INTRODUCTION

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Programmed cell death, or apoptosis, is a physiological process of cellular autodestruction. Apoptosis plays critical roles in development, maintenance of homeostasis, and host defense in multicellular organisms. Dysregulation of this process is implicated in various diseases ranging from cancer and autoimmune disorders to neurodegenerative diseases and ischemic injuries (210).

Diverse cell types can be triggered to undergo apoptosis by various signals derived from either the extracellular or intracellular milieu. Cells undergoing apoptosis exhibit a series of characteristic morphological changes, including plasma membrane blebbing, cell body shrinkage, and formation of membrane-bound apoptotic bodies, which in vivo are quickly engulfed by neighboring healthy cells (103). Thus, during apoptosis, intracellular contents are not released and potentially harmful inflammatory responses are prevented. Apoptosis is also accompanied by certain biochemical changes, notably the appearance of discrete DNA fragments on conventional gel electrophoresis (due to cleavage between nucleosomes), the flipping of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane, and limited cleavage of various cellular proteins.

These stereotypic changes are manifestations of an intrinsic suicide machinery that has been conserved through evolution. The core component of this machinery is a proteolytic system involving a family of proteases known as caspases. The term caspase denotes two key characteristics of these proteases: (i) they are cysteine proteases and use cysteine as the nucleophilic group for substrate cleavage and (ii) they are aspases and cleave the peptide bond C-terminal to aspartic acid residues (2). That caspases play an essential role in apoptosis is based on three observations. First, synthetic or natural inhibitors of caspases effectively abrogate apoptosis induced by diverse apoptotic stimuli. Second, animals lacking certain caspases show profound defects in apoptosis. Third, caspases are responsible for most proteolytic cleavages that lead to apoptosis. It is noteworthy that mammalian caspases have evolved additional roles in inflammatory responses.

Synthesized as latent precursors or procaspases, caspases are converted to active proteases during apoptosis through an intricately regulated proteolytic process. The proteolytic processing occurs at critical aspartic acid residues that conform to the caspase substrate recognition consensus. Consequently, caspases often function in cascades. In such a caspase cascade, an upstream caspase (initiator caspase) is activated by its interaction with a caspase adapter(s). This activation represents a key regulatory step in apoptosis and is controlled by both pro- and antiapoptotic proteins. Once activated, the initiator caspase processes and activates one or more downstream caspases (effector or executioner caspases). The activated effector caspases then cleave various cellular proteins, leading to apoptotic cell death. In this review, we will discuss various aspects of this emerging family of proteases, with particular emphasis on recent developments.

CASPASE STRUCTURE AND ENZYMATIC ACTIVITY

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Structures of Procaspases

The first caspase, caspase-1, also known as ICE (interleukin-1 converting enzyme), was identified due to its ability to convert the precursor of interleukin-1 (IL-1) to its mature form, a potent mediator of inflammation (17, 211). Subsequent cloning of ced-3 (ced, cell death abnormal), a proapoptotic gene in Caenorhabditis elegans (see below), revealed that it encodes a protein highly homologous to ICE (254). Since then, at least 14 mammalian caspases and five Drosophila caspases have been cloned. Structurally, all procaspases contain a highly homologous protease domain, the signature motif of this family of proteases (Fig. 1). This domain can be further divided into two subunits, a large subunit of approximately 20 kDa (p20) and a small subunit of approximately 10 kDa (p10). In some procaspases, there is a short linker (about 10 amino acids) between the large and small subunits. Based on the sequence similarity among the protease domains, caspases can be divided into three groups. The first group consists of inflammatory caspases: caspase-1, -4, -5, -11, -12, -13, and -14. The second group contains caspase-2 and -9. The rest of the caspases form the third group (Fig. 1).

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Mammalian caspase family and C. elegans caspase CED-3. All mammalian caspases are of human origin except for murine caspase-11 and -12, for which no human counterparts have been identified yet. Phylogenetic relationships are based on sequence similarity among the protease domains. Alternative names are listed in parentheses after each caspase. Dotted box, DED domains; wavy boxe, CARD domain. Substrate preferences at the P1 to P4 positions are indicated. Based on the substrate specificity, caspases are divided into three groups (indicated in parentheses) (212).

Each procaspase also contains a prodomain or NH₂-terminal peptide of variable length. Initiator apoptotic caspases and inflammatory caspases contain prodomains of over 100 amino acids, while the prodomains in effector caspases are usually less than 30 amino acids. The long prodomains contain distinct motifs, notably the death effector domain (DED) and the caspase recruitment domain (CARD). A novel motif termed the death-inducing domain (DID) was recently identified. Procaspase-8 and -10 each contain two tandem copies of DEDs (8, 54, 145), whereas the CARD domain is found in caspases-1, -2, -4, -5, and -9 as well as in CED-3 (86). DID is present in the Drosophila caspase DREDD (92). These domains mediate homophilic interaction between procaspases and their adapters and play important roles in procaspase activation (see below). In contrast, the short prodomains of executioner caspases are unlikely to mediate protein-protein interactions. Rather, they seem to inhibit caspase activation (see below).

The three-dimensional structure of a procaspase has not been determined. However, the structures of caspase prodomains, particularly those of the DED and the CARD domains, have already been solved (31, 48, 159). DED, CARD, and a related domain, DD (death domain, present in cell death adapter proteins), all consist of six alpha-helices and have similar overall folds. Nonetheless, there are some major differences among them. While charge-charge interactions govern CARD-CARD and DD-DD association, hydrophobic interactions govern DED-DED interaction.

Early studies showed that active caspase-1 and -3 consist of the large and small subunits, which are released from the procaspases through proteolytic processing, and that both subunits are required for the protease activity (151, 211). Subsequently, the three-dimensional structures of these two caspases as well as that of caspase-8, each complexed with their corresponding tetrapeptide aldehyde inhibitors, have been determined (7, 169, 224, 230, 235). These structures reveal that a mature caspase is a tetramer (homodimer of the p20 and p10 heterodimers arranged in twofold rotational symmetry), with the two adjacent small subunits surrounded by two large subunits (Fig. 2). Each p20-p10 heterodimer forms a single globular domain, and the core of the globular domain is a six-stranded beta-sheet flanked on either side by alpha-helices. These two heterodimers associate with each other primarily through the interaction between the p10 subunits.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Structure of the caspase-3 tetramer in complex with Ac-DEVD-CHO. The p17 subunits are shown in blue and green, the p12 subunits in red and pink, and the bound inhibitors in yellow. The C termini of p17 and N termini of p12 are indicated. Reproduced with permission from J. Rotonda et al., Nature Structure Biology 3:619-625, 1996.

