk1 / Tnfr1 / mice crossed to the necroptosis-deficient Mlkl / and Rip3 / mice were not fully protected from lethality. However, Ripk1 / Tnfr1 / Ripk3 / or Mlkl / mice survived longer than Ripk1 / Tnfr1 / mice, indicating that a form of TNFindependent necroptosis was kept in check by RIPK1 (164, 165). Similar findings were reported by Kaiser et al. (166) using Tnf / mice. Only Ripk1 / Ripk3 / Caspase-8 / mice were fully rescued from lethality and systemic inflammation, indicating that RIPK1 functioned to suppress both apoptosis and necroptosis in vivo (164–166). More evidence came from Dannappel et al. (167) and Takahashi et al. (168) who utilized RIPK1 conditional knockout mice to show that RIPK1 suppresses both apoptosis and necroptosis in the skin and intestine, and work from Rickard et al. (164) and Roderick et al. (169) showed that RIPK1 played an important survival role in hematopoiesis. Consistent with these in vivo observations, in vitro studies have also indicated that RIPK1 can suppress RIPK3-dependent necroptosis (170, 171). These recent experiments reaffirmed the initial observation by

154

Kelliher et al. (16) that RIPK1 has a prosurvival function. This further highlighted the dual functionality of RIPK1, reflecting the dual functionality of TNF. The molecular mechanism underlying the prosurvival function of RIPK1 remains to be fully understood. One mechanism is via its role in the induction of NF-jB, but this is likely not the only mechanism. O’Donnell et al. (52) had shown that ubiquitination of RIPK1 serves as an early NFjB-independent prosurvival signal. Furthermore, Ripk1 / MEFs were more sensitive than Ripk1+/+ MEFs to apoptosis induced by TNF and cycloheximide (172). The presence of the cycloheximide in the experiment demonstrates that RIPK1 possesses a post-translational mechanism to suppress death, and it is very likely that this prosurvival function of RIPK1 will require its ubiquitination. Whether and how this translates into suppression of Caspase-8 and RIPK3 remains to be fully elucidated. The behavior of RIPK1 may be shaped by outside influences: regulation by cross-talk between pathways TNF expression is rapidly induced in innate and adaptive cells following an encounter with pathogens. During this response, multiple receptor pathways are activated including pattern recognition receptors, antigen receptors, other TNFR family members, and receptors for other cytokines. Therefore, it is likely that the output from TNFR1 will be subjected to regulation by these other pathways and the early ubiquitin-dependent checkpoint represents an ideal point to cross-talk. Two examples of this have already been discussed. One: TRIF-coupled TLRs and the TCR are able to induce the cleavage of CYLD (D. Legarda, S. J. Justus and A. T. Ting, unpublished data). This would lead to more RIPK1 ubiquitination and therefore would be a prosurvival event. Two: TNFR2 stimulation results in the degradation of the TRAF2/cIAP1/2 E3 ligase complex (83, 85, 86). This would lead to less RIPK1 ubiquitination and therefore would be prodeath. Other TNFR family members that have been shown to induce TRAF2 degradation include CD30 (173), CD40 (174, 175), and FN14 (176, 177), which would also switch the TNFR1 response from survival to RIPK1-dependent death. Another group of cytokines that is present during an infection are interferons (IFNs). Type I IFN has been implicated in positively regulating necroptosis in several studies. It can prime fibroblasts for necroptosis (156, 178) and also appears to be required for necroptosis in macrophages. An early study by Kim et al. (149) showed that STAT1 was © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

