The Drosophila caspase DRONC is regulated by DIAP1
2000; Springer Nature; Volume: 19; Issue: 4 Linguagem: Inglês
10.1093/emboj/19.4.598
ISSN1460-2075
AutoresPascal Meier, John Silke, Sally J. Leevers, Gérard I. Evan,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoArticle15 February 2000free access The Drosophila caspase DRONC is regulated by DIAP1 Pascal Meier Pascal Meier Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author John Silke John Silke The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Sally J. Leevers Sally J. Leevers Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT UK Search for more papers by this author Gerard I. Evan Corresponding Author Gerard I. Evan Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 5BT UK Present address: UCSF Cancer Center, 2340 Sutter Street, San Francisco, 94143-0128 USA Search for more papers by this author Pascal Meier Pascal Meier Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author John Silke John Silke The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Sally J. Leevers Sally J. Leevers Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT UK Search for more papers by this author Gerard I. Evan Corresponding Author Gerard I. Evan Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 5BT UK Present address: UCSF Cancer Center, 2340 Sutter Street, San Francisco, 94143-0128 USA Search for more papers by this author Author Information Pascal Meier1, John Silke2, Sally J. Leevers3 and Gerard I. Evan 1,4,5 1Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 2The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, 3050 Australia 3Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1P 8BT UK 4Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 5BT UK 5Present address: UCSF Cancer Center, 2340 Sutter Street, San Francisco, 94143-0128 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:598-611https://doi.org/10.1093/emboj/19.4.598 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction In multicellular organisms, homeostasis is established and maintained by a dynamic balance between cell proliferation and cell death. Programmed cell death (PCD) is used as a means to eliminate damaged or supernumerary cells and to sculpt and whittle structures during development (Evan and Littlewood, 1998; Tschopp et al., 1998; Vaux and Korsmeyer, 1999). In addition, PCD provides an important defence against viral infection and the emergence of cancer (Thompson, 1995). PCD, usually called apoptosis in complex metazoans, is an active process implemented by a machinery that is evolutionarily conserved amongst nematodes, insects and vertebrates. Apoptosis involves execution of a complex and co-ordinated series of events culminating in activation of a family of cysteine proteases called caspases (cysteinyl aspartate-specific proteases) (Thornberry and Lazebnik, 1998). Caspases are expressed as pro-enzymes with little or no intrinsic catalytic activity that comprise three nascent domains: an N-terminal pro-domain, a large subunit containing the catalytically active cysteine (∼20 kDa) and a C-terminal small subunit (∼10 kDa). They are activated by proteolytic cleavages at sites located between these domains that abscize the pro-domain and release the large and small subunits, which then form the active (p20/p10)2 caspase hetero-tetramer. The inter-domain sites for proteolytic activation of caspases are themselves caspase consensus cleavage sites, indicating that caspases reside in cascades of auto- and trans-activation that are typically initiated by activation of initiating or ‘apical’ caspases (Alnemri, 1997). Once activated, caspases cleave various cellular substrates, such as lamins, kinases, DNA repair enzymes and proteins involved in mRNA splicing and DNA replication, and this is presumed to trigger many of the morphological processes of cell death defined as apoptosis (Thornberry and Lazebnik, 1998). Genetic studies in the nematode Caenorhabditis elegans provided the first direct evidence for the importance of caspases in PCD. Inactivating mutations in the nematode caspase CED-3 block all of the 131 developmental cell deaths that occur during C.elegans development (Ellis and Horvitz, 1986). Later studies indicated analogous requirements for caspases in PCD in Drosophila and in mammals. In Drosophila, RPR (reaper), GRIM and HID (head involution defective) proteins have been identified as key activators of the apoptotic machinery (White et al., 1994; Grether et al., 1995; Chen et al., 1996). Embryos with a chromosomal deletion that includes the rpr, grim and hid loci show