Each caspase tetramer has two cavity-shaped active sites formed by amino acids from both the p20 and p10 subunits, and these two active sites are likely to function independently. In the active site, a cysteine (Cys-285 in the p20 of caspase-1) is positioned close to the imidazole of a histidine (His-237, p20), which attracts the proton from the cysteine and enhances its nucleophilic property. The side chains of Arg-179 (p20) and Arg-341 (p10) in the S1 site participate in a direct charge-charge interaction with the aspartic acid of a substrate, contributing to the selective recognition of Asp at the P1 position of a substrate. The amino acids from the small subunit form the S2 and S3 sites. The side chains of amino acids at P2 and P3 (the second and third amino acids N-terminal to the cleavage site Asp) are mainly exposed to solvent, consistent with the less stringent requirement at these two positions in caspase substrates. One of the major differences among caspases is the S4 subsite. For example, in caspase-1, the S4 subsite is a large and shallow hydrophobic depression, and the corresponding site in caspase-3 is a narrow pocket, which accommodate a P4 tyrosyl and aspartyl side chain, respectively.

Because the small and large caspase subunits are derived from a single procaspase molecule following cleavages at the critical aspartic acid residues, the structures of active caspases are instrumental in understanding how caspases are activated (Fig. 2). The most significant features are the relative distance between the p20 C terminus and the p10 N terminus in cis (the p20-p10 pairs that form an active site) versus that in trans (the pairs that do not). The latter is only 5.3 Å, whereas the former is approximately 65 Å, too far apart for the linker region to span. Based on this critical structural feature, two alternative models have been put forth to explain the activation mechanism (224, 235). The process and association model postulates that the p20-p10 heterodimer is derived from the same procaspase molecule. This model requires an extensive reordering of the proenzyme to reconcile the fact that the p20 C terminus and the p10 N terminus in cis are distant from each other. Subsequent removal of the linker and prodomains allows the two heterodimers to form the tetrameric structure. In contrast, the association and process model predicts that the two molecules of zymogen associate and interdigitate, naturally forming a mature caspase-like intermediate. The linker and prodomain are subsequently removed to form a mature enzyme. The latter model fits well with the finding that procaspases are activated by induced proximity of two or more procaspase molecules (see below).

Early enzyme inhibition studies revealed that caspase-1 is a cysteine protease. This caspase was potently and irreversibly inhibited by a peptide substrate carrying diazomethylketone, a highly specific modifying group for cysteine proteases (211). The caspase-1 activity was also attenuated by other cysteine-modifying reagents such as N-ethylmaleimide and iodoacetamide, but not by inhibitors of serine, aspartate, or metalloproteases. In addition, caspase-1 was readily labeled by [¹⁴C]iodoacetate, a reagent that is commonly used to identify the active-site cysteine (211). The results from these enzymatic studies were subsequently confirmed by site-directed mutagenesis studies and the three-dimensional structures of caspase-1, -3, and -8. These structures reveal a catalytic diad of Cys and His in the active site (see above). Similar to other cysteine proteases, caspases are sensitive to transition metal ions such as Zn²⁺ due to the interaction of these ions with the catalytic thiol. This property of caspases may account for the observation that Zn²⁺ can prevent apoptosis (157, 204). In contrast, Ca²⁺ does not appear to affect caspase activity despite its reported role in apoptosis.

Caspases are among the most specific endopeptidases. An aspartic acid residue is almost absolutely required at the P1 position. For example, any substitution in this position in the caspase-1 substrates led to a >100-fold decrease in cleavage activity (88, 187). The P2 to P4 positions also show high preference for certain amino acids. Based on their substrates, the preferred recognition sequences for caspase-1 and -3 were determined to be Tyr-Val-Ala-Asp (P4 to P1) and Asp-Glu-Val-Asp, respectively (151, 211).

The substrate specificity of 10 human caspases was determined using a systematic approach involving combinatory peptide fluorogenic substrates (212). Based on these results, caspases are divided into three groups (Fig. 1). The optimal recognition motif for the first group is WEHD. This group includes the inflammatory caspase-1 and its close homologues caspase-4 and -5. The second group prefers the sequence DEXD (where X is V, T, or H), with a high selection for Asp at the P4 position. This second group consists of apoptotic caspases with either long or short domains (caspase-2, -3, and -7). DEXD is often found in proteins that are cleaved by caspases during apoptosis, consistent with the role of caspase-3 and -7 as effector caspases. The inclusion of caspase-2, which contains a long prodomain, may imply that it can function as both an initiator and an effector caspase. For caspase-2, the amino acid at the P5 position also affects the efficiency of cleavage (205). The third group (caspase-6, -8, -9, and -10) preferentially recognize (L/V)EXD, which resembles the processing site for effector caspases and is consistent with caspase-8, -9, and -10 being initiator caspases. Categorizing caspases based on their substrate specificity gives a different result than doing so based on sequence homology among the caspases (see above). This difference is related to the fact that only small numbers of amino acids determine the substrate specificity.

In contrast to the preference for the P1 to P4 position, a methylamine group at P1' (the position immediately C-terminal to the cleavage site) is sufficient for cleavage (211). Further analysis of protein substrates has revealed that small amino acids such as Ala, Ser, and Asn are preferred at P1'. It is noteworthy, however, that the in vitro caspase substrate specificity, determined by the tetrapeptide derivatives, may differ from that in vivo. In vivo, caspase specificity can be influenced by other factors, such as the context of the recognition sequence in the target proteins and the proximity of caspases and the target proteins. The high specificity of caspases allows them to perform limited and selective cleavages, often in the interdomain regions of the target proteins, which can lead to protein activation as well as inactivation (see below).

The enzymatic activities of the caspases within the same group vary substantially despite their similar substrate preference. For example, the peptide-based inhibitor WEHD-aldehyde has a K_i of 0.056 and 97 nM for caspase-1 and caspase-4, respectively, indicating that caspase-1 is much more active than caspase-4 (63). A similar study also revealed that caspase-8 is substantially more active than its close homologue caspase-10 (63). The significance of such differences is not clear, although they may reflect distinct roles of these caspases in apoptosis and inflammation. One possibility is that the precursor of a weak caspase may be activated before that of a more active one, leading to the amplification of death signals.

MECHANISMS OF PROCASPASE ACTIVATION

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Initiator Caspases

The identification of the first caspase (caspase-1 or ICE) came with the realization that a mature caspase is generated by proteolytic processing at critical aspartic acids and hence a mature caspase may activate its precursor as well as other procaspases (211). While this observation could readily explain the activation of effector caspases (see below), how is the first initiator caspase processed from its precursor? Early studies showed that expression of caspase precursors in Escherichia coli led to their full and precise activation (154, 241, 242), supporting a self-activation model. However, the zymogen concentration in these studies was much higher than that in vivo, and the physiological relevance of this activation was unclear.