required for LPS and zVAD-induced necroptosis in peritoneal macrophages, but this death did not depend on the acute production of IFNb as an IFNb neutralizing antibody was not able to prevent cell death. This was supported by a finding by He et al. (152), demonstrating that interferon regulatory factor 3 (IRF3) was dispensable for TLR-induced necroptosis in BMDMs. However, two recent studies by Sad and colleagues (179, 180) have established an essential requirement for type I IFN in macrophage necroptosis. Infection of BMDMs with Salmonella enterica serovar Typhimurium (S. Typhimurium) could induce necroptosis through the downregulation of Caspase-8 and this was dependent on a subunit of the type I interferon receptor (IFNAR1), the kinase activity of RIPK1, and RIPK3 (179). A follow-up study showed that the production of IFNb and signaling through IFNAR1 were required for TLR-induced necroptosis in macrophages (180). TLR-induced interferon production was required for the formation of the ISGF3 complex (STAT1-STAT2-IRF9), consistent with the original finding from Kim et al. showing that STAT1 was required for LPS-induced necroptosis (149, 180). ISGF3-mediated gene transcription was shown to be needed to stabilize the RIPK1/RIPK3 necrosome complex (180), but the precise mechanism remains unclear. Interestingly, TNF-induced necroptosis was also abrogated in the Ifnar1 / BMDM. As Trif / cells were protected from LPS- and poly(I:C)induced necroptosis and Irf1 / cells were protected from TNF-induced necroptosis, it was proposed that the IFN produced could directly induce necroptosis (180, 181). Rather than a direct role in triggering necroptosis in BMDM, we found that type I IFN can play a licensing role in necroptosis. Similar to McComb et al. (180), we found that necroptosis induced by TNF (or doses of LPS where death is TNF-dependent) requires IFNAR1 (D. Legarda, S. J. Justus and A. T. Ting, unpublished data). Since TNFR2 has been shown to be upregulated in activated immune cells and has been shown to have a pronecroptotic function (73, 182–184), we examined whether its expression may be compromised in Ifnar1 / BMDMs. This was indeed the case and consistent with the resistance of Ifnar1 / BMDMs to TNF-induced necroptosis, Tnfr2 / BMDMs exhibited a similar behavior (D. Legarda, S. J. Justus and A. T. Ting, unpublished data). These observations provide an example of how other cytokine signaling pathways can indirectly regulate the ubiquitin-dependent Checkpoint 1 via the expression of receptors that alter the ubiquitin-modifying machinery. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

The murder weapon used by RIPK1 may be dependent on how its ubiquitin chains are broken The consequence of disrupting Checkpoint 1 is RIPK1-mediated apoptosis or necroptosis. But, which form of death is triggered may be dependent on how the checkpoint is disrupted. If the checkpoint is disrupted directly by preventing the ubiquitin-dependent binding of RIPK1 to its partners, for instance by the use of SM or via the loss of a binding partner like NEMO, RIPK1 has the possibility of either associating with Caspase-8 to initiate apoptosis (Fig. 2A) or with RIPK3 to initiate necroptosis (Fig. 3A). In most cellular models examined, apoptosis is the dominant response under these circumstances and it is not clear why this is the case. Apoptosis may occur if RIPK3 is not expressed or if its kinase activity is disabled (94, 108, 185), although it was later shown that in this latter context, RIPK3 induces apoptosis due to a conformational change in RIPK3 and not due to the specific inhibition of kinase activity of RIPK3 (186). In addition, following the disruption of the ubiquitination of RIPK1, the rapid association of RIPK1 with Caspase-8 induces the formation of the p18/p10 tetrameric Caspase-8 (52), which could then cleave CYLD, RIPK1, and RIPK3 to disable necroptosis. Checkpoint 1 can also be disrupted solely by blocking the prosurvival Caspase-8 and in this case, the only option available for RIPK1 is RIPK3-dependent necroptosis by virtue of the loss of Caspase-8’s catalytic activity (Fig. 3B). Finally, it has also been suggested that the ratio of the cFLIP isoforms may also be an important regulatory factor in determining whether cell death proceeds via apoptosis or necroptosis (75). Since cell survival and death is central to the response to microbial infection, it is not surprisingly that the ubiquitindependent Checkpoint 1 can be affected by microbial encoded products. The best-characterized examples of pathogens that affect Checkpoint 1 are viruses. Poxviruses encode serpins that inhibit caspases to prevent apoptosis and induction of the inflammasome (187). Infection of cells by wildtype vaccinia, but not by a mutant lacking the serpin B13R/SPI-2, caused cells to die by necroptosis (73, 93). It was later found that murine cytomegalovirus and herpes simplex virus I also encode caspase inhibitors and have the potential of inducing necroptosis (159, 188). The data indicate that necroptosis may have evolved as an antiviral mechanism, and to counter this new line of defense viruses have evolved various strategies to inhibit necroptosis (73, 159). Most recently, it was shown that in the context of human immunodeficiency virus (HIV) infection, rapid turnover of

155

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

active Caspase-8 in CD8+ T cells in HIV chronic progressors may be sufficient to relieve Caspase-8’s necroptosis-suppressing function, inducing necroptosis and depletion of CD8+ T cells (189). In addition, a number of viruses have been shown to antagonize NEMO, although whether or not infection with these viruses can sensitize cells to RIPK1mediated death is not known (190–193). Examples of bacterial infection affecting Checkpoint 1 are less well characterized. Robinson et al. (179) reported that S. Typhimurium was able to induce necroptosis of macrophages by suppressing the expression of Caspase-8, although the underlying mechanism is unclear. Interestingly, there are descriptions of bacteria-encoded proteins that affect components of the ubiquitin-dependent checkpoint. For instance, Shigella flexneri encodes IpaH9.8, an E3 ligase that degrades NEMO (194), and ORF169b/OspI, which deaminates and inactivates UBC13, the E2 enzyme shown to be important for K63-linked ubiquitination (195). Enteropathogenic Escherichia coli encodes NleE, which methylates and disrupts the ubiquitin-sensing function of TAB2/3 (196), and NleF, which can inhibit Caspase-8 (197). It remains to be seen whether any of these bacterial encoded proteins can affect Checkpoint 1 and RIPK1-dependent cell death. If these pathogen-encoded molecules disrupt Checkpoint 1 and lead to the induction of necroptosis, an inflammatory form of cell death, it would suggest that disruption of Checkpoint 1 could act as a danger signal and have a pathogen-sensing role. The future looks bright for the killer/savior RIPK1 Since its initial description in 2007, the basic framework of this early ubiquitin-dependent, transcription-independent,