essentially no PCD during ontogeny (White et al., 1994). Ectopic expression of RPR, GRIM or HID in the developing Drosophila eye results in a highly efficient and dose-dependent ablation of eye structures. This occurs through activation of a caspase-dependent apoptotic machinery, since PCD induced by each of these pro-apoptotic proteins is blocked by expression of the baculovirus protein p35, a promiscuous caspase inhibitor (Grether et al., 1995; Chen et al., 1996; White et al., 1996). In Drosophila, four caspases have been identified thus far: drICE, DCP-1, DCP-2/DREDD and, most recently, DRONC (Fraser and Evan, 1997; Inohara et al., 1997; Song et al., 1997; Chen et al., 1998; Dorstyn et al., 1999). Both drICE and DCP-1 possess short pro-domains typical of ‘downstream’ or ‘effector’ caspases, such as mammalian caspases-3, -6 and -7, which are activated via proteolytic cleavage by ‘upstream’ caspases. In contrast, DCP-2/DREDD and DRONC contain extensive pro-domains, characteristic of ‘upstream’ or ‘apical’ caspases. The DRONC pro-domain contains a caspase recruiting domain (CARD), whereas the pro-domain of DREDD shares no significant homology, as judged by Pfam analysis (Bateman et al., 1999), with either the CARD or death effector domains (DEDs) found in other caspases. Ectopic expression of RPR, GRIM or HID leads to proteolytic cleavage and activation of drICE, DCP-1 and DCP-2/DREDD. However, nothing is known about the hierarchy of caspase activation, nor how RPR, GRIM and HID engage the apoptotic machinery. Intriguingly, expression of RPR, GRIM or HID leads to proteolytic cleavage of DREDD even in the presence of the caspase inhibitor p35 (Chen et al., 1998). Since drICE, DCP-1 and DREDD are each inhibited by p35, this suggests that DREDD is activated by a p35-resistant protease. The role of the recently reported caspase DRONC in PCD of Drosophila has not been established. During development, DRONC is ubiquitously expressed during embryogenesis as well as in the developing eye, brain and adult egg chambers, all places where PCD naturally occurs. Interestingly, in late third instar larvae, DRONC is dramatically up-regulated in salivary glands and mid-gut before histolysis of these tissues occurs during metamorphosis. Exposure of these tissues to ecdysone leads to a significant increase in dronc mRNA levels, suggesting that DRONC may be an ecdysone-inducible caspase (Dorstyn et al., 1999). The inhibitor of apoptosis protein (IAP) family comprises proteins conserved amongst a wide range of eukaryotic species that suppress apoptosis induced by a variety of stimuli (Uren et al., 1998; Deveraux and Reed, 1999). In Drosophila, ectopic expression in the developing eye of the cellular IAPs, DIAP1 or DIAP2, suppresses cell death induced by RPR or HID (Hay et al., 1995). Furthermore, genetic studies of DIAP1 in the eye and ovary suggest that DIAP1 is essential for ‘normal’ survival of these cell types. However, the mechanisms by which IAPs suppress cell death are poorly understood. In lepidopteran cells, DIAP1 and DIAP2 interact physically with, and block, the pro-apoptotic activity of RPR, GRIM and HID (Vucic et al., 1997, 1998a). In addition, DIAP1 inhibits the proteolytic activity of active drICE and DCP-1 in vitro (Kaiser et al., 1998; Hawkins et al., 1999). At present, however, it is unclear how effector caspases become activated in Drosophila, or how the pro-apoptotic proteins RPR, HID and GRIM promote caspase activation, and the DIAP proteins suppress it. To address these issues, we have searched for proteins that interact with the ‘effector’ caspase drICE and have identified DRONC. We show that DRONC has proteolytic activity that, unlike other caspases, is not blocked by p35. In addition, we show that DIAP1 interacts with the pro-domain of DRONC and appears to be a critical regulator of activation of this ‘apical’ caspase in vivo. Furthermore, we provide evidence that supports the idea that DRONC is a rate-limiting caspase in the RPR and HID pathway. Results DRONC interacts with the effector caspase drICE In Drosophila melanogaster, the pro-apoptotic proteins RPR, GRIM and HID induce cell death via activation of caspases. However, thus far, little is known concerning how RPR, GRIM or HID trigger caspase activation. To study the mechanisms underlying caspase activation, we sought to identify molecules that interact with pro-caspases and, hence, might be involved in their regulation and activation. As a target pro-caspase we chose the Drosophila caspase drICE, which has been shown to be required for execution of apoptosis in certain