More definitive evidence for the self-activation of procaspase came from the studies on procaspase-8 (also known as FLICE, MACH, and Mch4), a caspase linked to cell surface death receptors such as Fas (see below). When Fas is aggregated by the Fas ligand, procaspase-8 is recruited to the death receptor and subsequently becomes activated (8, 145), suggesting that activation of initiator procaspases may be triggered by their oligomerization. Controlled oligomerization of procaspase-8 as well as procaspase-1 indeed leads to enhancement of their cell death activity in mammalian cells (133, 147, 244). Procaspase-8 possesses weak protease activity (147), which may allow it to cleave other procaspase-8's when in close proximity. The most convincing evidence so far for the oligomerization model is perhaps the finding that, in vitro, induced proximity of procaspase-8 triggers the otherwise stable zymogen to undergo autocleavage, generating mature caspase-8 (244) (Fig. 3). Subsequent experiments suggested that initiator procaspases linked to mitochondria (e.g., CED-3 and caspase-9) are also activated by adapter protein-mediated oligomerization (193, 245, 269) (see below). Oligomerization is now recognized as a universal mechanism of initiator caspase activation. Interestingly, this mechanism is reminiscent of the activation of receptor tyrosine kinases by ligand-mediated oligomerization, and it appears that oligomerization is a general mechanism for activating signal transduction pathways.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Activation of the caspase cascade. Apoptotic signals trigger oligomerization of death adapter proteins (e.g., CED-4, Apaf-1, and FADD). Death adapter oligomers in turn oligomerize procaspases, which leads to their autoproteolytic activation. Active initiator caspases then process and activate effector procaspases. Effector caspases cleave various death substrates to induce apoptosis.

As elegant as the oligomerization model is, exactly how oligomerization leads to caspase activation remains an outstanding question. Recent studies showed that procaspase-9 mutants lacking processing sites could still induce apoptosis (198) and that activated caspase-9 retains its prodomain and may function as a holoenzyme complexed with adapter proteins (167). These results suggest that caspase self-cleavage may not be the primary event in the oligomerization-mediated caspase activation. In addition, induced proximity of two procaspase-9 mutants, one lacking caspase activity but containing intact processing sites while the other has intact caspase activity but lacks processing sites, generated no processing (269). This result is contradictory to the simple model that procaspase molecules process each other when in close proximity.

While the initiator procaspases are activated by oligomerization, effector procaspases are often activated by other proteases, most commonly by initiator caspases but also by other proteases (trans activation) (Fig. 3). Although the extent to which this trans activation occurs in vivo is not fully understood, at least in vitro procaspase-3 and -7 can be activated by caspase-6, -8, -9, and -10 (54, 124, 146, 154, 199, 247). In vivo, the best illustrations of caspase cascades come from studies on Fas and mitochondrial apoptosis pathways (see below). Effector caspases as well as some initiator caspases can also be activated in vitro by granzyme B, an Asp-specific serine protease (47, 54, 160, 206, 247, 265). Granzyme B is an effector for cytotoxic T lymphocytes (CTL) and natural killer cells and is delivered into virally infected cells during CTL- and natural killer cell-mediated apoptosis.

The trans activation appears to be a two-step process, as illustrated in the activation of caspase-3 by granzyme B (135). The first cleavage is carried out by granzyme B and occurs in the linker region between the large and small subunits to generate a partially active intermediate that may resemble the mature caspase in structure. This linker apparently plays a critical role in preventing caspase self-activation. It is too short to allow the large and small subunits within a procaspase molecule to form a heterodimer (see above). It may also prevent the association of two procaspase molecules to spontaneously form a tetramer. Artificially reversing the order of the large and small subunits within a procaspase, which is likely to ameliorate the structural constraint imposed by the linker, results in an active enzyme (194).

In the second step, the partially active intermediate processes itself to generate the fully active, mature caspase. This self-cleavage severs the short inhibitory prodomain from the large subunit. The short prodomains present in the Drosophila effector caspases drICE and DCP-1 also play an inhibitory role in caspase activation, and deletion of these prodomains enhances the caspase apoptotic activity (57, 191). The importance of the second step is underlined by the observation that certain members of a group of apoptotic inhibitors known as inhibitors of apoptosis (IAPs) (see below) prevent apoptosis by impeding this autocleavage (40). In addition to caspase-3 autoprocessing, the cleavage that separates the prodomain from the large subunit may be carried out in trans by other caspases, as in tumor necrosis factor alpha (TNF-)-mediated apoptosis (156). Engagement of TNF receptors by TNF- activates both caspase-8 and caspase-9 (see below). While caspase-8 functions similarly to granzyme B and severs the linker between the large and small domains, caspase-9 removes the prodomain. Interference with caspase-9 activation by the adenovirus protein E1B prevents the completion of caspase-3 processing and apoptosis.

Interestingly, procaspase-3 can also be activated by short peptides containing the arginine-glycine-aspartate (RGD) motif (12). This motif, present in many integrin ligands, binds to integrin extracellularly. RGD peptides were found to directly induce procaspase-3 autoprocessing and subsequent apoptosis, independent of the integrin-binding ability of RGD. The mechanism of this activation is not fully understood. However, procaspase-3 contains an RGD sequence near the active site and an RGD-binding motif (aspartate-aspartate-methionine [DDM]) at the linker region. Thus, these two motifs may interact to keep procaspase-3 in a self-inhibitory conformation.

Similar to apoptotic caspases, the caspases acting in inflammation, notably caspase-1, can also undergo full activation when expressed in Escherichia coli or refolded from denaturing conditions (161, 242). This activation is self-catalyzed, because mutations of the active-site cysteine abolish procaspase-1 processing. Activation of caspase-1 appears to be a quite complicated process and involves a series of processing intermediates with increasing enzymatic activity (242). Procaspase-1 also possesses a protease activity that is weaker than those of the intermediates. What initiates procaspase-1 activation in vivo is still unsettled. However, like procaspase-8, forced oligomerization of procaspase-1 leads to its activation (244), and thus there may be an adapter protein(s) facilitating procaspase-1 oligomerization in vivo.

Murine caspase-11 also plays a critical role in the activation of caspase-1. Mice deficient in caspase-11, similar to those deficient in caspase-1, fail to produce mature interleukin-1 (226). Activation of procaspase-1 is impaired in cells derived from these mice. Similarly, caspase-4 (likely a human homologue of caspase-11) enhances activation of human caspase-1. Caspase-11 and caspase-1 were found in the same complex (226). Identification of other components in this complex should help clarify the exact mechanism of procaspase-1 activation.

REGULATION OF CASPASES

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Natural Inhibitors

CrmA is a serpin that directly targets the active site of mature caspases (163). It has an active-site loop that is easily accessible to caspases. After being cleaved by a caspase, however, CrmA stays bound to the caspase and blocks the active site (5). The early experiments using this suicide substrate of caspases provide compelling evidence that caspases play a central role in apoptosis (62, 207). CrmA is limited to the group I and group III caspases (except for caspase-6) and granzyme B (266). The dissociation constants range from 0.01 nM for caspase-1 and 0.34 nM for caspase-8 to over 1 µM for the group II caspases. Similar to CrmA, the baculovirus protein p35 also targets mature caspases and serves as a suicide substrate (13, 240). The inhibition requires a substrate-like sequence containing Asp-Gln-Met-Asp-87-Gly that fits well with the caspase active site (55). p35 is a broad-spectrum caspase-specific inhibitor; it inhibits human caspase-1, -3, -6, -7, -8, and -10 with K_is of from less than 0.1 to less than 9 nM (264). However, it does not inhibit granzyme B.