Checkpoint 1 is now established. The ubiquitination of RIPK1 prevents RIPK1 from engaging Caspase-8 to induce apoptosis or RIPK3 to initiate necroptosis. The ubiquitin chains of RIPK1 are reinforced by NEMO and TAK1, and loss of ubiquitin or its binding partners allows RIPK1 to break free of its restraints to initiate its cell death program. However, a number of questions remain outstanding. It is likely that this checkpoint is regulated by many different post-translational mechanisms and this remains to be fully inventoried. The role of linear and unanchored K63-linked ubiquitin chains in promoting the prosurvival function of RIPK1 and the suppression of its prodeath tendencies remains to be fully characterized. In this regard, the role of other proteins with ubiquitin-binding UBAN domains, such as optineurin and ABIN-1 (128), in regulating this checkpoint has not been examined. Another question revolves around how Caspase-8, the upstream regulator of CYLD and RIPK1, is itself regulated. Modulation of the activity of Caspase-8 by ubiquitination (198–200), phosphorylation (201, 202), degradation (200, 203), binding partners such as cFLIP (122, 204, 205), and how they affect CYLD/RIPK1dependent death are poorly understood. A role for this ubiquitin-dependent checkpoint in signaling pathways other than that of TNF, and the potential for regulation by crosstalk between these pathways, are beginning to be explored. Finally, the understanding of the in vivo roles of this checkpoint in various tissues during inflammatory and immune responses remains in its infancy. Although this should now be facilitated by the availability of floxed alleles of Casp8, Fadd, Cyld, Ripk1, and other genes, a great deal remains to be discovered about this determinant of cell fate.

References 1. Golstein P, Ojcius DM, Young JD. Cell death mechanisms and the immune system. Immunol Rev 1991;121:29–65. 2. Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449–1456. 3. Hohmann HP, Remy R, Brockhaus M, van Loon AP. Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha). J Biol Chem 1989;264:14927–14934. 4. Brockhaus M, Schoenfeld HJ, Schlaeger EJ, Hunziker W, Lesslauer W, Loetscher H. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc Natl Acad Sci USA 1990;87:3127–3131. 5. Allen RD, Marshall JD, Roths JB, Sidman CL. Differences defined by bone marrow transplantation suggest that lpr and gld are mutations of genes encoding an interacting pair of molecules. J Exp Med 1990;172:1367–1375.

156

6. Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992;356:314–317. 7. Takahashi T, et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 1994;76:969–976. 8. Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wallach D. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem 1995;270:7795–7798. 9. Stanger BZ, Leder P, Lee TH, Kim E, Seed B. RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 1995;81:513–523. 10. Hsu H, Xiong J, Goeddel DV. The TNF receptor 1-associated protein TRADD signals cell death

11.

12.

13.

14.

15.

and NF-kappa B activation. Cell 1995;81:495– 504. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 1995;81:505–512. Schulze-Osthoff K, Krammer PH, Dr€ oge W. Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J 1994;13:4587– 4596. Tartaglia LA, Ayres TM, Wong GH, Goeddel DV. A novel domain within the 55 kd TNF receptor signals cell death. Cell 1993;74:845–853. Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV. TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 1996;4:387–396. nos FX, Seed B. RIP Ting AT, Pimentel-Mui~ mediates tumor necrosis factor receptor 1

