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Proteolytic signaling by TNFα: caspase activation and IκB degradation
www.elsevier.com/locate/jnlabr/ycyto Cytokine 21 (2003) 286–294
Proteolytic signaling by TNFa: caspase activation and IjB degradation Xiaotang Hu Hematologic Malignancies Program (MDC44, MCC3142), Department of Interdisciplinary Oncology, Department of Internal Medicine, University of South Florida College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA Received 20 November 2002; received in revised form 5 February 2003; accepted 15 March 2003
Abstract Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IjBa. Caspases are activated using mechanisms of oligomeration and Ôselfcontrolled proteolysisÕ. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-jB is dependent on the degradation of IjBa. IjB kinase (IKK) phosphorylates IjBa at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-jB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-jB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-jB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die. Ó 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Tumor necrosis factor (TNF) alpha is a protein synthesized and secreted by mononuclear phagocytes in response to stimulation with bacterial endotoxin and other inducers [1]. TNF has been demonstrated to affect a wide range of biological activities of many cell types, including mediating the inflammatory response, regulating immune function, and triggering apoptosis of certain tumor cells. Among these biological activities TNFinduced apoptosis and activation of nuclear factor-kappa B (NF-jB) [2–5] have gained particular attention in recent years. Apoptosis, or programed cell death, plays a central role in development, maintenance of homeostasis, and host defense. Apoptotic cell death is characterized by cytoskeletal disruption, cell shrinkage, membrane Tel.: +1-813-979-6721; fax: +1-813-972-8468. E-mail address: hu@moffitt.usf.edu
blebbing, and nuclear DNA fragmentation as a result of the activation of endogenous proteases [6]. NF-jB has been reported to be an important mediator of inflammation, immune response, and cell differentiation through the regulation of multiple target genes [7]. After initiating TNF stimulation NF-jB translocates into the nucleus, where NF-jB binds to jB sequences in the promoters of target genes (cytokines, chemokines, and cell adhesion molecules) and stimulates transcription. One important progress in NF-jB studies is the finding that activated NF-jB is able to protect cells from TNFainduced cell killing [8–10]. Both apoptosis signal and activation of NF-jB are triggered by the TNF receptor following TNF binding, which are regulated by a group cysteine proteases caspases and ubiquitin-dependent degradation, respectively. In this mini review, I will attempt to summarize our current understanding of the proteolytic signaling in TNFmediated activation of caspase and NF-jB pathways.
1043-4666/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-4666(03)00107-8
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2. TNF death receptor signaling
3. Proteolytic signaling in caspase activation
In humans and mice, two TNF receptors have been identified, with molecular masses of 55 kDa (TNF-R1) and 75 kDa (TNF-R2). It has been shown that TNF-R1 are predominately expressed in human cells of epithelial origin, whereas TNF-R2 are the major receptors expressed in human hematopoietic cell lines [11,12]. Even so, TNF-R1, but not TNF-R2, plays an essential role in TNF-induced cell death in these cells [12]. Subsequent experiments demonstrated that expression of these two receptors on the cell surface is independently regulated. The cytoplasmic portion of the TNF-R1 has a domain approximately 80 amino acids long, referred to as the death domain for its role in TNF-mediated cell death, whereas the cytoplasmic portion of the TNF-R2 lacks this death domain [13]. The receptors contains Ôdeath domainÕ are thus referred to as death receptors. Besides TNF-R1 the other death receptors are CD95 (Fas or Apo1), DR3 (TRAMP or Apo3), DR4 (TRAIL-R1), and DR5 (Apo2, or TRAIL-R2). All these death receptors belong to the members of TNF superfamily receptors, which are defined by similar, cysteine-rich extra cellular domains [14–20]. In 1999, an inhibitory protein termed silencer of death domains (SODDs), which is associated with death domain of TNF-R1, but not with those of TNF-R2, was identified [21]. Following binding its death receptor on the plasma membrane, TNF induces the receptor trimerization and the release SODD from the intracellular domain (ICD) of TNF-R1. The resulting aggregated ICD is recognized by an adapter protein, TNF receptor-associated death domain (TRADD) [22]. TRADD functions as a platform that recruits additional adapter proteins including receptor-interacting protein (RIP) [23], TNF-R-associated factor 2 (TRAF2) [24], Fas-associated death domain (FADD) [25], and cellular inhibitor of apoptosis protein (IAP) (Fig. 1, also see the Section 6). FADD contains two functionally and structurally distinct domains: a C-terminal DD that is necessary for its recruitment of the TNF-R1 complex and an N-terminal death effector domain (DED) that promotes activation of a downstream proteolytic reaction and caspase cascade, which finally lead to apoptosis. In contrast, TRAF2 and RIP activate the NF-jB-inducing kinase (NIK) [26], which in turn activates IjB kinase (IKK) [27–29] (Fig. 1). IKK complex consists of two catalytic subunits IKKa and IKKb, and a regulatory subunit IKKc. Activation of the IKK complex requires phosphorylation of two serine residues located in the Ôactivation loopÕ within the kinase domain of IKKa and IKKb. The activated IKK complex phosphorylates IjBa, causing IjBa degradation by proteasome and allowing NF-jB to translocate to the nucleus to activate transcription (Fig. 1).