fly cells in vitro (Fraser et al., 1997). A catalytically inactive mutant of drICE, in which the active site cysteine has been changed to alanine (drICE C→A), was used as bait in a yeast two-hybrid assay to screen a 0–24 h Drosophila embryonic cDNA library. From 2 × 106 yeast transformants, we isolated 34 clones encoding potential drICE interactors. Three of these clones were drICE-derived; one encoded full-length drICE (1–339) whereas the other two encoded N-terminal truncations of drICE (40–339 and 43–339, respectively). Interaction with these latter two suggests that pro-drICE can dimerize via its core region (the protein region without the pro-domain), even in its inactive pro-form. We further assessed the physical interaction between DRONC and drICE by testing for the ability of the two proteins to co-immunoprecipitate from cell extracts. FLAG-tagged, full-length, catalytically inactive DRONC (pro-DRONC C→A, 1–451) was co-expressed in 293T cells together with Myc-tagged catalytically inactive pro-drICE C→A (1–339), ΔN drICE C→A (29–339) or Bcl-10 (Figure 1C). The mammalian protein Bcl-10 that contains an N-terminal CARD was used as the control in the co-immunoprecipitation experiments. Pro-DRONC specifically co-immunoprecipitated both pro-drICE and ΔN drICE, but not Bcl-10, indicating that DRONC and drICE form a stable complex in cell extracts. Figure 1.DRONC is a drICE-interacting caspase. (A) The dendrogram shows the phylogenetic relationships of the core region of caspase family members (i.e. the protein sequence without the pro-domain). ClustalX was used for the sequence analysis. (B) Yeast two-hybrid analysis showing that DRONC and drICE interact with each other through their core regions. The extent of the β-galactosidase staining, as detected in filter tests, is indicated: +++, intense blue staining of large colonies; ++, light blue staining of medium size colonies. (C) Co-immunoprecipitation from 293T cell extracts. FLAG-tagged full-length DRONC (pro-DRONC C→A) and Bcl-10 (control) were co-expressed together with either Myc-tagged pro-drICE C→A, ΔN drICE C→A or Bcl-10 (control). Cell lysates were incubated with M2 anti-FLAG monoclonal antibody resin, washed, and the co-immunoprecipitated Myc epitope-tagged proteins were detected by immunoblot analysis using anti-Myc monoclonal antibody (9E10). Expression of FLAG-tagged and Myc-tagged proteins was confirmed. Molecular mass markers in kDa are shown. Download figure Download PowerPoint Ectopic expression of DRONC is toxic to S.pombe and induces apoptosis in Rat-1 cells The fission yeast Schizosaccharomyces pombe is devoid of caspase homologues or caspase-like activities. However, because many active caspases have been demonstrated to be toxic when expressed in yeast, S.pombe has emerged as a useful and facile model system in which to assess caspase functionality (Ekert et al., 1999). We inserted sequences encoding pro-DRONC and ΔN DRONC into the S.pombe expression vector pNeu under the control of a thiamine-repressible promoter. In the presence of thiamine, yeast transformed with either of the two constructs grew normally. However, both pro-DRONC and ΔN DRONC expression proved toxic and resulted in a time-dependent inhibition of yeast growth (Figure 2A). This toxicity is dependent on DRONC enzymatic activity since catalytically inactive DRONC mutants (pro-DRONC C→A or ΔN DRONC C→A) had no effect on yeast growth. Both pro-DRONC and ΔN DRONC underwent catalytic autoprocessing to a similar extent in S.pombe, as judged by immunoblotting of DRONC from cell extracts. However, when expressed at approximately similar levels, pro-DRONC appeared somewhat more toxic than ΔN DRONC. Figure 2.Ectopic expression of DRONC induces cell death in yeast and in mammalian Rat-1 cells. (A) Expression of DRONC is toxic to S.pombe. For cytotoxicity assays, yeast from two independent colonies were grown to log phase, the OD595 of the culture determined and the yeast then plated in serial 10-fold dilutions on selective, inducing media. Western blot analysis with anti-FLAG M2 antibody was used to confirm expression and autoproteolytic cleavage of C-terminally tagged DRONC. (B) Transient transfection of dronc leads to induction of apoptosis in mammalian Rat-1 fibroblast cells. Various expression constructs were co-transfected with a CMV-lacZ reporter plasmid in a ratio of 10:1. At 24 h post-transfection, cells were fixed and examined for β-galactosidase activity. Shown are the percentage of β-galactosidase-positive cells with apoptotic morphology from three independent experiments (mean ± SD). (C) DRONC