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

activation of NF-kappaB but not Fas/APO-1initiated apoptosis. EMBO J 1996;15:6189–6196. Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P. The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 1998;8:297–303. Lee TH, Shank J, Cusson N, Kelliher MA. The kinase activity of Rip1 is not required for tumor necrosis factor-alpha-induced IkappaB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2. J Biol Chem 2004;279:33185–33191. Liu Z-G, Hsu H, Goeddel DV, Karin M. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 1996;87:565–576. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 1996;274:787– 789. Beg AA, Baltimore D. An essential role for NFkappaB in preventing TNF-alpha-induced cell death. Science 1996;274:782–784. Wang CY, Mayo MW, Baldwin AS. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996;274:784–787. Cusson N, Oikemus S, Kilpatrick ED, Cunningham L, Kelliher M. The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor type 2-induced cell death. J Exp Med 2002;196:15–26. Pimentel-Mui~ nos FX, Seed B. Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity 1999;11:783–793. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADDTRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 1996;84:299–308. Muzio M, et al. FLICE, a novel FADDhomologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death– inducing signaling complex. Cell 1996;85:817– 827. Chinnaiyan AM, et al. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J Biol Chem 1996;271:4961–4965. Varfolomeev EE, et al. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 1998;9:267–276. Zhang J, Cado D, Chen A, Kabra NH, Winoto A. Fas-mediated apoptosis and activation-induced Tcell proliferation are defective in mice lacking FADD/Mort1. Nature 1998;392:296–300. Yeh WC, et al. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 1998;279:1954–1958. Lin Y, Devin A, Rodriguez Y, Liu ZG. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev 1999;13:2514–2526.

31. Martinon F, Holler N, Richard C, Tschopp J. Activation of a pro-apoptotic amplification loop through inhibition of NF-kappaB-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett 2000;468:134–136. 32. Shu HB, Takeuchi M, Goeddel DV. The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc Natl Acad Sci USA 1996;93:13973–13978. 33. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 2003;114:181–190. 34. Irmler M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997;388:190– 195. 35. Kreuz S, Siegmund D, Scheurich P, Wajant H. NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol 2001;21:3964– 3973. 36. Micheau O, Lens S, Gaide O, Alevizopoulos K, Tschopp J. NF-kappaB signals induce the expression of c-FLIP. Mol Cell Biol 2001;21:5299–5305. 37. Zhang SQ, Kovalenko A, Cantarella G, Wallach D. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon receptor stimulation. Immunity 2000;12:301–311. 38. Legler DF, Micheau O, Doucey M-A, Tschopp J, Bron C. Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NFkappaB activation. Immunity 2003;18:655–664. 39. Park S-M, Yoon J-B, Lee TH. Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro. FEBS Lett 2004;566:151–156. 40. Wertz IE, et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature 2004;430:694–699. 41. Newton K, et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 2008;134:668–678. 42. Xu M, Skaug B, Zeng W, Chen ZJ. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta. Mol Cell 2009;36:302– 314. 43. Gerlach B, et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 2011;471:591–596. 44. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 2001;412:346–351. 45. Kanayama A, et al. TAB 2 and TAB 3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell 2004;15:535– 548. 46. Chen ZJ. Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 2005;7:758–765. 47. Ea C-K, Deng L, Xia Z-P, Pineda G, Chen ZJ. Activation of IKK by TNFalpha requires sitespecific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 2006;22:245–257. 48. Li H, Kobayashi M, Blonska M, You Y, Lin X. Ubiquitination of RIP is required for tumor

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

necrosis factor alpha-induced NF-kappaB activation. J Biol Chem 2006;281:13636–13643. Ruff MR, Gifford GE. Rabbit tumor necrosis factor: mechanism of action. Infect Immun 1981;31:380–385. Lee SY, Reichlin A, Santana A, Sokol KA, Nussenzweig MC, Choi Y. TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyte proliferation and survival. Immunity 1997;7:703–713. Yeh WC, et al. Early lethality, functional NFkappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 1997;7:715–725. O’Donnell MA, Legarda-Addison D, Skountzos P, Yeh W-C, Ting AT. Ubiquitination of RIP1 regulates an NF-kappaB-independent cell-death switch in TNF signaling. Curr Biol 2007;17:418– 424. O’Donnell MA, Ting AT. Chronicles of a death foretold: dual sequential cell death checkpoints in TNF signaling. Cell Cycle 2010;9:1065–1071. Zheng L, et al. Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1. Mol Cell Biol 2006;26:3505–3513. Ermolaeva MA, et al. Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses. Nat Immunol 2008;9:1037–1046. Pobezinskaya YL, et al. The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Tolllike receptors. Nat Immunol 2008;9:1047– 1054. Alvarez SE, et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 2010;465:1084–1088. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A small molecule Smac mimic potentiates TRAIL- and TNFalphamediated cell death. Science 2004;305:1471– 1474. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000;102:33–42. Verhagen AM, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000;102:43–53. Chauhan D, et al. Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM). Blood 2007;109:1220–1227. Mizukawa K, et al. Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines. J Neurooncol 2006;77:247–255. Bockbrader KM, Tan M, Sun Y. A small molecule Smac-mimic compound induces apoptosis and sensitizes TRAIL- and etoposide-induced apoptosis in breast cancer cells. Oncogene 2005;24:7381–7388. Petersen SL, et al. Autocrine TNFalpha signaling renders human cancer cells susceptible to Smacmimetic-induced apoptosis. Cancer Cell 2007;12:445–456.