3.1. Caspase family and caspase activation
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Based on our current knowledge, caspases play an essential role during TNF-induced apoptosis. Although many molecular events may involve in cell death, activation of caspase cascade has been consistently involved in apoptosis. The term caspase resulted from their two key characteristics: cysteine proteases and aspartases. The caspase family was initially discovered following a search of human cDNA libraries for sequences homologous to ced-3, a cell death gene described in the nematode worm, Caenorhabditis elegans [30,31]. The first mammalian homologue of ced-3 identified is interleukin-1b converting enzyme (ICE) [32,33]. Subsequently, a number of mammalian ced-3 homologues (at least 14 mammalian members of the caspase family) were discovered [34,35]: caspase-1 (ICE), caspase-2 (ICH-1), caspase-3 (CPP32), caspase-4 (TX, ICH-2, ICErel-ll), caspase-5 (ICErel-lll), caspase-6 (Mch2), caspase-7 (mch3, ICE-LAP3, CMH-1), caspase-8 (MACH, FLICE, Mch5), caspase-9 (ICE-LAP6, Mch6), caspase-10 (Mch4, FLICE2), caspase-11 (ICH3), caspase-12, caspase-13 (ERICE), and caspase-14 (MICE). Based on their structures and functions caspases can be divided into three subgroups. Group I caspases are ICE related family (caspase-1, -4, -5, -11, -12, -13, and -14), which mainly involve in inflammation response, while group II (caspase-2, -3, and -7), and group III (caspase-6, -8, -9, and -10) are mostly linked to apoptosis in general. Caspases are synthesized as inactive precursors termed procaspases, which are converted to active proteases during apoptosis through auto-regulated proteolytic process. Most long prodomains in the group III can mediate oligomerization of the procaspase molecules and recruit caspase precursors to the specific death domain complexes, resulting in activation of caspase activation. Therefore, group III caspases are referred to as initiators or upstream caspases and group II caspases are called executive, effector, or downstream caspases. Caspase X-ray crystal structure shows that the caspase tetramer is comprised of two large subunits and two small subuints [36–38]. Both the large and the small subunits can be liberated from the proenzyme by cleavage at Asp-X bonds and become active form of the enzyme [39–41] (Fig. 2). Binding of TNFa to its death receptor recruits procaspase to an oligomeric activation complex using the adapter protein FADD. Following recruitment of multiple caspase proenzyme to a common oligomerization site, the low level of endogenous catalytic activity accumulated in the death receptor complex is sufficient to initiate full catalytic activation by proteolysis of the Asp-X site [39,40] (Fig. 1). The first study implying
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Fig. 1. An overview of TNF-induced proteolytic signals and their interactions. Binding of TNF to its death receptor, resulting in the release of SODD and recruiting several cytoplasmic adaptor proteins to form a receptor complex. The complex initiates activation of caspases and NF-jB, which lead to apoptosis and cell proliferation (anti-apoptosis), respectively. There are numerous crosstalk between caspase activation and NF-jB pathways.
oligomerization in caspase activation was involved in activation of caspase-1 [43]. Subsequently, similar results were obtained with caspase 2 [44] and several other caspases [42]. Additional support for the oligomerization is from the observation that caspase-3, -8, and -9 were activated by proteolysis in vitro when oligomerization was induced [39–41]. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site as described before. The activated upstream and downstream caspases then cleave various cellular proteins, leading to apoptotic cell death (Fig. 1).
3.2. Caspase-8 and caspase-3 Among the caspases, caspase-8 and -3 may play key role in apoptosis cascade [45]. Caspase-8 is an upstream kinase, which has been demonstrated by a number of experiments. First, recombinant active caspase-8 does not induce DNA fragmentation in a cell-free system, suggesting that caspase-8 itself is not able to induce apoptosis. However, in the presence of cell extracts from unstimulated cells, caspase-8 induced a complete DNA fragmentation [46]. Therefore, some cytosolic factors in the extracts are required for caspase-8-initiated
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Fig. 2. Procaspase structure and cleavage sites. Procaspases contain two large and two small subunits. TNF recruits formation of death receptor complex, which in turn triggers oligomerization of procaspases. The latter leads to procaspases self-proteolysis by cleavage of the Asp-X site at the junction between the large and small subunits. Active upstream caspases then process and activate downstream procaspases.
