DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. Here, we show that Drosophila melanogaster inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. We find that DIAP2 regulates the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. Our data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, our data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.     

Dissection of DIAP1 functional domains via a mutant replacement strategy

Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of active caspases. Drosophila IAP1 (DIAP1) activity is required to keep cells from undergoing apoptosis. The central cell death regulators Reaper and Hid induce apoptosis very rapidly by inhibiting DIAP1 function. We have developed a system for replacing endogenous DIAP1 with mutant forms of the protein, allowing us to examine the roles of various domains of the protein in living and dying cells. We found that DIAP1 is cleaved by a caspase early after the initiation of apoptosis. This cleavage is required for DIAP1 degradation, but Rpr and Hid can still initiate apoptosis in the absence of cleavage. The cleavage of DIAP1 promotes DIAP1 degradation in a manner dependent on the function of the ubiquitin ligase function of the DIAP1 ring domain. This ring domain function is required for Hid-induced apoptosis. We propose a model that synthesizes our data with those of other laboratories and provide a consistent model for DIAP1 function in living and dying cells.     

Down-regulation of DIAP1 triggers a novel Drosophila cell death pathway mediated by Dark and DRONC

Members of the inhibitor of apoptosis protein (IAP) family can inhibit caspases and cell death in a variety of insect and vertebrate systems. Drosophila IAP1 (DIAP1) inhibits cell death to facilitate normal embryonic development. Here, using RNA interference, we showed that down-regulation of DIAP1 is sufficient to induce cell death in Drosophila S2 cells. Although this cell death process was accompanied by elevated caspase activity, this activation was not essential for cell death. We found that DIAP1 depletion-induced cell death was strongly suppressed by a reduction in the Drosophila caspase DRONC or the Drosophila apoptotic protease-activating factor-1 (Apaf-1) homolog, Dark. RNA interference studies in Drosophila embryos also demonstrated that the action of Dark is epistatic to that of DIAP1 in this cell death pathway. The cell death caused by down-regulation of DIAP1 was accelerated by overexpression of DRONC and Dark, and a caspase-inactive mutant form of DRONC could functionally substitute the wild-type DRONC in accelerating cell death. These results suggest the existence of a novel mechanism for cell death signaling in Drosophila that is mediated by DRONC and Dark.     

The Drosophila caspase DRONC is regulated by DIAP1

We have isolated the recently identified Drosophila caspase DRONC through its interaction with the effector caspase drICE. Ectopic expression of DRONC induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue DRONC-induced cell death in vivo and is not cleaved by DRONC in vitro, making DRONC the first identified p35-resistant caspase. The DRONC pro-domain interacts with Drosphila inhibitor of apoptosis protein 1 (DIAP1), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-DRONC expression. In contrast, DIAP1 fails to rescue eye ablation induced by DRONC lacking the pro-domain, indicating that interaction of DIAP1 with the pro-domain of DRONC is required for suppression of DRONC-mediated cell death. Heterozygosity at the diap1 locus enhances the pro-DRONC eye phenotype, consistent with a role for endogenous DIAP1 in suppression of DRONC activation. Both heterozygosity at the dronc locus and expression of dominant-negative DRONC mutants suppress the eye phenotype caused by reaper (RPR) and head involution defective (HID), consistent with the idea that DRONC functions in the RPR and HID pathway.     

Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis

Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases.     

Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.     

Drosophila Morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis

In Drosophila melanogaster, apoptosis is controlled by the integrated actions of the Grim-Reaper (Grim-Rpr) and Drosophila Inhibitor of Apoptosis (DIAP) proteins (reviewed in refs 1 4). The anti-apoptotic DIAPs bind to caspases and inhibit their proteolytic activities. DIAPs also bind to Grim-Rpr proteins, an interaction that promotes caspase activity and the initiation of apoptosis. Using a genetic modifier screen, we identified four enhancers of grim-reaper-induced apoptosis that all regulate ubiquitination processes: uba-1, skpA, fat facets (faf), and morgue. Strikingly, morgue encodes a unique protein that contains both an F box and a ubiquitin E2 conjugase domain that lacks the active site Cys required for ubiquitin linkage. A reduction of morgue activity suppressed grim-reaper-induced cell death in Drosophila. In cultured cells, Morgue induced apoptosis that was suppressed by DIAP1. Targeted morgue expression downregulated DIAP1 levels in Drosophila tissue, and Morgue and Rpr together downregulated DIAP1 levels in cultured cells. Consistent with potential substrate binding functions in an SCF ubiquitin E3 ligase complex, Morgue exhibited F box-dependent association with SkpA and F box-independent association with DIAP1. Morgue may thus have a key function in apoptosis by targeting DIAP1 for ubiquitination and turnover.     