157

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

65. Varfolomeev E, et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 2007;131:669–681. 66. Vince JE, et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 2007;131:682–693. 67. Eckelman BP, Salvesen GS. The human antiapoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases. J Biol Chem 2006;281:3254–3260. 68. Bertrand MJM, et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell 2008;30:689–700. 69. Mahoney DJ, et al. Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation. Proc Natl Acad Sci USA 2008;105:11778–11783. 70. Varfolomeev E, et al. c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. J Biol Chem 2008;283:24295–24299. 71. Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. Cell 2008;133:693–703. 72. Wright A, et al. Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD. Dev Cell 2007;13:705–716. 73. Chan FK-M, et al. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J Biol Chem 2003;278:51613–51621. 74. O’Donnell MA, et al. Caspase 8 inhibits programmed necrosis by processing CYLD. Nat Cell Biol 2011;13:1437–1442. 75. Feoktistova M, et al. cIAPs block ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms. Mol Cell 2011;43:449–463. 76. Tenev T, et al. The ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs. Mol Cell 2011;43:432– 448. 77. Holler N, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 2000;1:489–495. 78. Vercammen D, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 1998;187:1477–1485. 79. Khwaja A, Tatton L. Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/caspasedependent signaling pathway. J Biol Chem 1999;274:36817–36823. 80. Li M, Beg AA. Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virusinfected cells. J Virol 2000;74:7470–7477. 81. Denecker G, et al. Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria. Cell Death Differ 2001;8:829–840.

158

82. Cauwels A, Janssen B, Waeytens A, Cuvelier C, Brouckaert P. Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat Immunol 2003;4:387–393. 83. Chan FK, Lenardo MJ. A crucial role for p80 TNF-R2 in amplifying p60 TNF-R1 apoptosis signals in T lymphocytes. Eur J Immunol 2000;30:652–660. 84. Rothe M, Wong SC, Henzel WJ, Goeddel DV. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 1994;78:681–692. 85. Fotin-Mleczek M, et al. Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1dependent activation of caspase-8. J Cell Sci 2002;115:2757–2770. 86. Li X, Yang Y, Ashwell JD. TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2. Nature 2002;416:345–347. 87. Hitomi J, et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 2008;135:1311–1323. 88. Degterev A, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 2008;4:313–321. 89. Degterev A, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005;1:112–119. 90. Vanlangenakker N, Vanden Berghe T, Krysko DV, Festjens N, Vandenabeele P. Molecular mechanisms and pathophysiology of necrotic cell death. Curr Mol Med 2008;8:207–220. 91. Henriquez M, Armisen R, Stutzin A, Quest AFG. Cell death by necrosis, a regulated way to go. Curr Mol Med 2008;8:187–206. 92. Kim Y-S, Morgan MJ, Choksi S, Liu Z-G. TNFinduced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death. Mol Cell 2007;26:675–687. 93. Cho Y, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137:1112–1123. 94. He S, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNFalpha. Cell 2009;137:1100–1111. 95. Zhang D-W, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009;325:332–336. 96. Sun X, Yin J, Starovasnik MA, Fairbrother WJ, Dixit VM. Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J Biol Chem 2002;277:9505– 9511. 97. Li J, et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 2012;150:339– 350. 98. Vandenabeele P, Declercq W, Van Herreweghe F, Vanden Berghe T. The role of the kinases RIP1

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

and RIP3 in TNF-induced necrosis. Sci Signal 2010;3:re4. Wang Z, Jiang H, Chen S, Du F, Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 2012;148:228–243. Sun L, et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012;148:213–227. Zhao J, et al. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 2012;109:5322–5327. Cai Z, et al. Plasma membrane translocation of trimerized MLKL protein is required for TNFinduced necroptosis. Nat Cell Biol 2013;16:55– 65. Chen X, et al. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res 2014;24:105–121. Wang H, et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 2014;54:133–146. Dondelinger Y, et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 2014;7:971–981. Wu J, et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res 2013;23:994–1006. Murphy JM, et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 2013;39:443– 453. Remijsen Q, et al. Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis 2014;5:e1004. Sakamaki K, et al. Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart. Cell Death Differ 2002;9:1196– 1206. Kang T-B, et al. Caspase-8 serves both apoptotic and nonapoptotic roles. J Immunol 2004;173:2976–2984. Salmena L, et al. Essential role for caspase 8 in Tcell homeostasis and T-cell-mediated immunity. Genes Dev 2003;17:883–895. Ch’en IL, Tsau JS, Molkentin JD, Komatsu M, Hedrick SM. Mechanisms of necroptosis in T cells. J Exp Med 2011;208:633–641. Kovalenko A, et al. Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease. J Exp Med 2009;206:2161–2177. Lee P, Lee D-J, Chan C, Chen S-W, Ch’en I, Jamora C. Dynamic expression of epidermal caspase 8 simulates a wound healing response. Nature 2009;458:519–523. Kang T-B, et al. Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice. J Immunol 2008;181:2522–2532.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