apoptosis. Second, recombinant caspase-8 can directly cleave caspase-3, -4, -7, -9, and -10 in the absence of cytosolic extracts. In contrast, caspase-2 and -6 are efficiently cleaved only in the presence of the extract. These observations suggest that caspase-8 is proapoptotic protease. Caspase-8 not only participates in the caspase cascade pathway, but also involve in cytochrome c-mediated cell death. It has been reported that cytochrome c, an essential component of the respiratory chain for the generation of ATP, also plays an important role in apoptosis. Cytochrome c normally resides in the space between the outer and inner membrane of mitochondria and its release to cytosol is closely related to apoptotic process. Cytochrome c activates Apaf-1, in the presence of dATP, which in turn activates procaspase-9. Activated caspase-9 then cleaves downstream caspases and leads to apoptosis [47]. Release of cytochrome c is regulated by the Bcl family proteins. Bid, a BH3 domain-containing proapoptotic Bcl-2 family protein, is activated by caspase-8 in response to TNFR1/Fas death receptor activation [48–50]. Activated Bid is then translocated to mitochondria and induces cytochrome c release. This Bid-mediated pathway is critical in some cell apoptosis (such as hepatocyte) induced by TNF-R1 signal, because direct activation of cytosolic caspase cascade appears inefficient in these cells. Caspase-3 has been implicated as a major and key protease that is activated during the early stages of apoptosis [51]. Active caspase-3 consists of a heterodimer of 17 and 12 kDa subunits, which are derived from the 32 kDa proenzyme by proteolysis [52–54]. In its active form, caspase-3 proteolytically cleaves and activates other caspases, as well as many relevant targets (see below) in the cytoplasm. Therefore, caspase-3 is an important mediator for triggering caspase cascade and scleaving substrates. 3.3. Cleavage of caspase substrates A number of proteins have been shown to be substrates of caspases during apoptotic cell death [55]. These proteins involve in DNA repair processes, cell
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cycle progression, or as components of apoptosis inhibitors. Caspases either inactivate the normal biochemical function of their substrates or activate them by removal of regulatory domains. Alternatively, caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold. For example, nuclear DNA fragmentation is a result of chromatin condensation and DNA brokerage into small fragments. These processes are regulated by caspase-regulated DNA fragmentation factor (DFF) and acinus. DFF is composed of caspaseactivated DNase (CAD) and its inhibitor ICAD [56]. During apoptotic process ICAD is cleaved by caspase-3 and -7, releasing CAD to degrade nuclear DNA [57]. Acinus (apoptotic chromatin condensation inducer in the nucleus) is a nuclear protein that contains a potential DNA/RNA-binding motif. Acinus has been reported to be activated by proteolytic cleavage of caspase-3 and activated acinus induces chromatin condensation without effecting DNA fragmentation [58]. Caspases also cleave many cytoskeletal proteins. Lamins, which are required for the nuclear envelope, are cleaved by caspase-6, leading to nuclear fragmentation [59,60]. Gelsolin, a cytoplasmic F actin-depolymerizing enzyme, which is required for protection of cells from membrane blebbing, is cleaved by caspase-3 to yield a fragment with constitutive activity [61]. Actin is highly conserved proteins that is expressed in all eukaryotic cells. Actin filaments can form both stable and labile structures, and is crucial components of microvilli and the contractile apparatus of cells. During apoptosis actin is cleaved by effector caspases in certain cells [55]. PAK2, a serine/threonine kinse downstream of Rac and cdc42, is cleaved by caspase-3. Poly ADP-ribose polymerase (PARP) is a nuclear enzyme that senses DNA nicks and catalyzes the ADP-ribosylation of histone and other nuclear proteins in order to facilitate DNA repair. Studies from several laboratories demonstrated that PARP is cleaved at a single site by caspase-3, which separates the N-terminal DNA-binding domain from the catalytic domain and inactivates the enzymatic activity [62–64]. More information about caspase substrates can be found in several review papers [65,66].
4. Proteolytic signaling in IjB phosphorylation and degradation 4.1. Degradation of IjB is required for NF-jB activation NF-jB is a transcription factor first identified in B cells and is crucial for lg gene function [67,68]. It was considered as a B cell specific transcription factor. However, it soon became evident that NF-jB exists in many cell types, defining NF-jB as an ubiquitous transcription factor involved in the regulation of a wide
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variety of genes through their jB sequences. NF-jB is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 (RelA) subunits. More recently the anti-apoptotic effects of NF-jB in TNF-induced apoptosis have been well established. For example, treatment of p65 deficient mouse fibroblasts and macrophages with TNFa resulted in a significant reduction in viability, whereas p65 +/+ cells were unaffected [8]. Inhibition of NF-jB nuclear translocalization enhanced apoptic killing but not by apoptotic stimuli that do not activate NF-jB [9]. In quiescent cells, NF-jB resides in the cytosol (except B cells) as a complex form, bound to an inhibitor protein IjBa. Stimulation of cells with various inducers, including TNFa triggers a series of signaling events that ultimately leads to the phosphorylation and proteolytic degradation of IjBa and liberation of NF-jB [3,4]. In the presence of proteasome inhibitors, N-terminally phosphorylated IjBa accumulates very rapidly, which suggests that its phosphorylation precedes its degradation. Thus, one wondered whether phosphorylation or degradation of IjBa is a direct reason for the dissociation of NF-jB from IjBa. By using several protease inhibitors, such as TPCK and TLCK, a number of laboratories have demonstrated that degradation, but not phosphorylation, is directly related to the activation of NF-jB, because these inhibitors can inhibit IjBa degradation and consequently inhibit NF-jB activation [69–71]. Although phosphorylation of IjBa does not cause its dissociation from NF-jB, recent studies have shown that the phosphorylated form of IjBa is preferentially degraded (see below). Phosphorylation of IjBa is regulated by the upstream kinase IKK complex [27–29] (Fig. 1). IKKa and IKKb preferentially form heterodimers and both can directly phosphorylate the critical serine sites of IjBa. Phosphopeptide mapping and sitedirected mutagenesis suggested that two residues, serines 32 and 36 of IjBa were phosphorylated in response to TNF treatment and activation of IKK (Fig. 1). Substitution of either the serine 32 or 36 blocks or slows down induction of IjBa degradation. 4.2. Ubiquitin–proteasome is required for IjBa degradation As discussed before IjBa is degraded by proteasome in response to TNF treatment. Proteasomes are large multi-subuint protease complexes that are localized in the nucleus and cytosol, and selectively degraded intracellular proteins. Proteasomes play a major role in the degradation of many proteins involved in cell cycling, proliferation, and apoptosis. Degradation by this pathway must first be recognized as substrates by components of the ubiquitin system that functions to mark proteins. Ubiquitin is first covalently ligated to target proteins by a multi-enzymatic system consisting