In contrast to CrmA and p35, IAPs are not active-site-specific inhibitors, and their inhibition of apoptosis does not require cleavage by caspases. The baculovirus IAPs, Op-IAP and Cp-IAP, were identified by their ability to functionally replace p35 (6, 35). While no cellular homologues of CrmA and p35 have been identified so far, the cellular homologues of IAPs constitute a major family of caspase regulators. The first human IAP, the neuronal apoptosis inhibitor protein, was cloned based on its frequent deletion in a neurodegenerative disorder, spinal muscular atrophy (125). To date, at least four other mammalian IAPs (XIAP/MIHA, c-IAP-1/MIHB, c-IAP-2/MIHC, and survivin) and two Drosophila IAPs (DIAP-1 and -2) have been identified (reviewed in reference 39). Each IAP protein contains at least one but often two to three copies of the characteristic BIR sequence (baculovirus IAP repeat), which are required for their function. In addition, several IAPs, including IAP-1 and XIAP, also have a zinc ring domain that controls the degradation of these proteins (see below).

Human XIAP and c-IAP-1 and -2 were found to inhibit mature caspase-3 and -7 (40, 41, 170). However, the inhibitory effect is substantially weaker than that of CrmA and p35. While CrmA and p35 reach maximal inhibitory effect when present at an equal molar ratio to caspases, IAPs need to be in molar excess to achieve a reasonable level of apoptosis inhibition. The exact mechanism of caspase inhibition by IAPs is not fully understood. However, IAPs may target the early steps of caspase activation, as shown by the observation that IAPs block activation of Drosophila caspases in vivo and activation of mammalian procaspase-9 in a cell-free system (40, 178). Evolutionarily ancient IAPs such as survivin may also be involved in cytokinesis and link caspase activation to cell cycle progression (120, 121). The level and activity of IAPs may determine the sensitivity of cells to apoptotic stimuli, and both can be downregulated during apoptosis to ensure effective cell killing. For example, during thymocyte death, c-IAP-1 and x-IAP are degraded in proteasomes after autoubiquitination, which is catalyzed by the ubiquitin ligase activity of their zinc ring domains (248). The pathway leading to this autoubiquitination remains to be determined. In addition, the activity of IAPs is regulated by the Drosophila cell death inducers Reaper, Grim, and HID and mammalian protein Smac/DIABLO (see below).

v-FLIPs represent another group of viral apoptotic inhibitors. v-FLIPs contain two DEDs that are similar to those in the N-terminal region of procaspase-8 (also known as FLICE and MACH). They inhibit apoptosis mediated by death receptors through competition with procaspases for recruitment to the death receptor complex. Cellular homologues of v-FLIP have been identified, and they come as both a long form and a short form, termed c-FLIP_L and c-FLIP_S, respectively. c-FLIP_S is similar to v-FLIP and contains only the DEDs. Overexpression of this form inhibits apoptosis mediated by Fas and related death receptors. In contrast, c-FLIP_L is strikingly similar to procaspase-8 and -10, comprising two NH₂-terminal DEDs and a COOH-terminal caspase-like domain. The role of c-FLIP_L in apoptosis still remains controversial (see below). Similar to c-FLIP_S, proteins derived from alternatively spliced caspase-2 and -9 transcripts also function as apoptotic inhibitors and are named caspase-2S and caspase-9S, respectively. Both inhibitors lack the large caspase subunit and may compete with the corresponding caspases for binding to adapters.

As a major form of posttranslational modification, phosphorylation is also employed to modulate caspase activity. One example is the phosphorylation of caspase-9 by Akt (14), a serine-threonine protein kinase downstream of phosphatidylinositol 3-kinase, which is implicated in apoptosis suppression mediated by growth factor receptors. Phosphorylation of caspase-9 by Akt inhibits caspase activity in vitro and caspase activation in vivo. The phosphorylation occurs at a consesus substrate recognition sequence of Akt (RxRxxS/T), which is away from the enzymatic active site. The phosphorylation may affect assembly of the caspase tetramer. Alternatively, it may regulate caspase activity allosterically. However, this regulation is not conserved through evolution, because mouse caspase-9 does not contain the recognition site serine and is not phosphorylated by Akt (60).

Another way to modify caspases posttranslationally is by S-nitrosylation. Nitric oxide (NO) and related molecules have been found to inhibit apoptosis. A study showed that in unstimulated human cells, the active site of endogenous procaspase-3 is S-nitrosylated, but during Fas-mediated apoptosis, it becomes denitrosylated (131). The denitrosylation enhances mature caspase-3 activity, although it does not affect procaspase-3 processing. The pathway that leads to denitrosylation has not been defined, nor is it clear whether other caspases are regulated by a similar mechanism. Nevertheless, this study suggests that caspase S-nitrosylation and denitrosylation is a dynamic process during apoptosis.

Different procaspases may be present at different intracellular compartments, and their localizations may change during apoptosis. One of the best examples is that, during Fas-mediated apoptosis, procaspase-8 is recruited from the cytosol to the Fas receptor complex and becomes activated (see below). This translocation is mediated by the homotypic interaction between the DEDs in the prodomain region of procaspase-8 and that in a Fas-associated adapter protein named FADD. In addition, during TNF-induced apoptosis in HeLa cells, procaspase-1 translocates from the cytoplasm to the nucleus and becomes activated there (132). Procaspase-2 is also present in the nucleus as well as the cytoplasm (33). In both cases, the prodomains are required for nuclear localization. These findings suggest a previously unsuspected function of the nucleus in the initiation of apoptosis. Caspase-12 resides in the endoplasmic reticulum (ER) and responds specifically to ER stress (149). Furthermore, in mouse liver, procaspase-3 is present in both cytosol and mitochondria, while procaspase-7 is found only in the cytosol. During Fas-induced apoptosis, caspase-3 is confined primarily to the cytosol, whereas caspase-7 is found in microsomal fractions (20). Therefore, these two effector caspases may cleave protein substrates in different compartments. Procaspases may normally be compartmentalized away from their substrates to prevent accidental apoptosis; during apoptosis, the activation and coordinate translocation of caspases allow them to move close to their targets.

During the development of the hermaphroditic nematode C. elegans, 1,090 cells are generated, and 131 of them undergo programmed cell death. Genetic studies have identified mutations that either abrogate or enhance apoptosis in C. elegans (reviewed in references 87 and 140). These and subsequent genetic analyses have helped establish the paradigm that apoptosis is an active process of cell autodestruction and is genetically programmed. Four genes are found to be at the core of this program and affect apoptosis in all cells: three of them (egl-1, ced-3, and ced-4) are required for cell death, and one (ced-9) inhibits cell death (34, 80, 253, 254). Additional genes affect apoptosis in a subset of cells, and those that control the engulfment of cell corpses have also been identified.