116. Oberst A, et al. Catalytic activity of the caspase8–FLIPL complex inhibits RIPK3-dependent necrosis. Nature 2011;471:363–367. 117. Zhang H, Zhou X, McQuade T, Li J, Chan FK-M, Zhang J. Functional complementation between FADD and RIP1 in embryos and lymphocytes. Nature 2011;471:373–376. 118. Kaiser WJ, et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 2011;471:368–372. 119. Laouar Y, Ezine S. In vivo CD4+ lymph node T cells from lpr mice generate CD4 CD8 B220+ TCR-beta low cells. J Immunol 1994;153:3948– 3955. 120. Yeh WC, et al. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. Immunity 2000;12:633–642. 121. Dillon CP, et al. Survival function of the FADDCASPASE-8-cFLIP(L) complex. Cell Rep 2012;1:401–407. 122. Pop C, et al. FLIP(L) induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity. Biochem J 2011;433:447–457. 123. Weinlich R, et al. Protective roles for Caspase-8 and cFLIP in adult homeostasis. Cell Rep 2013;5:340–348. 124. Lin Y, et al. Tumor necrosis factor-induced nonapoptotic cell death requires receptorinteracting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem 2004;279:10822–10828. 125. Feng S, et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell Signal 2007;19:2056–2067. 126. Lu JV, et al. Complementary roles of Fasassociated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity. Proc Natl Acad Sci USA 2011;108:15312–15317. 127. Legarda-Addison D, Hase H, O’Donnell MA, Ting AT. NEMO/IKKc regulates an early NFjB-independent cell-death checkpoint during TNF signaling. Cell Death Differ 2009;16:1279– 1288. 128. Wagner S, et al. Ubiquitin binding mediates the NF-kappaB inhibitory potential of ABIN proteins. Oncogene 2008;27:3739–3745. 129. O’Donnell MA, Hase H, Legarda D, Ting AT. NEMO Inhibits programmed necrosis in an NFjB-independent manner by restraining RIP1. PLoS ONE 2012;7:e41238. 130. Morioka S, Omori E, Kajino T, Kajino-Sakamoto R, Matsumoto K, Ninomiya-Tsuji J. TAK1 kinase determines TRAIL sensitivity by modulating reactive oxygen species and cIAP. Oncogene 2009;28:2257–2265. 131. Omori E, Morioka S, Matsumoto K, NinomiyaTsuji J. TAK1 regulates reactive oxygen species and cell death in keratinocytes, which is essential for skin integrity. J Biol Chem 2008;283:26161– 26168. 132. Vanlangenakker N, Vanden Berghe T, Fulda S, Vandenabeele P. cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

150.

RIP3-dependent reactive oxygen species production. Cell Death Differ 2010;18:656–665. C ß €ol Arslan S, Scheidereit C. The prevalence of TNFa-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay. PLoS ONE 2011;6:e26069. Yan J, et al. Inactivation of BAD by IKK inhibits TNFa-induced apoptosis independently of NF-jB activation. Cell 2013;152:304–315. Tokunaga F, et al. SHARPIN is a component of the NF-jB-activating linear ubiquitin chain assembly complex. Nature 2011;471:633–636. Ikeda F, et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-jB activity and apoptosis. Nature 2011;471:637–641. Seymour RE, et al. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun 2007;8:416–421. Kirisako T, et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J 2006;25:4877–4887. Kumari S, et al. Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis. eLife 2014;3:e03422. Rickard JA, et al. TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice. eLife 2014;3:e03464. Berger SB, et al. Cutting edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J Immunol 2014;192:5476–5480. Komander D, Reyes-Turcu F, Licchesi JDF, Odenwaelder P, Wilkinson KD, Barford D. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep 2009;10:466–473. Rahighi S, et al. Specific recognition of linear ubiquitin chains by NEMO is important for NFkappaB activation. Cell 2009;136:1098–1109. Xia Z-P, et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 2009;461:114–119. Kim SO, Han J. Pan-caspase inhibitor zVAD enhances cell death in RAW246.7 macrophages. J Endotoxin Res 2001;7:292–296. Kim SO, Ono K, Han J. Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages. Am J Physiol Lung Cell Mol Physiol 2001;281:L1095–L1105. Kim SO, Ono K, Tobias PS, Han J. Orphan nuclear receptor Nur77 is involved in caspaseindependent macrophage cell death. J Exp Med 2003;197:1441–1452. Franchi L, Cond o I, Tomassini B, Nicolo C, Testi R. A caspaselike activity is triggered by LPS and is required for survival of human dendritic cells. Blood 2003;102:2910–2915. Kim HS, Lee M-S. Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway. Mol Cell Biol 2005;25:6821–6833. Xu Y, Kim SO, Li Y, Han J. Autophagy contributes to caspase-independent macrophage cell death. J Biol Chem 2006;281:19179–19187.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