is a cysteine protease that cleaves drICE C→A, lamin DmO and DREP-1 but not p35 in vitro. In vitro translated substrates were incubated with (1) control (no protease added); (2) pro-DRONC C→A purified from yeast; (3) pro-DRONC purified from yeast; and (4) purified bacterially expressed drICE. The unprocessed substrate is indicated by an arrow and the cleavage product is denoted by an asterisk. Download figure Download PowerPoint Many caspases induce apoptosis when expressed in mammalian cells. We therefore asked whether pro-DRONC, ΔN DRONC or the catalytically inactive mutant of ΔN DRONC (ΔN DRONC C→A) killed Rat-1 fibroblasts (Figure 2B). Expression of ΔN DRONC, which lacks its pro-domain, was very effective at inducing cell death, as was expression of either of the positive controls, caspase-8 and the Fas pathway adaptor FADD. However, in complete contrast, expression of full-length DRONC exerted no lethal effect. DRONC therefore resembles caspases-4 and -5 (Munday et al., 1995), both of which kill mammalian cells only when expressed without their respective pro-domains. As in S.pombe, the catalytically inactive ΔN DRONC C→A mutant had no effect on Rat-1 cell viability, consistent with a requirement for the caspase activity of DRONC to induce Rat-1 cell death. The lack of toxicity of full-length DRONC in Rat-1 cells is in stark contrast to the situation in S.pombe in which both pro-DRONC and ΔN DRONC are toxic and undergo autocatalytic activation. One possible explanation for this discrepancy is that mammalian cells contain cellular factors that suppress pro-DRONC activation by binding its pro-domain. If true, deletion of the pro-domain in ΔN DRONC would then render the caspase no longer inhibitable by such putative factors, resulting in the spontaneous activation of ΔN DRONC and consequent cell death. Cell line-specific variations in levels of such putative inhibitory factors might explain why the efficacy with which pro-DRONC induces cell death is variable amongst different cell types. In this context, it is noteworthy that although pro-DRONC does not induce cell death in Rat-1 cells, it is lethal to NIH 3T3 cells (Dorstyn et al., 1999). As DRONC interacts with drICE, we next assayed the ability of active DRONC to cleave drICE C→A, lamin DmO (Gruenbaum et al., 1988), the DNA fragmentation factor DREP-1 (Inohara et al., 1998) and the baculovirus caspase inhibitor p35 (Figure 2C). Both DRONC and drICE cleaved drICE C→A, lamin DmO and DREP-1. The cleavage products generated by DRONC and drICE were clearly different, indicating that DRONC and drICE each cleave lamin DmO and DREP-1 at different sites. Unlike drICE, however, DRONC was unable to cleave p35. Together, these results indicate that dronc encodes a catalytically active protease and that its unique active site PFCRG pentapeptide confers upon it a different substrate specificity from classical caspases such as drICE that share the QAC(R/Q/G)(G/E) active site pentapeptide consensus. Ectopic expression of DRONC driven by an eye-specific promoter induces an eye ablation phenotype in Drosophila To determine whether ectopic expression of DRONC can induce cell death in D.melanogaster, we used the GAL4/UAS system to express various forms of DRONC in the developing Drosophila compound eye. Independent transgenic Drosophila lines were generated carrying pro-dronc, ΔN dronc, pro-dronc C→A, ΔN dronc C→A or dronc-card (the pro-domain of DRONC on its own) under the control of GAL4-upstream activating sequences (UAS). These flies were then crossed with Drosophila strains expressing GAL4 under the control of the glass multimer reporter (GMR-gal4; Hay et al., 1994) in differentiating photoreceptors and pigment cells posterior to the morphogenetic furrow in the eye imaginal disc (Ellis et al., 1993). The DRONC-induced phenotypes that we observed were of variable severity, depending on the insertion line used, presumably because of insertion site-specific effects on the transgene expression level (Spradling and Rubin, 1983). Accordingly, one representative weak UAS-pro-dronc (pro-droncW) and one representative strong UAS-pro-dronc line (pro-droncS) were selected for further characterization, along with one UAS-ΔN dronc line. Pro-droncW flies carrying one copy of the transgene exhibited a ‘spotted eye’ phenotype when crossed with GMR-gal4 flies: although pro-droncW flies are white+, and should therefore have red eyes, their eyes appeared white with occasional red spots (Figure 3B). Such eyes have an essentially normal external morphology and size [compare Figure 3A, F and K (control) with B, G