Genetic analysis revealed the functional relationship between the apoptotic genes of C. elegans (Fig. 4A). Specifically, loss of ced-9 function, which results in excessive cell death, is suppressed by mutations in either ced-3 or ced-4 but not by those in egl-1 (egg laying defective). Therefore, genetically, egl-1 functions upstream of ced-9 to inhibit its function, while ced-3 and -4 act downstream of ced-9 to promote apoptosis. The functional relationship between ced-3 and ced-4 was determined by overexpression experiments. Increased expression of ced-3 leads to cell death in ced-4 mutant cells, while increased expression of ced-4 in ced-3-deficient cells fails to induce cell death. Thus, ced-4 acts upstream of ced-3. Furthermore, overexpression of egl-1 can kill cells that normally live, and this killing requires the function of ced-3 and ced-4. The last experiment placed egl-1 in the same pathway as and upstream of ced-3 and ced-4.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   (A) C. elegans apoptosis pathway and mammalian homologues of C. elegans cell death proteins. (B) CED-4 oligomerization as a unifying mechanism in the C. elegans cell death pathway.

Molecular cloning of ced-3 revealed that it encodes a homologue of mammalian ICE or caspase-1, which provided the first evidence that caspases play critical roles in apoptosis (254). Since then, additional caspases have been identified in C. elegans, and the function of these caspases remains to be determined (180). The CED-9 protein was found to be homologous to mammalian Bcl-2 family proteins, especially Bcl-2 and Bcl-x_L, two predominant antiapoptotic proteins in this family (80). Subsequent experiments showed that Bcl-2 could substitute for CED-9 in C. elegans to prevent apoptosis (80). In addition, EGL-1 has been characterized as a BH3 domain-containing protein homologous to a subfamily of the mammalian Bcl-2 protein (34). A mammalian CED-4 homologue, Apaf-1, was also identified (268) (Fig. 4A). These results have enforced the idea that the apoptotic program is evolutionarily conserved.

The activation of the CED-3 caspase is regulated by the EGL-1, CED-9, and CED-4 proteins. Results from several groups suggested that CED-9, CED-4, and CED-3 coexist in a complex known as the apoptosome, with CED-4 being the crux of this complex and interacting with both CED-9 and CED-3 (28, 192, 237). EGL-1 can bind to and displace CED-9 from this complex, thereby allowing CED-4 to activate CED-3 (34). Inhibition of CED-4 by CED-9 and activation of CED-3 by CED-4 appear to occur at distinct intracellular compartments, because CED-4 and CED-9 colocalize to mitochondria in living cells, whereas CED-4 assumes a perinuclear localization in dying cells (23). How does CED-4 activate CED-3? CED-4 was found to form homooligomers both in vitro and in vivo. This oligomerization in turn aggregates the CED-4-associated CED-3 precursors, leading to CED-3 self-activation (245). Furthermore, CED-4 oligomerization is inhibited by CED-9 (245). These results suggest that CED-4 oligomerization is a unifying mechanism that connects the functions of these C. elegans apoptotic proteins (Fig. 4B) and hinted at the function of Apaf-1 (see below).

In recent years, Drosophila melanogaster has also become a model genetic system for the study of programmed cell death. In screening for chromosomal deletions that impair embryonic apoptosis, one such deletion in the region of 75C1,2 was identified (233). Subsequent analysis revealed that this region encompasses at least three genes: reaper, grim, and hid (head involution defective) (24, 73, 233). reaper and grim encode small proteins of only 65 and 138 amino acids, respectively, while hid encodes a protein of 410 amino acids. The proteins all have a short stretch of amino acids at their N terminus which appears to be important for their apoptotic function. Transcription of these three genes is upregulated in cells induced to die, and ectopic expression of each protein causes cell death in Drosophila as well as in mammalian cells, supporting their roles as cell death inducers. Reaper, Grim, and Hid-induced apoptosis is blocked by caspase inhibitors such as p35, indicating that these proteins engage caspases to kill cells.

At least five caspases have been identified in Drosophila. Two of them (DREDD and DRONC) contain long prodomains and are likely to be initiator caspases. DREDD is similar to mammalian procaspase-8 and contains two tandem DED domains in its N-terminal region (25). DRONC has a CARD domain (44). The other three caspases (DCP-1, drICE, and DECAY), with only short prodomains, bear strong similarity to mammalian effector caspases such as caspase-3 and -7 (45, 56, 191). Like mammalian caspase-3 and -7, DCP-1 and drICE are inhibited by IAPs, particularly DIAP-1.

Reaper, Grim, and Hid seem to activate caspases through inhibition of DIAP-1. In vitro, recombinant DIAP-1 inhibits the protease activity of drICE, and this effect can be reversed by recombinant Hid (227). In yeast cells which lack endogenous caspases but are sensitive to caspase cleavage, expression of drICE causes cell death. The lethality of drICE is inhibited by coexpression of DIAP-1, but the expression of reaper, hid, or grim reverses this inhibitory effect (227). In another study, DIAP-1 gain-of-function mutations were identified and strongly suppressed reaper-, hid-, and grim-induced apoptosis (68). These mutations were single amino acid changes in the BIR region that decreased the interaction between DIAP-1 and Reaper, Grim, and Hid. These three proteins have homology in their first 14 amino acids, which are critical for interaction with and inhibition of DIAP-1. Together, these results support the notion that the three Drosophila apoptotic inducers activate caspases through direct inhibition of DIAP-1 (Fig. 5). This apoptotic pathway is distinct from the classic pathways mediated by death adapters such as CED-4, Apaf-1, and FADD (see the sections above and below). A similar pathway appears to exist in mammalian cells. Smac, a recently identified mammalian functional homologue of Reaper, Grim, and Hid, promotes caspase-9 and -3 activation by eliminating the inhibitory effect of mammalian IAPs (46, 71, 221). The N-terminal regions of Smac, like those in Reaper, Grim, and Hid, are indispensable for its function (18). However, unlike Reaper, Grim, and Hid, Smac resides in mitochondria in living cells and translocates to the cytosol during apoptosis to exert its effect.

View larger version (9K): [in this window] [in a new window]   FIG. 5.   Three Drosophila apoptotic pathways converge at caspase activation. Reaper, Grim, and Hid activate caspases through inhibition of DIAP-1. Caspases can also be activated by an Apaf-1-like pathway (Dapaf-1/HAC-1/Dark) and a FADD-like pathway (dFADD).

A Drosophila protein similar to CED-4 and its mammalian homologue Apaf-1 (see below) was identified and named HAC-1 (Dapaf-1, Dark) (101, 166, 263). Dapaf-1 is more closely related to Apaf-1 than CED-4 in that both Dapaf-1 and Apaf-1 have long C-terminal regions with WD-40 repeats and may require cytochrome c for activation (see below). Dapaf-1 was found to interact with DRONC in one study but with DREDD in another (101, 166), and the real target caspase remains to be determined. A death adapter, dFADD, which is homologous to the mammalian protein FADD that activates caspase-8 (see below) was also identified (92). dFADD binds to DREDD through the DID, a novel domain involved in caspase-adapter interactions, and promotes DREDD cell death activity and autoprocessing. Thus, multiple Drosophila apoptosis pathways converge on caspase activation (Fig. 5).