of Ubq-activating (E1), Ubq-conjugating (E2), and the Ubq-ligating (E3) enzymes [78–81]. The E1 hydrolyses ATP and forms a high-energy thioester between a systeine of its active site and the COOH terminus of ubiquitin. Activated E1 is then transferred to a reactive cysteine residue of the E2 enzyme, which forms thioester-linked complexes with ubiquitin in a similar fashion. The final transfer of ubiquitinated proteins is escorted to the proteasome where it undergoes final degradation and the ubquitin is released and recycled. E3 is generally required for the formation of multi-ubiquitin chains on the substrate, as step that facilitates efficient recognition of the substrate by the proteasome. The E3 is very heterogeneous, and most of its members are poorly characterized. The proteasomes are highly conserved and may exist in different forms in the cytosol of eukaryotic cells. At the core of these forms are the 20S and 26S proteasome. Only 26S proteasome can hydrolyze ubquitinated proteins. Once IjB is phosphorylated, it is subsequently polyubiquitinated at lysine 21 or 22 and degraded by the 26S proteasomes [72–75]. In contrast, some specific proteasome inhibitors such as lactacystin and PS-34 can inhibit the degradation of IjB and block the activation of NF-jB [75–78]. Besides the ubiquitin–proteasome pathway, there is another protease pathway, the calcium-activated neutral protease (calpain)-calpastatin system. This pathway is initially thought to be important in regulating turnover of protein kinases and key structural proteins in the cells. By using a specific fluorescent assay, Han et al. [82] observed a rapid increase in cytosolic calpain activity in TNF-treated cells. They subsequently showed that pretreatment with calpain or proteasome selective inhibitors only partially blocks up to 50% of TNF-inducible IjB proteolysis. However, pretreatment with both completely blocked IjB degradation, which suggests that calciumdependent calpastatin may also involve in the degradation of IjBa.
5. Crosstalk of proteolytic signaling between caspase activation and NF-jB pathway It is conceivable that there are numerous crosstalk between caspase activation and NF-jB pathway. It has been reported that in the absence of NF-jB activity, cellular susceptibility to TNF-induced apoptosis increases, whereas enforced activation of NF-jB protects cells against apoptosis [8,10]. Recent studies demonstrated that caspase-8 can proteolytically cleave RIP in its intermediate domain, result in an inhibition of NF-jB activity and enhance TNF-induced apoptosis [83–85]. In contrast, activation of NF-jB in response to TNF stimulation inhibits activation of caspase-8 through the activation of a group of gene products (TRAF1, TRAF2, cIAP1, and cIAP2) that functions
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cooperatively at the earliest checkpoint [86]. In addition, ubiquitin–proteasome has also been repeatedly reported to involve in the inhibition of caspases. For example, degradation of caspase-8 by ubiqutin–proteasome has been observed in p53-mediated apoptosis [87]. Finally, one of ubiquitin-protein ligase (Nedd4) has been recently identified to be cleaved by caspase-1, -3, -6, and -7 in TNF-induced apoptosis [88]. All these studies suggest that crosstalk between the two pathways may play an important role to maintain the balance between cell death and life.
6. Conclusion Proteolysis has been implicated in the control of many important biological functions initiated by TNF stimulation. It has become clear that caspases and ubiquitin– proteasome-mediated protein degradation have became major and fundamental ways to regulate TNF-induced apoptosis and NF-jB activation, respectively. Following TNF binding to its death receptor, caspases are activated by oligomeration and self-controlled proteolysis. Activated caspases cleaved substrates and led to cell death. Whereas activation of NF-jB is dependent on the degradation of IjBa. The latter is mainly regulated by ubiquitin–proteasome pathways. However, there is extensive crosstalk between the apoptotic and NF-jB signaling pathways. These crosstalks may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die. Although we have accumulated considerable knowledge of proteolysis involved in these two pathways, the mechanisms that shift the balance from one side to another are still largely unknown. The challenge in the future might be to understand the molecular event around the TNF receptor. One of important regulatory molecules associated with the receptor complex is the IAP [89–91]. In human, IAP is consisted of four members: c-IAP1, c-IAP2, XIAP, and NAIP. It has been reported that c-IAP1, c-IAP2, and XIAP have ubiquitin (E3) activity, which can bind to and inhibit specific caspases that function in the distal portions of the proteolytic cascases involved in apoptosis, such as caspase-3, -7, and -9[92,93]. On the other hand, IAP itself can be ubiquitined and degraded by proteasome and can enhance apoptosis [94]. Currently, it is not clear that what mechanism regulates ubiquitination process by IAP or the degradation process of IAP. Therefore, unraveling the molecular detail of IAP events will help to interpret the complexity of TNF signaling and the balance between the apoptosis and anti-apoptosis pathways. In addition, although inhibition of caspase activity is one of the mechanisms accounting for the anti-apoptotic effect of NF-jB in TNF-induce apoptosis, we have little knowledge on the target genes of NF-jB, which
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directly regulate cell survival. Thus, identification of target genes involved in cell cycle or differentiation may provide a new approach to understand how disregulation of proteolysis can lead to cancers. The TNF-induced signals and their crosstalk have been summarized in Fig. 1.
Acknowledgements I thank Dr Kenneth Zuckerman for helpful discussions and excellent suggestions. This work was supported by the Elsa U. Pardee Foundation Research Grant and the American Cancer Society’s Institutional Research Grant #93-032-07.
