Interactive Fly, Drosophila

Targets of caspases (part 1/2)

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. Caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. These results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function (H. Li, 1997).

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Specific and ordered changes in the actin cytoskeleton take place during apoptosis. In this context, one of the first hallmarks in cell death has been isolated for study: the severing of contacts among neighboring cells. A mechanism has been demonstrated that may contribute to the disassembly of cytoskeletal organization at the level of cell-cell adhesion. Beta-catenin (Drosophila homolog: Armadillo), a known regulator of cell-cell adhesion, is proteolytically processed in different cell types after the induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated beta-catenin into a form that is similar in size to that observed in cells undergoing apoptosis. beta-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting beta-catenin product is unable to bind alpha-catenin, which is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at the level of cell-cell contacts could be dismantled during apoptosis. These observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton that occur during cell death via cleavage of different regulators of the microfilament system (Brancolini, 1997).

Recently, human interleukin 18 (hIL-18) cDNA has been cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence has been generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, natural hIL-18 was purified from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibits a molecular mass of 18.2 kDa; the NH2-terminal amino acid is Tyr37. Biological activities of the purified protein are identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-gamma production by human peripheral blood mononuclear cells. Two precursor hIL-18 (prohIL-18)-processing activities have been found in the cytosol of THP.1 cells. These activities are blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes have shown that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77, to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells (Akita, 1997).

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. At the early phase of Fas ligand (FasL)-induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins is suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins is blocked. The interleukin-1beta-converting enzyme (ICE) protease inhibitors suppress the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade is first activated, and then ERM proteins are dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. To examine the subsequent events in microvillar breakdown, plasma membranes were prepared with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments are densely detected, whereas those from FasL-treated cells are free from ERM proteins or actin filaments. It is concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes (Kondo, 1997b).

Previous work identified a developmental timer that controls the stability of cyclin A (See Drosophila CyclinA) protein in interphase-arrested Xenopus embryos. Cyclins A1 and A2 abruptly become unstable in hydroxyurea-treated embryos at the time that untreated embryos are beginning gastrulation, termed early gastrulation transition (EGT). Cyclins A1 and A2 are degraded at the equivalent of the EGT by the ICE-like caspases that are responsible for programmed cell death or apoptosis. The cleavage site is identified as DEPD, located between residues 87 to 90. Analysis of embryos treated with hydroxyurea or cycloheximide showed widespread cellular apoptosis coincident with cyclin A cleavage. These data further indicate that the apoptotic pathway is present in Xenopus embryos prior to the EGT; however, it is maintained in an inactive state in early cleaving embryos by maternally encoded inhibitors. In normal embryos, suppression of apoptosis is timed to begin with the initiation of zygotic transcription, at the mid-blastula transition (MBT). The decreased biosynthetic capacity of embryos treated with hydroxyurea or cycloheximide most likely interferes with the ability to maintain sufficient levels of apoptotic inhibitors and results in widespread apoptosis. These results suggest a scenario whereby the apoptotic pathway is suppressed in the early cleaving embryo by maternally contributed inhibitors. Degradation at the EGT of maternal RNAs encoding these inhibitors is compensated for by new zygotic transcription beginning at the MBT. This indicates that the interval between the MBT and the EGT represents a critical developmental period during which the regulation of embryonic cellular processes is transferred from maternal to zygotic control (Stack, 1997).

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. These results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities (Zhou, 1998).

Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease (1) cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis; (2) is inhibitable by the CrmA protein, and (3) is distinct from ICE. A ced-3/ICE-related gene was cloned (designated Yama) that encodes a protein identical to CPP32 beta. Purified Yama is a zymogen; when activated, itcleaves PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama is inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocks cleavage of PARP in cells undergoing apoptosis. It is proposed that Yama may represent an effector component of the mammalian cell death pathway and it is suggested that CrmA blocks apoptosis by inhibiting Yama (Tewari, 1995a).

Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that trigger apoptotic cell death when stimulated with ligand or cross-linking antibody -- the mechanism involved has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor than can inhibit interleukin-1 beta converting enzyme (ICE) and ICE-like proteases. Expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways. The 70-kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage is inhibited by expression of CrmA, but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway (Tewari, 1995b).

Programmed cell death is mediated by members of the interleukin 1-beta convertase family of proteases, which are activated in response to diverse cell death stimuli. However, the key substrates of these proteases that are responsible for apoptotic cell death have not been identified. MDM2 is a negative regulator of the tumor suppressor p53, which induces both apoptosis and cell cycle arrest. MDM2 oncoprotein is cleaved by members of the CPP32 subfamily of interleukin 1-beta convertase proteases both in vitro and in vivo, resulting in the disappearance of MDM2 from apoptotic cells. This cleavage of MDM2 by CPP32-like proteases may result in deregulation of p53 and contribute directly to the process of apoptotic cell death (Erhardt, 1997).

The Fas receptor mediates a signaling cascade that results in programmed cell death (apoptosis) within hours of receptor cross-linking. Fas activates the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlates temporally with the onset of apoptosis, and transfection of constitutively active MKK3(glu), an upstream regulator of p38, potentiates Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1-converting enzyme) family proteases to induce apoptosis. The cowpox ICE inhibitor protein CrmA antagonizes, and the synthetic tetrapeptide ICE inhibitor YVAD-CMK and the tripeptide pan-ICE inhibitor Z-VAD-FMK completely inhibit Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members. Conversely, the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. The ICE/CED-3 family-p38 regulatory relationship indicates that in addition to the destructive cleavage of substrates (such as poly[ADP ribose] polymerase, lamins, and topoisomerase), the apoptotic cysteine proteases also function to regulate stress kinase signaling cascades (Juo, 1997).

The fate of the nuclear envelope (NE) has been studied in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), a selective and conserved cleavage of LB, LAP2 and Nup153 is observed. In lymphoid cells, the rate of cleavage of these markers is independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 is probably caspase 3, since (1) cleavage of both proteins is specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. Since LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, it is suggested that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study (Buendia, 1999).

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis is blocked in hsp70-expressing cells; this is associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death is correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK is strongly inhibited in cells in which hsp70 is induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation is not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule generated by apoptosis-inducing treatments and linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance (Mosser, 1997).

Claspin (potential Drosophila homolog: CG32251) is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide causes activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin is cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that each contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress (Clarke, 2005).

Table of contents

Death caspase-1: Biological Overview | Developmental Biology | Effects of Mutation | References

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