The C. elegans CED-4 pathway for caspase activation is conserved in mammalian cells. Components in this mammalian pathway were identified through biochemical purification of proteins that participate in dATP-induced caspase activation in a cell-free system (126). One such protein, Apaf-1 (apoptosis protease-activating factor-1), is homologous to CED-4 (268). Like CED-4, Apaf-1 contains an N-terminal CARD domain that can interact with the CARD domain in the N terminus of procaspase-9 (124). In addition, Apaf-1 also forms oligomers, likely octamers, in a dATP- and cytochrome c-dependent manner (193, 245, 269). The oligomerization is mediated by a CED-4-homologous region C-terminal to the CARD domain (193, 245). Unlike CED-4, however, Apaf-1 also harbors a long C-terminal region with about a dozen WD-40 repeats, a motif found in many proteins and presumably involved in protein-protein interactions. The WD-40 repeats have a self-inhibitory effect on the rest of the protein, and binding of dATP and cytochrome c to Apaf-1 antagonizes this inhibitory effect and allows Apaf-1 self-aggregation as well as Apaf-1 binding to procaspase-9 (124, 193, 269) (Fig. 6A). Apaf-1 and procaspase-9 form a complex at a 1:1 ratio (269). Thus, procaspase-9 molecules are brought into close proximity by Apaf-1 and activated by autocleavage (Fig. 6A). Active procaspase-9 then cleaves and activates procaspase-3 (124). There may be a positive feedback loop between these two caspases, as active caspase-3 cleaves partially processed caspase-9 to generate the final form of the mature caspase-9 (193). Apaf-1 was found to possess intrinsic dATPase activity that promotes oligomer formation (269). CED-4 also requires the binding of nucleotide to activate CED-3, and mutations that alter the conserved nucleotide-binding site abolish CED-4 function (27, 179). However, a dATPase activity in CED-4 is yet to be determined. CED-4 does not contain WD-40 repeats, to which cytochrome c binds, and it remains to be determined whether CED-4 requires cytochrome c for its function.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Mammalian apoptosis pathways. Procaspase-8 and -9 are activated by oligomerized FADD and Apaf-1, respectively. Upon activation, these two caspases cleave effector caspases such as caspase-3 (see Fig. 3). The red circles indicate cytochrome c.

The finding that cytochrome c, an essential component of the respiratory chain for the generation of ATP, also plays a critical role in apoptosis came as a surprise to a field in which cell life and death were generally regarded as mutually exclusive processes. The dual role of cytochrome c may be rooted in an essential logic for multicellular organisms: the purpose of individual cells' existence is the well-being of the organism. Using an essential component for cell survival as an apoptotic inducer provides an effective way to ensure that all cells that grow can undergo apoptosis when needed. Other cell death proteins, including the death adapter protein FADD (see below), may also play a dual role in cell life and death.

Cytochrome c normally resides in the space between the outer and inner membrane of mitochondria. Its release is triggered by various cellular stresses, including cytotoxic drugs, growth factor withdrawal, and DNA damage. Notably, p53-mediated apoptosis employs this mitochondrial cell death pathway (190). Cytochrome c release is also induced by development cues, and mice deficient in either Apaf-1 or caspase-9 show profound apoptosis defects during development (see below). Release of cytochrome c is regulated by the Bcl-2 family proteins (reviewed in references 1 and 72). Specifically, the antiapoptotic members of this family such as Bcl-2 and Bcl-x_L prevent cytochrome c release, while the proapoptotic members such as Bax and Bid promote it (Fig. 6A) (100, 108, 122, 130, 243). Many of the Bcl-2 family proteins attach to or, during apoptosis, translocate to the outer member of mitochondria. Unlike the C. elegans Bcl-2 homologue CED-9, which prevents apoptosis through direct inhibition of CED-4, endogenous Bcl-2 family proteins are not found to physically interact with Apaf-1 (143).

The exact mechanisms by which the Bcl-2 family proteins regulate cytochrome c release are still in debate. One hypothesis states that a mitochondrial conductance channel known as the permeability transition (PT) pore opens during apoptosis. Due to the high osmolarity of the matrix, the opening of the PT pore causes swelling of the mitochondrial matrix, leading to outer membrane rupture and cytochrome c release. The dissipation of the mitochondrial inner membrane potential (_m) (hypopolarization), an indication of the opening of the PT pore, is observed in many apoptotic models (137, 255). Bcl-2 prevents and Bax promotes opening of the PT pore (239, 256). Another hypothesis postulates that during apoptosis, a transient hyperpolarization of the mitochondrial inner membrane causes high osmolarity in the intermembrane space and subsequent rupture of the outer membrane. Such hyperpolarization is observed in a variety of apoptosis scenarios and is suppressed by Bcl-x_L (215, 216).

Both hypotheses assume that a major morphological change of mitochondria (i.e., rupture of the outer membrane) precedes cytochrome c release. However, cytochrome c release and caspase activation can occur before any detectable changes in mitochondrial morphology (72). Caspase activation can also induce cytochrome c release. It is thus possible that cytochrome c release occurs in two steps: at first only some cytochrome c molecules are released due to outer membrane rupture in a small number of mitochondria, and these cytochrome c molecules lead to caspase activation. Active caspases then cause changes in most mitochondria, and consequently almost all cytochrome c molecules are released. It is also possible that cytochrome c release occurs through a special channel instead of via outer membrane rupture. In this regard, it is intriguing that the three-dimensional structure of Bcl-x_L resembles those of pore-forming bacterial toxins such as diphtheria toxin, and that Bcl-2, Bcl-x_L, and Bax can each form ion channels in a synthetic membrane (141, 144). However, the channel-forming ability of these proteins alone is unlikely to account for the opposite effects of these proteins on cytochrome c release. The Bcl-2 family proteins may interact with channel-forming proteins on mitochondria to modulate cytochrome c release, and a few studies suggest that Bax and Bid promote while Bcl-x_L inhibits opening of the PT channel (150, 183).

Cytochrome c is not the only harmful protein hidden in mitochondria. Other cell death effectors in mitochondria include procaspases (202) (also see above), an apoptosis-inducing factor (AIF), and the caspase coactivator Smac (also called DIABLO). AIF is a flavoprotein, and like cytochrome c, it is normally confined to mitochondria. During apoptosis, AIF translocate to the nucleus, where it induces chromatin condensation and large DNA fragmentation via a caspase-independent mechanism (203). Smac promotes activation of caspase-9 and -3 by binding to IAPs and eliminating their inhibitory effect on the caspases (18, 46, 71, 221).

Fas death pathway. While the cell death pathways identified so far in C. elegans and D. melanogaster are initiated by developmental cues within individual cells, mammals have clearly evolved a mechanism by which a cell is instructed to die. This type of cell death is critical in regulating the immune response, as illustrated by the functions of a group of death receptors in the TNF receptor (TNFR) superfamily (3, 188). These receptors include Fas, TNFR1, and death receptors 3, DR-4, and -5 and contain the death domain in their intracellular region to recruit downstream apoptotic proteins.