Baculovirus caspase inhibitors P49 and P35 block virus-induced apoptosis downstream of effector caspase DrICE activation in Drosophila melanogaster cells

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of Drosophila melanogaster. Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of Drosophila DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD(230)-G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD(28)-A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.     

Multiple Apoptotic Caspase Cascades Are Required in Nonapoptotic Roles for Drosophila Spermatid Individualization

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.     

The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated,processed form of the apical caspase DRONC

Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment resulted in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc or dark largely suppressed this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibited DRICE processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induced DRONC processing, which occurred in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulated when dark was silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 resulted in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing was observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase.     

A biochemical analysis of the activation of the Drosophila caspase DRONC

The activation of caspases is the principal event in the execution of apoptosis. Initiator caspases are activated through an autocatalytic mechanism often involving dimerisation or oligomerisation. In Drosophila, the only initiator caspase DRONC, is tightly inhibited by DIAP1 and removal of DIAP1 permits activation of DRONC by the Drosophila Apaf-1-related killer, ARK. ARK is proposed to facilitate DRONC oligomerisation and autoprocessing at residue E352. This study examines whether autoprocessing of DRONC is required for its activation and for DRONC-mediated cell death. Using purified recombinant proteins, we show here that while DRONC autocleaves at residue E352, mutation of this site did not abolish enzyme activation, DRICE-induced cleavage of DRONC or DRONC-mediated activation of DRICE. We performed a detailed mutational analysis of DRONC cleavage sites and show that overexpression of DRONC cleavage mutants in Drosophila cells retain pro-apoptotic activity. Using an in vitro cell-free assay, we found ARK alone did not activate DRONC and demonstrate a requirement for an additional cytosolic factor in ARK-mediated DRONC activation. These results suggest that, similar to mammalian caspase-2 and caspase-9, the initial cleavage of DRONC is not essential for its activation and suggest a mechanism of ARK-mediated DRONC activation different from that proposed previously.     

Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery

Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE.     

The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis

Members of the Inhibitor of Apoptosis Protein (IAP) family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper (Rpr), Grim and Hid (head involution defective) all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. Here, we show that in vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, we show that the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. Our data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination.     

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.     

STRICA, a novel Drosophila melanogaster caspase with an unusual serine/threonine-rich prodomain, interacts with DIAP1 and DIAP2

The recently published genome sequence of Drosophila melanogaster predicts seven caspases in the fly. Five of these caspases have been previously characterised. Here, we describe the Drosophila caspase, STRICA. STRICA is a caspase with a long amino-terminal prodomain that lacks any caspase recruitment domain or death effector domain. Instead, the prodomain of STRICA consists of unique serine/threonine stretches. Low levels of strica expression were detected in embryos, larvae, pupae and adult animals. STRICA is a cytoplasmic protein that, upon overexpression, caused apoptosis in cultured Drosophila SL2 cells that was partially suppressed by DIAP1. Interestingly, unlike other fly caspases, STRICA showed physical association with DIAP2, in cotransfection experiments. These results suggest that STRICA may have a unique cellular function.     

The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID.

Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.     

IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding

The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways.     

IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKK epsilon is a member of the IkappaB kinase family, and its developmental role has not been clear. Drosophila IKK epsilon was localized to the ruffling membrane of cultured cells and was required for F actin turnover at the cell margin. In IKK epsilon mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibited aberrantly branched morphology. These phenotypes were sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKK epsilon destabilized F actin-based structures. Expression of a dominant-negative form of IKK epsilon increased the amount of DIAP1. The results suggest that at the physiological level, IKK epsilon acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKK epsilon function involves the negative regulation of the nonapoptotic activity of DIAP1.