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Proteolytic signaling by TNFa: caspase activation and IjB degradation Xiaotang Hu Hematologic Malignancies Program (MDC44, MCC3142), Department of Interdisciplinary Oncology, Department of Internal Medicine, University of South Florida College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA Received 20 November 2002; received in revised form 5 February 2003; accepted 15 March 2003
Abstract Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IjBa. Caspases are activated using mechanisms of oligomeration and Ôselfcontrolled proteolysisÕ. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-jB is dependent on the degradation of IjBa. IjB kinase (IKK) phosphorylates IjBa at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-jB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-jB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-jB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die. Ó 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Tumor necrosis factor (TNF) alpha is a protein synthesized and secreted by mononuclear phagocytes in response to stimulation with bacterial endotoxin and other inducers [1]. TNF has been demonstrated to affect a wide range of biological activities of many cell types, including mediating the inflammatory response, regulating immune function, and triggering apoptosis of certain tumor cells. Among these biological activities TNFinduced apoptosis and activation of nuclear factor-kappa B (NF-jB) [2–5] have gained particular attention in recent years. Apoptosis, or programed cell death, plays a central role in development, maintenance of homeostasis, and host defense. Apoptotic cell death is characterized by cytoskeletal disruption, cell shrinkage, membrane Tel.: +1-813-979-6721; fax: +1-813-972-8468. E-mail address: hu@moffitt.usf.edu
blebbing, and nuclear DNA fragmentation as a result of the activation of endogenous proteases [6]. NF-jB has been reported to be an important mediator of inflammation, immune response, and cell differentiation through the regulation of multiple target genes [7]. After initiating TNF stimulation NF-jB translocates into the nucleus, where NF-jB binds to jB sequences in the promoters of target genes (cytokines, chemokines, and cell adhesion molecules) and stimulates transcription. One important progress in NF-jB studies is the finding that activated NF-jB is able to protect cells from TNFainduced cell killing [8–10]. Both apoptosis signal and activation of NF-jB are triggered by the TNF receptor following TNF binding, which are regulated by a group cysteine proteases caspases and ubiquitin-dependent degradation, respectively. In this mini review, I will attempt to summarize our current understanding of the proteolytic signaling in TNFmediated activation of caspase and NF-jB pathways.
1043-4666/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-4666(03)00107-8
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2. TNF death receptor signaling
3. Proteolytic signaling in caspase activation
In humans and mice, two TNF receptors have been identified, with molecular masses of 55 kDa (TNF-R1) and 75 kDa (TNF-R2). It has been shown that TNF-R1 are predominately expressed in human cells of epithelial origin, whereas TNF-R2 are the major receptors expressed in human hematopoietic cell lines [11,12]. Even so, TNF-R1, but not TNF-R2, plays an essential role in TNF-induced cell death in these cells [12]. Subsequent experiments demonstrated that expression of these two receptors on the cell surface is independently regulated. The cytoplasmic portion of the TNF-R1 has a domain approximately 80 amino acids long, referred to as the death domain for its role in TNF-mediated cell death, whereas the cytoplasmic portion of the TNF-R2 lacks this death domain [13]. The receptors contains Ôdeath domainÕ are thus referred to as death receptors. Besides TNF-R1 the other death receptors are CD95 (Fas or Apo1), DR3 (TRAMP or Apo3), DR4 (TRAIL-R1), and DR5 (Apo2, or TRAIL-R2). All these death receptors belong to the members of TNF superfamily receptors, which are defined by similar, cysteine-rich extra cellular domains [14–20]. In 1999, an inhibitory protein termed silencer of death domains (SODDs), which is associated with death domain of TNF-R1, but not with those of TNF-R2, was identified [21]. Following binding its death receptor on the plasma membrane, TNF induces the receptor trimerization and the release SODD from the intracellular domain (ICD) of TNF-R1. The resulting aggregated ICD is recognized by an adapter protein, TNF receptor-associated death domain (TRADD) [22]. TRADD functions as a platform that recruits additional adapter proteins including receptor-interacting protein (RIP) [23], TNF-R-associated factor 2 (TRAF2) [24], Fas-associated death domain (FADD) [25], and cellular inhibitor of apoptosis protein (IAP) (Fig. 1, also see the Section 6). FADD contains two functionally and structurally distinct domains: a C-terminal DD that is necessary for its recruitment of the TNF-R1 complex and an N-terminal death effector domain (DED) that promotes activation of a downstream proteolytic reaction and caspase cascade, which finally lead to apoptosis. In contrast, TRAF2 and RIP activate the NF-jB-inducing kinase (NIK) [26], which in turn activates IjB kinase (IKK) [27–29] (Fig. 1). IKK complex consists of two catalytic subunits IKKa and IKKb, and a regulatory subunit IKKc. Activation of the IKK complex requires phosphorylation of two serine residues located in the Ôactivation loopÕ within the kinase domain of IKKa and IKKb. The activated IKK complex phosphorylates IjBa, causing IjBa degradation by proteasome and allowing NF-jB to translocate to the nucleus to activate transcription (Fig. 1).