TNFR1, DR3, DR4, and DR5 pathways. Two other death receptors, DR4 and DR5, resemble Fas structurally and functionally (3). They mediate apoptosis upon activation by their ligand TRAIL. However, unlike FasL, which is expressed only in activated T cells, natural killer cells, immune-privileged sites, and some tumor cells, TRAIL mRNA is detected in many tissues. Similarly, DR4 and DR5 transcripts are also found in many normal tissues, suggesting that TRAIL is nontoxic to these tissues. Significantly, TRAIL triggers apoptosis in many tumor cells while sparing normal tissues, and it may prove to be an effective anticancer reagent. The different responses towards TRAIL are in part due to the expression of decoy TRAIL receptors in normal but not in tumor cells (155, 181). The signaling pathways of DR4 and DR5 are similar to that of Fas and are mediated by FADD (107).

To date, more than 60 proteins have been shown to be substrates of one or more caspases in mammalian cells, and the list is still growing (201). These substrate proteins contain one or very few caspase cleavage sites in interdomain linker sequences. Substrate proteins are thus not degraded by caspase processing; instead, caspase cleavage may activate or inactivate the substrate protein's functions. Because of the strict requirement for an aspartate residue at P1 and distinct preferences for P2 to P4 residues, it is relatively straightforward to identify the likely caspase cleavage sites within a given substrate protein and the likely group of caspases that may recognize them. The substrates identified so far fall into two general groups: a large group of proteins thought to be involved in regulation and execution of apoptosis, and a small group of proinflammatory cytokine precursors (Table 1). For many of the identified substrates, the functional consequences of their cleavage have only been inferred from their normal functions. In other cases, the role of caspase cleavage has been experimentally assessed by expressing mutant substrate proteins that have altered caspase cleavage sites or by expressing protein fragments that represent caspase cleavage products. The evolutionary conservation of caspase substrate proteins, in particular their caspase cleavage sites, suggests that caspases were first employed for apoptosis and later coopted for cytokine processing in mammals.

Once it was appreciated that CED-3 is a homologue of ICE (interleukin-1-converting enzyme, now known as caspase-1) (254), the search began in earnest for key caspase substrates in apoptotic execution. The involvement of proteases in apoptosis was anticipated; after all, proteolysis must occur in the irreversible disintegration of the cell. However, apoptosis could not result from the indiscriminate digestion of all cellular proteins; instead, it must involve controlled activation and inactivation of certain choice proteins to carefully dismantle the cell. The search for caspase substrates has brought several major questions into focus. What are the minimal set of proteins that must be cleaved in order to induce the phenotypic features of apoptosis? How is apoptosis coordinated with other cellular processes? How is apoptosis regulated after the activation of caspases? Although the significance of caspase-mediated cleavage is not well understood for many of the substrates, the study of caspase substrates has already shed light on these questions. Here we highlight several themes that have emerged from the accumulating information regarding caspase substrates during apoptosis.

DNA metabolism. During apoptosis, nuclear DNA is condensed and degraded into large (50 to 300 kb) and subsequently into small oligonucleosomal fragments of several hundred base pairs. DNA degradation irreversibly dooms the cell and also destroys viruses or harmful mutations that may have found their way into the genetic blueprint. Biochemical purification by several groups led to the discovery of a caspase-regulated DNase complex termed DFF (DNA fragmentation factor) that is composed of a DNase termed CAD (caspase-activated DNase: also named DFF40 and CPAN) and its inhibitor ICAD (also named DFF45) (51, 128, 173). In healthy cells, CAD is complexed with ICAD and functionally inactive. In apoptotic cells, ICAD is cleaved by caspase-3 and -7, releasing CAD to degrade nuclear DNA (173). Two other regulators, CIDE-A and CIDE-B, have an N-terminal regulatory domain homologous to ICAD and CAD, which allows them to interact with and regulate the activity of the DFF complex (94, 129). DFF induces both chromatin condensation and DNA fragmentation in vitro (127), and cells from mice deficient for DFF activity exhibit neither of these classic apoptotic features when induced to die (259). Another caspase-3-activated factor, named acinus, induces chromatin condensation without affecting DNA fragmentation (172). Acinus contains a potential DNA/RNA-binding motif and is activated by concordant proteolytic cleavage of caspases and an unidentified serine protease activity (172). The task of DNA fragmentation appears to be separate from other phenotypic aspects of apoptosis. For example, when cells that overexpress caspase-resistant ICAD and thus lack CAD activity are induced to die, they exhibit neither large-scale nor nucleosomal DNA fragmentation, but these cells still die manifesting other features of apoptosis, such as cell body shrinkage and phosphatidylserine exposure (173). Mice deficient for DFF activity develop normally and have no obvious defects in homeostatic apoptosis (259). These results are consistent with previous genetic studies with the nuc-1 nuclease in C. elegans programmed cell death: DNA fragmentation is a downstream effect of caspase activation and is dispensable for cell killing (87). Instead, DNA fragmentation is a way of "hiding the body" and represents one of the multiple parallel execution pathways in apoptosis.

Cytoskeletal scaffold proteins. The cytoskeleton of an apoptotic cell undergoes profound changes as the nucleus fragments, the cell body shrinks, and the cell becomes detached from surrounding cells and the basal membrane. The molecular logic behind these changes is becoming clearer with the biochemical identification of many cytoskeletal proteins that are cleaved by caspases. Lamins, the intermediate filament scaffold proteins of the nuclear envelope, are cleaved mainly by caspase-6 and inactivated, leading to nuclear fragmentation in the final phases of apoptosis (115, 204). Overexpression of lamins with mutated caspase cleavage sites delays the onset of chromatin condensation and pyknosis during apoptosis, indicating that caspase cleavage of lamins facilitates these morphologic changes specifically (162). Apoptotic execution also takes advantage of endogenous regulatory mechanisms for disassembling the cytoskeleton. Gelsolin, a cytoplasmic F actin-depolymerizing enzyme, is cleaved by caspase-3 to yield a fragment with constitutive activity (109). Gelsolin-deficient neutrophils exhibit greatly delayed membrane blebbing during apoptosis, a classic morphological feature, implying that membrane blebbing requires actin reorganization mediated by caspase-activated gelsolin (109). Caspases attack multiple targets in the cortical actin architecture: -fodrin and focal adhesion kinase, two components of the focal adhesion complex, which links cortical actin filaments, plasma membrane, and transmembrane proteins to the extracellular matrix, are cleaved by caspases and may lead to cell body shrinkage and allow apoptotic cells to detach from neighboring cells and basement membrane (36, 136, 232). Actin itself is cleaved by effector caspases in certain cells, and the caspase-generated fragments may contribute directly to apoptotic changes in cell shape (201). In addition, PAK2, a serine/threonine kinase downstream of Rac and Cdc42, small GTPases that regulate actin dynamics, is cleaved by caspase-3 to generate a constitutively active kinase during apoptosis (171). Blocking PAK2 activity with a dominant negative kinase-dead PAK2 mutant inhibits the formation of apoptotic bodies (small sealed vesicles that contain condensed cytoplasmic material from fragmented apoptotic cells), but interestingly, nuclear features of apoptosis and phosphatidylserine externalization were not inhibited (171). From these examples, it is evident that multiple cytoskeletal proteins and their regulators are targeted by caspases to dismantle the cellular architecture, and perturbation of particular cytoskeletal targets may be responsible for specific morphological features in apoptosis. In general, preventing one substrate cleavage may delay or even abrogate a morphological feature of apoptosis but will not prevent cell death. The possibility of uncoupling morphological features in apoptosis should also caution investigators to identify clearly the microscopic or biochemical criteria used to measure apoptosis.