and L], in contrast to eyes expressing RPR under the control of GMR, which are severely reduced in size (Figure 3E, J and O). By comparison, pro-droncS and ΔN dronc transgenic flies exhibited dramatically ‘roughened eyes’ that were severely reduced in size (Figure 3C and D). Scanning electron microscopy (SEM) analysis of pro-droncS and ΔN dronc eyes confirmed that surface morphology was severely distorted, erupted and rough (Figure 3H and M, and I and N). As with pro-droncW flies, eyes from pro-droncS and ΔN dronc flies were white, not red. This consequence of DRONC expression in eyes is particularly intriguing given that expression of RPR dramatically reduces eye size yet has no effect on eye colour (compare Figure 3B–D with E, and L–N with O). The phenotypes induced by DRONC expression are a consequence of DRONC caspase activity since overexpression of catalytically inactive C→A mutants of DRONC exerted no detectable effect on eye development (data not shown). Figure 3.Ectopic expression of DRONC in the developing eye causes ablation of all retinal structures resulting in a hollow eye. Phenotypes were analysed by light microscopy of whole mounts (A–E), tangential thin sections of adult eyes (P–T), scanning electron microscopy (F–O) and acridine orange staining of eye discs of third instar larvae (U–W) and 60 h after puparium formation (X and Y). (A, F, K, P, U and X) Control flies (+/GMR-gal4). (B, G, L, Q, V and Y) The weak pro-droncW transgenic line (GMR-gal4/UAS-pro-droncW) displays a spotted eye phenotype (B) with an essentially normal eye morphology on the outside (G and L) but a severely malformed cell arrangement in the interior (Q). (C, H, M and R) Pro-droncS transgenic flies that show reduced eye size (C and H) with no defined interior eye structure (R) (GMR-gal4/UAS-pro-droncS). (D, I, N, S and W) Ectopic expression of ΔN DRONC (GMR-gal4/UAS-ΔN dronc) causes excessive cell death in the eye disc of third instar larvae (W) resulting in a small eye phenotype (D and I). (E, J, O and T) GMR-rpr flies display eyes of a reduced size (E and J) but unlike dronc transgenic fly eyes they are red instead of white (E) (GMR-rpr/+). (C, H, M and R) and (D, I, N and S) represent pictures from animals that were crossed with GMR-gal4 (815, weak) and raised at 18°C. All other images were obtained from animals crossed with GMR-gal4 (816, strong) and raised at 25°C. In this and the following figures, anterior is to the right and posterior to the left. Download figure Download PowerPoint To investigate in detail the consequences of DRONC expression on the survival of photoreceptor and pigment cells underlying the eye surface, we examined transverse sections of adult transgenic eyes. Surprisingly, even in the pro-droncW flies, no normal cellular structures of either pigment or photoreceptor cells were visible: only remnants of pigment cells and vacuole-like structures remained (compare Figure 3P with Q–S). These remnant pigment cells, containing the red pigment pteridine, were responsible for the red ‘spots’ observed in the pro-droncW fly eyes (Figure 3B). We therefore conclude that GMR-driven DRONC expression kills both pigment and photoreceptor cells. One possibility is that the ablation of internal eye structures seen in dronc transgenic flies may result from excess cell death in the developing eye disc. We therefore examined third instar larval eye discs for the appearance of apoptotic cells using acridine orange, which stains apoptotic cells (Abrams et al., 1993). Compared with controls, third larval instar eye discs expressing ΔN DRONC exhibited dramatic and supernumerary apoptosis posterior to the morphogenetic furrow (compare Figure 3U with W). In contrast, no such sign of excessive apoptosis was evident in eye discs from third instar larvae expressing full-length pro-droncW (Figure 3V). However, during later development (60 h after puparium formation), eye discs of pro-droncW pupae exhibited a dramatic increase in numbers of apoptotic cells (compare Figure 3X with Y). It is presumably this very late activation of apoptosis, essentially after the eye lens structure has formed, which gives the eyes of pro-droncW flies their characteristic morphology wherein the eyes show an essentially normal outer structure with internal ablation. In contrast, the devastating ‘small eye’ phenotype seen in pro-droncS, ΔN dronc or GMR-rpr transgenic flies (Figure 3C–E) is consistent with the observed induction of cell death much earlier during larval eye development. The pro-domain-less ΔN DRONC generates a consistently more severe eye ablation phenotype