3.1. Caspase family and caspase activation
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Based on our current knowledge, caspases play an essential role during TNF-induced apoptosis. Although many molecular events may involve in cell death, activation of caspase cascade has been consistently involved in apoptosis. The term caspase resulted from their two key characteristics: cysteine proteases and aspartases. The caspase family was initially discovered following a search of human cDNA libraries for sequences homologous to ced-3, a cell death gene described in the nematode worm, Caenorhabditis elegans [30,31]. The first mammalian homologue of ced-3 identified is interleukin-1b converting enzyme (ICE) [32,33]. Subsequently, a number of mammalian ced-3 homologues (at least 14 mammalian members of the caspase family) were discovered [34,35]: caspase-1 (ICE), caspase-2 (ICH-1), caspase-3 (CPP32), caspase-4 (TX, ICH-2, ICErel-ll), caspase-5 (ICErel-lll), caspase-6 (Mch2), caspase-7 (mch3, ICE-LAP3, CMH-1), caspase-8 (MACH, FLICE, Mch5), caspase-9 (ICE-LAP6, Mch6), caspase-10 (Mch4, FLICE2), caspase-11 (ICH3), caspase-12, caspase-13 (ERICE), and caspase-14 (MICE). Based on their structures and functions caspases can be divided into three subgroups. Group I caspases are ICE related family (caspase-1, -4, -5, -11, -12, -13, and -14), which mainly involve in inflammation response, while group II (caspase-2, -3, and -7), and group III (caspase-6, -8, -9, and -10) are mostly linked to apoptosis in general. Caspases are synthesized as inactive precursors termed procaspases, which are converted to active proteases during apoptosis through auto-regulated proteolytic process. Most long prodomains in the group III can mediate oligomerization of the procaspase molecules and recruit caspase precursors to the specific death domain complexes, resulting in activation of caspase activation. Therefore, group III caspases are referred to as initiators or upstream caspases and group II caspases are called executive, effector, or downstream caspases. Caspase X-ray crystal structure shows that the caspase tetramer is comprised of two large subunits and two small subuints [36–38]. Both the large and the small subunits can be liberated from the proenzyme by cleavage at Asp-X bonds and become active form of the enzyme [39–41] (Fig. 2). Binding of TNFa to its death receptor recruits procaspase to an oligomeric activation complex using the adapter protein FADD. Following recruitment of multiple caspase proenzyme to a common oligomerization site, the low level of endogenous catalytic activity accumulated in the death receptor complex is sufficient to initiate full catalytic activation by proteolysis of the Asp-X site [39,40] (Fig. 1). The first study implying
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Fig. 1. An overview of TNF-induced proteolytic signals and their interactions. Binding of TNF to its death receptor, resulting in the release of SODD and recruiting several cytoplasmic adaptor proteins to form a receptor complex. The complex initiates activation of caspases and NF-jB, which lead to apoptosis and cell proliferation (anti-apoptosis), respectively. There are numerous crosstalk between caspase activation and NF-jB pathways.
oligomerization in caspase activation was involved in activation of caspase-1 [43]. Subsequently, similar results were obtained with caspase 2 [44] and several other caspases [42]. Additional support for the oligomerization is from the observation that caspase-3, -8, and -9 were activated by proteolysis in vitro when oligomerization was induced [39–41]. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site as described before. The activated upstream and downstream caspases then cleave various cellular proteins, leading to apoptotic cell death (Fig. 1).
3.2. Caspase-8 and caspase-3 Among the caspases, caspase-8 and -3 may play key role in apoptosis cascade [45]. Caspase-8 is an upstream kinase, which has been demonstrated by a number of experiments. First, recombinant active caspase-8 does not induce DNA fragmentation in a cell-free system, suggesting that caspase-8 itself is not able to induce apoptosis. However, in the presence of cell extracts from unstimulated cells, caspase-8 induced a complete DNA fragmentation [46]. Therefore, some cytosolic factors in the extracts are required for caspase-8-initiated
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Fig. 2. Procaspase structure and cleavage sites. Procaspases contain two large and two small subunits. TNF recruits formation of death receptor complex, which in turn triggers oligomerization of procaspases. The latter leads to procaspases self-proteolysis by cleavage of the Asp-X site at the junction between the large and small subunits. Active upstream caspases then process and activate downstream procaspases.