Cell cycle regulators. Given some morphological similarities between apoptosis and mitotic catastrophe (in which cell division occurs without completion of S phase, leading to cell body shrinkage, DNA condensation, nuclear envelope breakdown, and eventual cell death), an early idea in the field was that apoptosis may be a kind of aborted or ectopic cell cycle. This idea was supported by the finding that cyclin-dependent kinase (Cdk) activity increases during apoptosis, and inhibition of Cdk activity attenuates apoptosis in several experimental systems (182). However, with the continued identification of caspase substrates, it is now clear that the induction of Cdk activity during apoptosis is a consequence of effector caspase activity. Cdc27, a component of the ubiquitin ligase complex that mediates the degradation of mitotic cyclins, and Weel, a kinase that provides an inhibitory phosphorylation on Cdks, are both cleaved and inactivated by caspase-3-like activities (262). Similarly, two Cdk inhibitors, p21Cipl and p27Kipl, can be cleaved by caspase-3, which decreases their association with Cdks and thus allows Cdk activity to accumulate during apoptosis (119). Because cleavage of cell cycle regulators occurs late in apoptosis by caspase-3-like activities in parallel with the dismantling of the transcription and translation machinery, caspase-activated Cdk activity cannot activate the normal mitotic program (262). For example, mitotic spindles do not form in apoptotic cells, distinguishing apoptosis from mitotic catastrophe. Instead, Cdk activity in apoptosis appears to be required for several aspects of apoptotic morphology, including DNA condensation and cell body shrinkage, but not others, such as loss of mitochondrial potential and externalization of plasma membrane phosphatidylserine (79). These results qualify caspase-mediated Cdk activation as another independent effector pathway for the execution of apoptosis. The mechanism by which activated Cdks carry out their jobs during apoptosis is unclear, but it is likely to be quite distinct from their normal targets during mitosis (262).

Repair and housekeeping enzymes. Historically, one of the first proteins identified as being cleaved during apoptosis by diverse stimuli was poly(ADP-ribose) polymerase (PARP); PARP cleavage activity was later used to identify and purify caspase-3 as an effector caspase (151, 208). PARP is a nuclear enzyme that senses DNA nicks and catalyzes the ADP-ribosylation of histones and other nuclear proteins in order to facilitate DNA repair. 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 (114). With subsequent identification of other DNA repair enzymes, such as DNA-dependent protein kinases, and housekeeping enzymes, such as the 70-kDa U1-snRNP protein involved in mRNA splicing, are cleaved during apoptosis, a hypothesis was forwarded that cleavage and inactivation of repair proteins may lead to lethal DNA damage and thus contribute to apoptosis. However, this hypothesis is probably incorrect given that enucleated cells (cytoplasts) can maintain metabolic homeostasis for some time and be triggered to undergo apoptosis with all the cytoplasmic features of apoptosis (99) and cells deficient for PARP or DNA repair enzymes have no general perturbation in apoptotic execution (228). Instead, new evidence suggests that PARP inactivation by caspase-3 is important for turning off an energetically expensive repair pathway following commitment to apoptosis. Activated PARP transfers up to 100 ADP-ribose moieties to each acceptor site in target proteins, and each cycle of ADP-ribosylation is coupled with consumption of 1 NAD⁺ molecule, which is metabolically equivalent to 4 ATP molecules. One can imagine that during apoptotic execution, DNA fragmentation by CAD can cause significant activation of PARP and quickly deplete cellular energy stores. In the absence of an energy pool sufficient to maintain ionic homeostasis, the cell can die quickly by default necrosis (258). Indeed, when cells engineered to express caspase-resistant PARP are induced to undergo apoptosis, they undergo more extensive apoptosis and a fraction of cells also undergo necrosis (11, 81). However, because PARP contributes to DNA base excision repair, its genetic absence or inactivation by caspase cleavage can sensitize cells to apoptosis induction by DNA-damaging agents (77, 152). Consistent with the postulated requirement of maintaining cellular energy during apoptosis, cells artificially manipulated to have low ATP pools undergo necrosis instead of apoptosis in stress conditions, like cells that are unable to cleave PARP (118). The biologic roles of DNA-dependent protein kinases and U1-snRNP cleavage in apoptosis have not been examined in detail, but they may represent a concerted strategy to prevent futile repair and synthetic efforts during apoptosis. Caspase-mediated inactivation of DNA repair and housekeeping enzymes reinforces the idea that apoptosis is a carefully orchestrated program of irreversible cell suicide, and the cell's metabolic efforts may be redirected to suit that goal.

Signaling molecules. A large number of signal transduction proteins have been found to be caspase substrates during apoptosis (Table 1). Most, if not all, of these proteins are cleaved by caspase-3-like effector caspases, in parallel with the proteolysis of housekeeping enzymes such as PARP. For several serine/threonine kinases, such as PAK2, the mitogen-activated kinase kinase kinase (MAP3Ks) MEKK1 and MST1, caspase-3 cleavage occurs between the N-terminal autoinhibitory domain and the catalytic kinase domain, resulting in kinase activation (15, 70, 171). Intriguingly, these kinases are all able to activate the JNK and p38 stress-activated protein kinase pathways, which can provide a mechanism of caspase-dependent JNK activation. As described above, activation of PAK2 is important for cytoskeletal reorganization and plasma membrane blebbing. In the case of MEKK-1, caspase-mediated kinase activation is thought to provide a positive feedback loop for signaling apoptosis, because expression of the caspase-cleaved kinase fragment induces caspase activation and apoptosis (15). Epithelial cells undergo apoptosis if they are detached from basement membrane, a process termed anoikis (Greek for homelessness)(59). MEKK1 is activated following epithelial cell detachment, and blockade of either MEKK1 or caspase activity blocks anoikis. MST1 cleavage by caspase-3 also yields a constitutive kinase and potent inducer of apoptosis (70). Apoptosis induction by activated MAP3Ks upstream of the JNK pathway may be explained in part by the ability of JNK to phosphorylate and inactivate Bcl-2 (138). Caspase-3-mediated cleavage and inactivation of the antiapoptotic kinases Akt and c-Raf provide two more examples of positive feedback loops in apoptosis (234).