151. Ma Y, Temkin V, Liu H, Pope RM. NF-kappaB protects macrophages from lipopolysaccharideinduced cell death: the role of caspase 8 and receptor-interacting protein. J Biol Chem 2005;280:41827–41834. 152. He S, Liang Y, Shao F, Wang X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc Natl Acad Sci USA 2011;108:20054–20059. 153. Meylan E, et al. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation. Nat Immunol 2004;5:503–507. 154. Kim SJ, Li J. Caspase blockade induces RIP3mediated programmed necrosis in Toll-like receptor-activated microglia. Cell Death Dis 2013;4:e716. 155. G€ unther C, et al. Caspase-8 controls the gut response to microbial challenges by Tnf-adependent and independent pathways. Gut 2015;64:601–610. 156. Kaiser WJ, et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3 and MLKL. J Biol Chem 2013;288:31268–31279. 157. Estornes Y, et al. dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8. Cell Death Differ 2012;19:1482–1494. 158. Maelfait J, et al. Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8. J Exp Med 2008;205:1967–1973. 159. Upton JW, Kaiser WJ, Mocarski ES. Virus inhibition of RIP3-dependent necrosis. Cell Host Microbe 2010;7:302–313. 160. Upton JW, Kaiser WJ, Mocarski ES. DAI/ZBP1/ DLM-1 complexes with RIP3 to mediate virusinduced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe 2012;11:290–297. 161. Moujalled DM, et al. TNF can activate RIPK3 and cause programmed necrosis in the absence of RIPK1. Cell Death Dis 2013;4:e465. 162. Cho Y, McQuade T, Zhang H, Zhang J, Chan FKM. RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation. PLoS ONE 2011;6:e23209. 163. Vanlangenakker N, Bertrand MJM, Bogaert P, Vandenabeele P, Vanden Berghe T. TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members. Cell Death Dis 2011;2:e230. 164. Rickard JA, et al. RIPK1 regulates RIPK3-MLKLdriven systemic inflammation and emergency hematopoiesis. Cell 2014;157:1175–1188. 165. Dillon CP, et al. RIPK1 blocks early postnatal lethality mediated by Caspase-8 and RIPK3. Cell 2014;157:1189. 166. Kaiser WJ, et al. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc Natl Acad Sci USA 2014;111:7753–7758. 167. Dannappel M, et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature 2014;513:90–94. 168. Takahashi N, et al. RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis. Nature 2014;513:95–99.

159

Justus & Ting  Ubiquitin-dependent regulation of RIPK1

169. Roderick JE, et al. Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis. Proc Natl Acad Sci USA 2014;111:14436–14441. 170. Orozco S, et al. RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ 2014;21:1511– 1521. 171. Kearney CJ, Cullen SP, Clancy D, Martin SJ. RIPK1 can function as an inhibitor rather than an initiator of RIPK3-dependent Necroptosis. FEBS J 2014;281:4921–4934. 172. Wong WW-L, Gentle IE, Nachbur U, Anderton H, Vaux DL, Silke J. RIPK1 is not essential for TNFR1-induced activation of NF-kappaB. Cell Death Differ 2010;17:482–487. 173. Csomos RA, Wright CW, Galban S, Oetjen KA, Duckett CS. Two distinct signalling cascades target the NF-kappaB regulatory factor c-IAP1 for degradation. Biochem J 2009;420:83–91. 174. Brown KD, Hostager BS, Bishop GA. Differential signaling and tumor necrosis factor receptorassociated factor (TRAF) degradation mediated by CD40 and the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1). J Exp Med 2001;193:943–954. 175. Brown KD, Hostager BS, Bishop GA. Regulation of TRAF2 signaling by self-induced degradation. J Biol Chem 2002;277:19433–19438. 176. Vince JE, et al. TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFalpha. J Cell Biol 2008;182:171–184. 177. Ikner A, Ashkenazi A. TWEAK induces apoptosis through a death-signaling complex comprising receptor-interacting protein 1 (RIP1), Fasassociated death domain (FADD), and caspase-8. J Biol Chem 2011;286:21546–21554. 178. Thapa RJ, et al. Interferon-induced RIP1/RIP3mediated necrosis requires PKR and is licensed by FADD and caspases. Proc Natl Acad Sci USA 2013;110:E3109–E3118. 179. Robinson N, McComb S, Mulligan R, Dudani R, Krishnan L, Sad S. Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium. Nat Immunol 2012;13:954–962. 180. McComb S, et al. Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages. Proc Natl Acad Sci USA 2014;111:E3206–E3213.

