Mechanisms of caspase activation and inhibition during apoptosis

Caspases are central components of the machinery responsible for apoptosis. Recent structural and biochemical studies on procaspases, IAPs, Smac/DIABLO, and apoptosome have revealed a conserved mechanism of caspase activation and inhibition. This article reviews these latest advances and presents our current understanding of caspase regulation during apoptosis.     

Mechanisms of caspase activation and inhibition during apoptosis

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. We determined the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage in between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180-degree-flipping of the N-terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of     

MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation

The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.     

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E(1)), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.     

Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1

Human neutrophils normally have a very short half-life and die by apoptosis. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay this apoptosis via increases in the cellular levels of Mcl-1, an anti-apoptotic protein of the Bcl-2 family with a rapid turnover rate. Here we have shown that inhibition of the proteasome (a) decreases the rate of Mcl-1 turnover within neutrophils and (b) significantly delays apoptosis. This led us to determine whether GM-CSF could enhance neutrophil survival by altering the rate of Mcl-1 turnover. Addition of GM-CSF to neutrophils enhanced Mcl-1 stability and delayed apoptosis by signaling pathways requiring PI3K/Akt and p44/42 Erk/Mek, because inhibitors of these pathways completely abrogated the GM-CSF-mediated effect on both Mcl-1 stability and apoptosis delay. Conversely, induction of Mcl-1 hyperphosphorylation by the phosphatase inhibitor, okadaic acid, significantly accelerated both Mcl-1 turnover and apoptosis. Neither the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, nor the pan caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone, had any effect on Mcl-1 stability under these conditions. These observations indicate that profound changes in the rate of neutrophil apoptosis following cytokine signaling occur via dynamic changes in the rate of Mcl-1 turnover via the proteasome.     

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.     

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP)

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.     

Sodium salicylate promotes neutrophil apoptosis by stimulating caspase-dependent turnover of Mcl-1

Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins that plays a central role in cell survival of neutrophils and other cells. The protein is unusual among family members in that it has a very short half-life of 2-3 h. In this report, we show that sodium salicylate (at 10 mM) greatly enhances the rate at which neutrophils undergo apoptosis and, in parallel, greatly accelerates the turnover rate of Mcl-1, decreasing its half-life to only 90 min. Whereas constitutive and GM-CSF-modified Mcl-1 turnover is regulated by the proteasome, the accelerated sodium salicylate-induced Mcl-1 turnover is mediated largely via caspases. Sodium salicylate resulted in rapid activation of caspase-3, -8, -9, and -10, and salicylate-accelerated Mcl-1 turnover was partly blocked by caspase inhibitors. Sodium salicylate also induced dramatic changes in the activities of members of the MAPK family implicated in Mcl-1 turnover and apoptosis. For example, sodium salicylate blocked GM-CSF-stimulated Erk and Akt activation, but resulted in rapid and sustained activation of p38-MAPK, an event mimicked by okadaic acid that also accelerates Mcl-1 turnover and neutrophil apoptosis. These data thus shed important new insights into the dynamic and highly regulated control of neutrophil apoptosis that is effected by modification in the rate of Mcl-1 turnover.     

FLICE-like inhibitory protein blocks transforming growth factor beta 1-induced caspase activation and apoptosis in prostate epithelial cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.     

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Direct inhibition of cytochrome c-induced caspase activation in vitro by Toxoplasma gondii reveals novel mechanisms of interference with host cell apoptosis

The intracellular parasite Toxoplasma gondii is known to inhibit apoptosis of its host cell. The molecular mechanisms of this interference are, however, not yet completely understood. We show here that viable parasites prominently inhibited the activation of caspase 3/7 induced by cytochrome c, dATP and dithiothreitol in cytosolic extracts of human-derived Jurkat leukemic T cells. In contrast, granzyme B-induced caspase activity was only slightly diminished. De novo protein biosynthesis by T. gondii was dispensable for the inhibition of cytochrome c-induced caspase activation. Furthermore, a complete parasite lysate or, more importantly, molecules released by extracellular parasites mediated the interaction with the caspase cascade. The cell-free system applied here is thus a valuable tool to study the interaction of T. gondii and possibly other intracellular pathogens with host cell apoptosis.     

Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.     

Mcl-1 is a key regulator of the ovarian reserve

A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.Estimates of the human primordial follicle (PMF) reservoir, the size of which dictates the extent of the ovarian reserve, indicates the presence of at least half a million oocytes per ovary at birth.^{1, 2} The essential decision that PMFs face is either long-term arrest with a possibility of recruitment toward the growing pool, or death. Even upon recruitment to the growing pool, intricately orchestrated crosstalk of survival signals between ovarian somatic cells and oocytes facilitate the ovulation of a single oocyte in human in each cycle. Hence, the default fate for millions of ovarian germ cells is death, as only a small fraction survive till ovulation.³ Insufficient endowment during fetal development or excessive oocyte loss during postnatal life further limits the ovarian reserve and can result in an untimely exhaustion of the follicle pool leading to premature ovarian failure (POF); a syndrome that affects around 1% of all women, with a higher prevalence (up to 30%) in families with heritable traits of this condition.^{4, 5} Mechanisms responsible for maintenance of the follicular reserve are poorly understood, however, biological assessments and mathematical modeling reveal that progressive loss of follicles with age is non-linear and accelerates, especially after 38 years.^{6, 7} With a declining ovarian reserve, poor oocyte quality is an additional factor that contributes to the reduced fertility associated with increased maternal age. Oocytes and resulting embryos of older mothers have increased rates of aneuploidies likely due to defects in chromosomal cohesion and meiotic spindle stability, decreased DNA repair capacity, altered gene expression, impaired mitochondrial function and elevated cellular redox, all contributing to increased rates of cell death.^{8, 9, 10}The marked decline of oocyte number in mammalian ovaries has been attributed to oocyte loss via stage-specific modes of death. As yet, perinatal PMF loss in mice most frequently engages apoptotic cell death,^{11, 12} whereas within the postnatal ovary, oocytes in growing follicles undergo atresia, a less ''molecularly'' defined death, carrying hallmarks of both apoptosis and autophagy.^{13, 14, 15} It is thus surprising that no member of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family has been identified with a definitive role in governing oocyte survival and the maintenance of the ovarian reserve. Bcl-2l2/Bcl-w and Bcl-2-l10/Diva deficiency had no apparent impact on the ovarian reserve, and although ablation of Bcl-2 led to a loss of one-third of the adult PMF pool, the growing follicle pool was not significantly impacted and these animals did not undergo POF.^{16, 17, 18, 19} Conditional Bcl-x (Bcl-2l1) inactivation led to increased primordial germ cell apoptosis in the embryo,²⁰ but postnatal inactivation of Bcl-x in oocytes did not compromise the ovarian reserve in young females.²¹ Bcl2a1a/Bfl-1/A1 was low to undetectable in fully grown germinal vesicle (GV) or ovulated murine oocytes,²² however, the impact of Bfl-1 deficiency on the ovarian reserve has not yet been analyzed to the best of our knowledge. Consequently, either various anti-apoptotic Bcl-2 members have overlapping roles in governing postnatal oocyte survival and maintenance of the adult ovarian reserve in mice, or the anti-apoptotic Bcl-2 member that regulates this decision has yet to be identified.     

Bistability analyses of a caspase activation model for receptor-induced apoptosis

Apoptosis is an important physiological process crucially involved in development and homeostasis of multicellular organisms. Although the major signaling pathways have been unraveled, a detailed mechanistic understanding of the complex underlying network remains elusive. We have translated here the current knowledge of the molecular mechanisms of the death-receptor-activated caspase cascade into a mathematical model. A reduction down to the apoptotic core machinery enables the application of analytical mathematical methods to evaluate the system behavior within a wide range of parameters. Using parameter values from the literature, the model reveals an unstable status of survival indicating the need for further control. Based on recent publications we tested one additional regulatory mechanism at the level of initiator caspase activation and demonstrated that the resulting system displays desired characteristics such as bistability. In addition, the results from our model studies allowed us to reconcile the fast kinetics of caspase 3 activation observed at the single cell level with the much slower kinetics found at the level of a cell population.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.