apoptosis. Second, recombinant caspase-8 can directly cleave caspase-3, -4, -7, -9, and -10 in the absence of cytosolic extracts. In contrast, caspase-2 and -6 are efficiently cleaved only in the presence of the extract. These observations suggest that caspase-8 is proapoptotic protease. Caspase-8 not only participates in the caspase cascade pathway, but also involve in cytochrome c-mediated cell death. It has been reported that cytochrome c, an essential component of the respiratory chain for the generation of ATP, also plays an important role in apoptosis. Cytochrome c normally resides in the space between the outer and inner membrane of mitochondria and its release to cytosol is closely related to apoptotic process. Cytochrome c activates Apaf-1, in the presence of dATP, which in turn activates procaspase-9. Activated caspase-9 then cleaves downstream caspases and leads to apoptosis [47]. Release of cytochrome c is regulated by the Bcl family proteins. Bid, a BH3 domain-containing proapoptotic Bcl-2 family protein, is activated by caspase-8 in response to TNFR1/Fas death receptor activation [48–50]. Activated Bid is then translocated to mitochondria and induces cytochrome c release. This Bid-mediated pathway is critical in some cell apoptosis (such as hepatocyte) induced by TNF-R1 signal, because direct activation of cytosolic caspase cascade appears inefficient in these cells. Caspase-3 has been implicated as a major and key protease that is activated during the early stages of apoptosis [51]. Active caspase-3 consists of a heterodimer of 17 and 12 kDa subunits, which are derived from the 32 kDa proenzyme by proteolysis [52–54]. In its active form, caspase-3 proteolytically cleaves and activates other caspases, as well as many relevant targets (see below) in the cytoplasm. Therefore, caspase-3 is an important mediator for triggering caspase cascade and scleaving substrates. 3.3. Cleavage of caspase substrates A number of proteins have been shown to be substrates of caspases during apoptotic cell death [55]. These proteins involve in DNA repair processes, cell
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cycle progression, or as components of apoptosis inhibitors. Caspases either inactivate the normal biochemical function of their substrates or activate them by removal of regulatory domains. Alternatively, caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold. For example, nuclear DNA fragmentation is a result of chromatin condensation and DNA brokerage into small fragments. These processes are regulated by caspase-regulated DNA fragmentation factor (DFF) and acinus. DFF is composed of caspaseactivated DNase (CAD) and its inhibitor ICAD [56]. During apoptotic process ICAD is cleaved by caspase-3 and -7, releasing CAD to degrade nuclear DNA [57]. Acinus (apoptotic chromatin condensation inducer in the nucleus) is a nuclear protein that contains a potential DNA/RNA-binding motif. Acinus has been reported to be activated by proteolytic cleavage of caspase-3 and activated acinus induces chromatin condensation without effecting DNA fragmentation [58]. Caspases also cleave many cytoskeletal proteins. Lamins, which are required for the nuclear envelope, are cleaved by caspase-6, leading to nuclear fragmentation [59,60]. Gelsolin, a cytoplasmic F actin-depolymerizing enzyme, which is required for protection of cells from membrane blebbing, is cleaved by caspase-3 to yield a fragment with constitutive activity [61]. Actin is highly conserved proteins that is expressed in all eukaryotic cells. Actin filaments can form both stable and labile structures, and is crucial components of microvilli and the contractile apparatus of cells. During apoptosis actin is cleaved by effector caspases in certain cells [55]. PAK2, a serine/threonine kinse downstream of Rac and cdc42, is cleaved by caspase-3. Poly ADP-ribose polymerase (PARP) is a nuclear enzyme that senses DNA nicks and catalyzes the ADP-ribosylation of histone and other nuclear proteins in order to facilitate DNA repair. Studies from several laboratories demonstrated that PARP is cleaved at a single site by caspase-3, which separates the N-terminal DNA-binding domain from the catalytic domain and inactivates the enzymatic activity [62–64]. More information about caspase substrates can be found in several review papers [65,66].
4. Proteolytic signaling in IjB phosphorylation and degradation 4.1. Degradation of IjB is required for NF-jB activation NF-jB is a transcription factor first identified in B cells and is crucial for lg gene function [67,68]. It was considered as a B cell specific transcription factor. However, it soon became evident that NF-jB exists in many cell types, defining NF-jB as an ubiquitous transcription factor involved in the regulation of a wide
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variety of genes through their jB sequences. NF-jB is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 (RelA) subunits. More recently the anti-apoptotic effects of NF-jB in TNF-induced apoptosis have been well established. For example, treatment of p65 deficient mouse fibroblasts and macrophages with TNFa resulted in a significant reduction in viability, whereas p65 +/+ cells were unaffected [8]. Inhibition of NF-jB nuclear translocalization enhanced apoptic killing but not by apoptotic stimuli that do not activate NF-jB [9]. In quiescent cells, NF-jB resides in the cytosol (except B cells) as a complex form, bound to an inhibitor protein IjBa. Stimulation of cells with various inducers, including TNFa triggers a series of signaling events that ultimately leads to the phosphorylation and proteolytic degradation of IjBa and liberation of NF-jB [3,4]. In the presence of proteasome inhibitors, N-terminally phosphorylated IjBa accumulates very rapidly, which suggests that its phosphorylation precedes its degradation. Thus, one wondered whether phosphorylation or degradation of IjBa is a direct reason for the dissociation of NF-jB from IjBa. By using several protease inhibitors, such as TPCK and TLCK, a number of laboratories have demonstrated that degradation, but not phosphorylation, is directly related to the activation of NF-jB, because these inhibitors can inhibit IjBa degradation and consequently inhibit NF-jB activation [69–71]. Although phosphorylation of IjBa does not cause its dissociation from NF-jB, recent studies have shown that the phosphorylated form of IjBa is preferentially degraded (see below). Phosphorylation of IjBa is regulated by the upstream kinase IKK complex [27–29] (Fig. 1). IKKa and IKKb preferentially form heterodimers and both can directly phosphorylate the critical serine sites of IjBa. Phosphopeptide mapping and sitedirected mutagenesis suggested that two residues, serines 32 and 36 of IjBa were phosphorylated in response to TNF treatment and activation of IKK (Fig. 1). Substitution of either the serine 32 or 36 blocks or slows down induction of IjBa degradation. 4.2. Ubiquitin–proteasome is required for IjBa degradation As discussed before IjBa is degraded by proteasome in response to TNF treatment. Proteasomes are large multi-subuint protease complexes that are localized in the nucleus and cytosol, and selectively degraded intracellular proteins. Proteasomes play a major role in the degradation of many proteins involved in cell cycling, proliferation, and apoptosis. Degradation by this pathway must first be recognized as substrates by components of the ubiquitin system that functions to mark proteins. Ubiquitin is first covalently ligated to target proteins by a multi-enzymatic system consisting