160

181. Yarilina A, Park-Min K-H, Antoniv T, Hu X, Ivashkiv LB. TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes. Nat Immunol 2008;9:378–387. 182. Tannenbaum CS, Major JA, Hamilton TA. IFNgamma and lipopolysaccharide differentially modulate expression of tumor necrosis factor receptor mRNA in murine peritoneal macrophages. J Immunol 1993;151:6833–6839. 183. de Kossodo S, Critico B, Grau GE. Modulation of the transcripts for tumor necrosis factor-alpha and its receptors in vivo. Eur J Immunol 1994;24:769–772. 184. Bethea JR, Ohmori Y, Hamilton TA. A tandem GC box motif is necessary for lipopolysaccharide-induced transcription of the type II TNF receptor gene. J Immunol 1997;158:5815–5823. 185. Newton K, et al. Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science 2014;343:1357–1360. 186. Mandal P, et al. RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell 2014;56:481–495. 187. Turner S, Kenshole B, Ruby J. Viral modulation of the host response via crmA/SPI-2 expression. Immunol Cell Biol 1999;77:236–241. 188. Wang X, et al. Direct activation of RIP3/MLKLdependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense. Proc Natl Acad Sci USA 2014;111:15438–15443. 189. Gaiha GD, et al. Dysfunctional HIV-specific CD8 (+) T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis. Immunity 2014;41:1001–1012. 190. Fliss PM, et al. Viral mediated redirection of NEMO/IKKc to autophagosomes curtails the inflammatory cascade. PLoS Pathog 2012;8: e1002517. 191. Wang D, et al. Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling. J Virol 2012;86:9311–9322. 192. Huang C, et al. Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-jB essential modulator. J Virol 2014;88:10934–10945.

193. Wang D, et al. Hepatitis A virus 3C protease cleaves NEMO to impair induction of beta interferon. J Virol 2014;88:10252–10258. 194. Ashida H, Kim M, Schmidt-Supprian M, Ma A, Ogawa M, Sasakawa C. A bacterial E3 ubiquitin ligase IpaH9.8 targets NEMO/IKKgamma to dampen the host NF-kappaB-mediated inflammatory response. Nat Cell Biol 2010;12:66–73. 195. Sanada T, et al. The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response. Nature 2012;483:623–626. 196. Zhang L, et al. Cysteine methylation disrupts ubiquitin-chain sensing in NF-jB activation. Nature 2012;481:204–208. 197. Blasche S, et al. The E. coli effector protein NleF is a caspase inhibitor. PLoS ONE 2013;8:e58937. 198. Gonzalvez F, et al. TRAF2 sets a threshold for extrinsic apoptosis by tagging caspase-8 with a ubiquitin shutoff timer. Mol Cell 2012;48:888– 899. 199. Jin Z, et al. Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling. Cell 2009;137:721– 735. 200. Peng C, et al. Phosphorylation of caspase-8 (Thr263) by ribosomal S6 kinase 2 (RSK2) mediates caspase-8 ubiquitination and stability. J Biol Chem 2011;286:6946–6954. 201. Alvarado-Kristensson M, Melander F, Leandersson K, R€ onnstrand L, Wernstedt C, Andersson T. p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils. J Exp Med 2004;199:449–458. 202. Cursi S, et al. Src kinase phosphorylates Caspase8 on Tyr380: a novel mechanism of apoptosis suppression. EMBO J 2006;25:1895–1905. 203. Hou W, Han J, Lu C, Goldstein LA, Rabinowich H. Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis. Autophagy 2010;6:891–900. 204. Bidere N, Snow AL, Sakai K, Zheng L, Lenardo MJ. Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NFkappaB activation. Curr Biol 2006;16:1666– 1671. 205. Kawadler H, Gantz MA, Riley JL, Yang X. The paracaspase MALT1 controls caspase-8 activation during lymphocyte proliferation. Mol Cell 2008;31:415–421.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 266/2015

Cloaked in ubiquitin, a killer hides in plain sight: the molecular regulation of RIPK1.

In the past decade, studies have shown how instrumental programmed cell death (PCD) can be in innate and adaptive immune responses. PCD can be a means...
488KB Sizes 0 Downloads 11 Views