Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Apoptotic cysteine–aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction.     

ER stress induces caspase-8 activation,stimulating cytochrome c release and caspase-9 activation

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.     

Interaction of influenza A virus M1 matrix protein with caspases

In this investigation, an ability of influenza A virus M1 matrix protein to bind intracellular caspases, the key enzymes of cell apoptosis, has been examined. Protein–protein binding on polystyrene plates and polyvinyl pyrrolidone membrane was employed for this purpose. Under a comparative study of caspases-3, -6, -7, -8 influenza virus M1 protein specifically bound caspase-8 and weakly bound caspase-7. Using a computer analysis of the N-terminal region of M1 protein, a site similar to the anti-caspase site of baculovirus p35 protein, which inhibits caspases and displays antiapoptotic activity, was identified. These results are in good agreement with the supposition that influenza virus M1 protein is involved in a caspase-8-mediated apoptosis pathway in influenza virus infected cells.     

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) do not inhibit caspase-3 and caspase-7 processing in cells exposed to pro-apoptotic inducing stimuli

We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.     

Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex.

In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active approximately 60-kDa heterotetramers. We now demonstrate that activation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized protein complexes, the "aposome" and the "microaposome". Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microaposome complexes with only small amounts of active caspase-3 present as its free approximately 60-kDa heterotetramer. The larger aposome complex (M(r) = approximately 700,000) contained Apaf-1 and processed caspase-9, -3, and -7. The smaller microaposome complex (M(r) = approximately 200,000-300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active caspase-9. Lysates isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an approximately 200-300-kDa complex, which did not have caspase cleavage (DEVDase) activity. Procaspase-3 and -9 were present as separate and smaller 60-90-kDa (dimer) complexes. During caspase activation, Apaf-1, caspase-9, and the effector caspases redistributed and formed the aposome. This resulted in the processing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Activation of the caspase cascade during Stx1-induced apoptosis in Burkitt's lymphoma cells

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1-mediated cytotoxicity, we observed that multiple caspases, including caspase-3, -7, and -8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and -7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-alpha, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with specific antibody is sufficient to induce activation of caspase-8. Our findings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.     

XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis

Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the 'native' apoptosome complex via a specific interaction with the small p12 subunit of processed caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAP's capacity to maintain inhibition of caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of caspase-9 or 3 is essential for XIAP's anti-apoptotic activity.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Caspase-2-induced apoptosis is dependent on caspase-9, but its processing during UV- or tumor necrosis factor-dependent cell death requires caspase-3

Mammalian caspases are a family of cysteine proteases that plays a critical role in apoptosis. We have analyzed caspase-2 processing in human cell lines containing defined mutations in caspase-3 and caspase-9. Here we demonstrate that caspase-2 processing, during cell death induced by UV irradiation, depends both on caspase-9 and caspase-3 activity, while, during TNF-alpha-dependent apoptosis, capase-2 processing is independent of caspase-9 but still requires caspase-3. In vitro procaspase-2 is the preferred caspase cleaved by caspase-3, while caspase-7 cleaves procaspase-2 with reduced efficiency. We have also demonstrated that caspase-2-mediated apoptosis requires caspase-9 and that cells co-expressing caspase-2 and a dominant negative form of caspase-9 are impaired in activating a normal apoptotic response and release cytochrome c into the cytoplasm. Our findings suggest a role played by caspase-2 as a regulator of the mitochondrial integrity and open questions on the mechanisms responsible for its activation during cell death.     

Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.     

Proteasomal degradation of caspase-6 in 17beta-estradiol-treated neurons

In primary cultures of human neurons, 17beta-estradiol (17beta-E2) prevents caspase-6-mediated cell death and induces a caspase inhibitory factor (CIF) inhibiting active caspase-6 (Csp-6) in vitro. Here, we show that treatment of neurons with 17beta-E2 results in a proteasomal-dependent but ubiquitin-independent degradation of endogenous and exogenous active Csp-6 in live neurons and in cell free assays, respectively. We further show that the proteasomal-dependent degradation of Csp-6 is not required for its inhibition. Using several protease inhibitors, we find that leupeptin, E-64, and ALLN prevent inhibition of recombinant active Csp-6 (R-Csp-6) in 17beta-E2-treated neuronal protein extracts. Because all three protease inhibitors have the ability to inhibit cysteine proteases, we believe that a cysteinyl protease activity may be required for 17beta-E2-mediated inhibition of active Csp-6. However, we exclude caspases, calpains, and cathepsins as potential cysteinyl proteases involved in the 17beta-E2-mediated Csp-6 inhibition. The results suggest that a proteolytic activity inhibited by leupeptin, E-64, and ALLN is needed to inhibit Csp-6 and that the inhibited Csp-6 is subsequently degraded by the proteasome. The mechanism of 17beta-E2-mediated inhibition of Csp-6 is different from the ubiquitin-dependent proteasomal degradation of Csp-3 and Csp-7 by XIAP and cIAP2 but consistent with the mechanism of Baculovirus p35 inhibition of caspases.     

Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-α induced apoptosis

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-α induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-α in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology. XiaoHe Yang: This work was supported in part by the Career Development Award DAMD17-99-1-9180 from Department of Defense to X.H.Y.     

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.     

Caspase-8 in apoptosis: the beginning of "the end"?

Caspase-8 is a member of the cysteine proteases, which are implicated in apoptosis and cytokine processing. Like all caspases, caspase-8 is synthesized as an inactive single polypeptide chain zymogen procaspase and is activated by proteolytic cleavage, through either autoactivation after recruitment into a multimeric complex or trans-cleavage by other caspases. Thus, ligand binding-induced trimerization of death receptors results in recruitment of the receptor-specific adapter protein Fas-associated death domain (FADD), which then recruits caspase-8. Activated caspase-8 is known to propagate the apoptotic signal either by directly cleaving and activating downstream caspases or by cleaving the BH3 Bcl2-interacting protein, which leads to the release of cytochrome c from mitochondria, triggering activation of caspase-9 in a complex with dATP and Apaf-1. Activated caspase-9 then activates further "downstream caspases," including caspase-8. Knockout data indicate that caspase-8 is required for killing induced by the death receptors Fas, tumor necrosis factor receptor 1, and death receptor 3. Moreover, caspase-8-/- mice die in utero as a result of defective development of heart muscle and display fewer hematopoietic progenitor cells, suggesting that the FADD/caspase-8 pathway is absolutely required for growth and development of specific cell types.