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.     

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.     

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.     

The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor.     

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.     

Cleavage of the apoptosis inhibitor DIAP1 by the apical caspase DRONC in both normal and apoptotic Drosophila cells

In Drosophila S2 cells, the apical caspase DRONC undergoes a low level of spontaneous autoprocessing. Unintended apoptosis is prevented by the inhibitor of apoptosis DIAP1, which targets the processed form of DRONC for degradation through its E3 ubiquitin protein ligase activity. Recent reports have demonstrated that shortly after the initiation of apoptosis in S2 cells, DIAP1 is cleaved following aspartate residue Asp-20 by the effector caspase DrICE. Here we report a novel caspase-mediated cleavage of DIAP1 in S2 cells. In both living and dying S2 cells, DIAP1 is cleaved by DRONC after glutamate residue Glu-205, located between the first and second BIR domains. The mutation of Glu-205 prevented the interaction of DIAP1 and processed DRONC but had no effect on the interaction with full-length DRONC. The mutation of Glu-205 also had a negative effect on the ability of overexpressed DIAP1 to prevent apoptosis stimulated by the proapoptotic protein Reaper or by UV light. These results expand our knowledge of the events that occur in the Drosophila apoptosome prior to and after receiving an apoptotic signal.     

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.     

The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.     

Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.     

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.     

An efficient method to express and refold a truncated human procaspase-9: a caspase with activity toward Glu-X bonds

A truncated form of human procaspase-9 missing the first 111 amino acids, and a variety of mutants derived therefrom, have been expressed in Escherichia coli inclusion bodies. Upon refolding to active enzymes, Delta(1-111) procaspase-9 and mutants were recovered at purity greater than 95% and with a final yield of 20-35 mg/L cell culture. Our active procaspase-9 retains its pro-segment, while undergoing major auto processing at Asp315 and a minor (20%) cleavage at Glu306. This unusual cleavage at a Glu-X bond also took place in the D315E mutant, and we describe herein the inhibitor Z-VAE-fmk that shows enhanced inactivation of procaspase-9 over caspases-3. The bond at Asp330, not processed by procaspase-9, is cleaved by caspase-3 and the resulting procaspase-9 variant, missing the 316-330 bridge, is six times as active as the non-mutated Delta(1-111) proenzyme. A deletion mutant lacking residues 316-330 underwent auto activation by cleavage at Asp315-Ala331 bond. Moreover, substitution of Glu306 by an Asp residue in this mutant led to rapid removal of the peptide spanning Ser307 to Asp330, and resulted in an enzyme that was 7.6 times as active as the non-mutated Delta(1-111) procaspase-9. Finally, replacing both Asp315 and Glu306 with Ala generated a procaspase-9 mutant incapable of auto processing. This single chain procaspase-9 was fully as active as the non-mutated Delta(1-111) enzyme processed at Asp315 or Glu306. Our demonstration that unprocessed procaspase-9 mutants are active as proteases with caspase-type specificity suggests that the role of procaspase-9 in cascade activation of executioner caspases might, in some circumstances, be carried out alone and without association of the apoptosome.     

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.     

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.     

A Substrate-Phage Approach for Investigating Caspase Specificity

We have developed a substrate-phage approach for examining the substrate specificities of an important group of proteases involved in apoptosis--the caspases. After establishing selection conditions with caspases-3 and caspase-8 vs control substrate-phage, we sorted X4 and X6 diversity libraries, identified consensus motifs that agree with previously defined caspase substrate motifs, confirmed the selection of active substrates using synthetic peptide rate assays under a range of buffer conditions, and compared kinetic parameters for selected substrates. The libraries produced some variations on the canonical motifs. From caspase-3 selections, a phage-derived synthetic peptide, DLVD, was hydrolyzed up to 170% faster than the canonical substrate DEVD. The P4 Asp residue was essential for good protease-sensitivity, but even substrates with substitutions at P4 were selected by phage and shown to be hydrolyzed. Caspase-8 selections, as expected, yielded predominantly clones containing a Glu at P3. In this case, the most frequent phage-derived peptide, LEVD, was cleaved at a rate of only 20% of the canonical caspase-8 substrate LETD. However, based on substitutions observed in the phage selectants at P4, a substrate peptide, AETD, was designed and shown to be hydrolyzed up to 160% faster than LETD. We consider factors that may contribute to differences in caspase substrate-phage selections vs synthetic peptide studies on the caspases, and suggest that the two approaches may offer complementary information.     

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.     

Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35

Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its ‘reactive site loop'' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.     

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).     

DRONC coordinates cell death and compensatory proliferation

Accidental cell death often leads to compensatory proliferation. In Drosophila imaginal discs, for example, gamma-irradiation induces extensive cell death, which is rapidly compensated by elevated proliferation. Excessive compensatory proliferation can be artificially induced by "undead cells" that are kept alive by inhibition of effector caspases in the presence of apoptotic stimuli. This suggests that compensatory proliferation is induced by dying cells as part of the apoptosis program. Here, we provide genetic evidence that the Drosophila initiator caspase DRONC governs both apoptosis execution and subsequent compensatory proliferation. We examined mutants of five Drosophila caspases and identified the initiator caspase DRONC and the effector caspase DRICE as crucial executioners of apoptosis. Artificial compensatory proliferation induced by coexpression of Reaper and p35 was completely suppressed in dronc mutants. Moreover, compensatory proliferation after gamma-irradiation was enhanced in drice mutants, in which DRONC is activated but the cells remain alive. These results show that the apoptotic pathway bifurcates at DRONC and that DRONC coordinates the execution of cell death and compensatory proliferation.