than does pro-DRONC. Indeed, all ΔN dronc transgenic lines die when crossed with GMR-gal4 (816, strong) and maintained at 25°C, although viability of some of these lines can be sustained by crossing them to a weak GMR-gal4 driver line (815, weak) and maintaining them at 18°C. The lethality is most likely not to be a trivial result of misexpression of GMR-gal4 in tissues other than the developing eye but, rather, to be due to the inability of ΔN DRONC flies to open the pupae case with their heads because of extreme head malformation. As a consequence, such flies die trapped in their pupae cases. In confirmation of this, we found that flies with severely deformed and black eyes could indeed be rescued by manually opening the puparium at the end of their development (data not shown). The pro-domain of DRONC interacts with DIAP1 The observed difference between the pro-apoptotic activity of pro-DRONC and pro-domain-lacking ΔN DRONC in Drosophila and mammalian cells raises the possibility that spontaneous activation of pro-DRONC is suppressed through interaction of its pro-domain with some putative cellular inhibitor. To identify such an inhibitor, we searched for Drosophila proteins that interact specifically with the DRONC pro-domain in a yeast two-hybrid assay using a 0–24 h Drosophila embryonic cDNA library. From 1 × 106 yeast transformants, we recovered 56 DRONC-interacting clones, of which 17 encoded DIAP1 (Figure 4A). The second BIR domain of DIAP1 was necessary and sufficient for the interaction with the pro-domain of DRONC (DRONC-CARD, Figure 4B). This is particularly intriguing since the BIR2 region of DIAP1 is also known to interact physically with, and block the pro-apoptotic activity of, RPR, GRIM and HID (Vucic et al., 1997, 1998a,b). Figure 4.DIAP1 physically interacts with DRONC. (A) Seventeen DRONC-interacting clones encoded full-length and N-terminal truncations of DIAP1 of which seven representative DIAP1 clones are indicated. The positions of the first amino acid of the clones relative to full-length DIAP1 are denoted on the left. (B) Various DIAP1 deletion mutants were used in a yeast two-hybrid assay to map the interaction domain between DIAP1 and the pro-domain of DRONC. The BIR2 region of DIAP1 was sufficient for the interaction with the pro-domain of DRONC (C) Co-immunoprecipitation of DRONC and DIAP1 from cellular extracts. 293T cells were transiently transfected with plasmids expressing FLAG-tagged DRONC, ΔN DRONC, DRONC-CARD or Bcl-10 (control) and Myc-tagged BIR1/2, BIR1, BIR2 or Bcl-10 in the indicated combinations. Cell lysates were immunoprecipitated with anti-FLAG and immunoblotted with anti-Myc as in Figure 1C. Epression of FLAG-tagged and Myc-tagged proteins was confirmed. Molecular mass markers in kDa are shown. Download figure Download PowerPoint To verify the observed interaction between DIAP1 and DRONC, we performed co-immunoprecipitation experiments on cellular extracts obtained from 293T cells (Figure 4C). FLAG-tagged pro-DRONC C→A, ΔN DRONC C→A and DRONC-CARD (the pro-domain of DRONC on its own) were each tested for interaction with Myc-tagged DIAP1 deletion mutants (BIR1/2, 1–341; BIR1, 1–146; and BIR2, 177–341; see schematic representation in Figure 4B). As expected, full-length DRONC and the isolated pro-domain of DRONC (DRONC-CARD) both co-immunoprecipitated with BIR1/2 and BIR2 but not with BIR1, consistent with our yeast two-hybrid data showing that the BIR2 domain of DIAP1 is required for the interaction with DRONC. Somewhat surprisingly, however, ΔN DRONC lacking the pro-domain also co-immunoprecipitated with DIAP1, although to a far lesser extent than full-length DRONC or DRONC-CARD. The BIR2 region of DIAP1 was required for this interaction between ΔN DRONC and DIAP1 since ΔN DRONC formed stable complexes only with BIR1/2 and BIR2 and not with BIR1. Taken together, these results indicate that DIAP1 physically interacts with unprocessed pro-caspase DRONC and that the BIR2 region of DIAP1 is able to bind both the pro-domain and the core region of DRONC. Expression of DIAP1 rescues the eye phenotype induced by ectopic expression of pro-DRONC but not ΔN DRONC To assess the ability of DIAP1 to modulate DRONC activation in vivo, we co-expressed DIAP1 with DRONC and ΔN DRONC (Figure 5F–I). Ectopic expression of DIAP1 in the developing eye of pro-droncW transgenic flies completely rescued the phenotype caused by ectopic expression of pro-DRONC (compare Figure 5B with G). Furthermore, GMR-diap1 also rescued the more severe small eye phenotype
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