of Ubq-activating (E1), Ubq-conjugating (E2), and the Ubq-ligating (E3) enzymes [78–81]. The E1 hydrolyses ATP and forms a high-energy thioester between a systeine of its active site and the COOH terminus of ubiquitin. Activated E1 is then transferred to a reactive cysteine residue of the E2 enzyme, which forms thioester-linked complexes with ubiquitin in a similar fashion. The final transfer of ubiquitinated proteins is escorted to the proteasome where it undergoes final degradation and the ubquitin is released and recycled. E3 is generally required for the formation of multi-ubiquitin chains on the substrate, as step that facilitates efficient recognition of the substrate by the proteasome. The E3 is very heterogeneous, and most of its members are poorly characterized. The proteasomes are highly conserved and may exist in different forms in the cytosol of eukaryotic cells. At the core of these forms are the 20S and 26S proteasome. Only 26S proteasome can hydrolyze ubquitinated proteins. Once IjB is phosphorylated, it is subsequently polyubiquitinated at lysine 21 or 22 and degraded by the 26S proteasomes [72–75]. In contrast, some specific proteasome inhibitors such as lactacystin and PS-34 can inhibit the degradation of IjB and block the activation of NF-jB [75–78]. Besides the ubiquitin–proteasome pathway, there is another protease pathway, the calcium-activated neutral protease (calpain)-calpastatin system. This pathway is initially thought to be important in regulating turnover of protein kinases and key structural proteins in the cells. By using a specific fluorescent assay, Han et al. [82] observed a rapid increase in cytosolic calpain activity in TNF-treated cells. They subsequently showed that pretreatment with calpain or proteasome selective inhibitors only partially blocks up to 50% of TNF-inducible IjB proteolysis. However, pretreatment with both completely blocked IjB degradation, which suggests that calciumdependent calpastatin may also involve in the degradation of IjBa.
5. Crosstalk of proteolytic signaling between caspase activation and NF-jB pathway It is conceivable that there are numerous crosstalk between caspase activation and NF-jB pathway. It has been reported that in the absence of NF-jB activity, cellular susceptibility to TNF-induced apoptosis increases, whereas enforced activation of NF-jB protects cells against apoptosis [8,10]. Recent studies demonstrated that caspase-8 can proteolytically cleave RIP in its intermediate domain, result in an inhibition of NF-jB activity and enhance TNF-induced apoptosis [83–85]. In contrast, activation of NF-jB in response to TNF stimulation inhibits activation of caspase-8 through the activation of a group of gene products (TRAF1, TRAF2, cIAP1, and cIAP2) that functions
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cooperatively at the earliest checkpoint [86]. In addition, ubiquitin–proteasome has also been repeatedly reported to involve in the inhibition of caspases. For example, degradation of caspase-8 by ubiqutin–proteasome has been observed in p53-mediated apoptosis [87]. Finally, one of ubiquitin-protein ligase (Nedd4) has been recently identified to be cleaved by caspase-1, -3, -6, and -7 in TNF-induced apoptosis [88]. All these studies suggest that crosstalk between the two pathways may play an important role to maintain the balance between cell death and life.
6. Conclusion Proteolysis has been implicated in the control of many important biological functions initiated by TNF stimulation. It has become clear that caspases and ubiquitin– proteasome-mediated protein degradation have became major and fundamental ways to regulate TNF-induced apoptosis and NF-jB activation, respectively. Following TNF binding to its death receptor, caspases are activated by oligomeration and self-controlled proteolysis. Activated caspases cleaved substrates and led to cell death. Whereas activation of NF-jB is dependent on the degradation of IjBa. The latter is mainly regulated by ubiquitin–proteasome pathways. However, there is extensive crosstalk between the apoptotic and NF-jB signaling pathways. These crosstalks may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die. Although we have accumulated considerable knowledge of proteolysis involved in these two pathways, the mechanisms that shift the balance from one side to another are still largely unknown. The challenge in the future might be to understand the molecular event around the TNF receptor. One of important regulatory molecules associated with the receptor complex is the IAP [89–91]. In human, IAP is consisted of four members: c-IAP1, c-IAP2, XIAP, and NAIP. It has been reported that c-IAP1, c-IAP2, and XIAP have ubiquitin (E3) activity, which can bind to and inhibit specific caspases that function in the distal portions of the proteolytic cascases involved in apoptosis, such as caspase-3, -7, and -9[92,93]. On the other hand, IAP itself can be ubiquitined and degraded by proteasome and can enhance apoptosis [94]. Currently, it is not clear that what mechanism regulates ubiquitination process by IAP or the degradation process of IAP. Therefore, unraveling the molecular detail of IAP events will help to interpret the complexity of TNF signaling and the balance between the apoptosis and anti-apoptosis pathways. In addition, although inhibition of caspase activity is one of the mechanisms accounting for the anti-apoptotic effect of NF-jB in TNF-induce apoptosis, we have little knowledge on the target genes of NF-jB, which
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directly regulate cell survival. Thus, identification of target genes involved in cell cycle or differentiation may provide a new approach to understand how disregulation of proteolysis can lead to cancers. The TNF-induced signals and their crosstalk have been summarized in Fig. 1.
Acknowledgements I thank Dr Kenneth Zuckerman for helpful discussions and excellent suggestions. This work was supported by the Elsa U. Pardee Foundation Research Grant and the American Cancer Society’s Institutional Research Grant #93-032-07.
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