Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).
Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).
In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).
The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).
Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).
Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).
The steroid hormone ecdysone has been shown to mediate apoptosis of larval tissues during pupariation. To investigate whether ecdysone also induces expression of dronc, the levels of dronc mRNA were examined after the addition of ecdysone to second instar larval midgut and salivary gland tissues, which normally show only low levels of dronc mRNA. After 1 hr exposure to ecdysone there was a several-fold increase in dronc mRNA levels in the early second instar larval midgut tissue, indicating that ecdysone can induce dronc expression in the midgut. However, salivary glands from early second instar larvae do not show dronc induction after ecdysone treatment. Because salivary glands normally undergo apoptosis later than midgut tissues, it is possible that the failure of ecdysone to induce dronc in salivary glands is the result of the absence of a developmentally controlled factor required for ecdysone-induced gene expression. For this reason, whether ecdysone could induce dronc expression in salivary glands at a later developmental stage was examined. Salivary glands from late second instar larvae, which normally only express very low levels of dronc, were found to strongly express dronc 1 hr after ecdysone treatment. Thus, ecdysone induces dronc expression in both midgut and salivary gland tissues (Dorstyn, 1999).
To check whether DRONC is indeed a caspase, recombinant DRONC was generated in E. coli and its proteolytic activity was assessed on synthetic fluorogenic peptide substrates. Expression of both the full-length DRONC precursor or truncated DRONC lacking the putative prodomain (residues 1-113) generated enzyme that showed low-level activity on a caspase-3 substrate DEVD-afc. However, DRONC activity on the caspase-2 pentapeptide substrate VDVAD-amc was 5-fold higher than on DEVD-afc, suggesting that, similar to caspase-2, the minimum substrate requirement for DRONC includes a P5 residue. Under identical conditions, recombinant DRONC and caspase-2 show approximately similar activities on VDVAD and DEVD substrates. No significant cleavage by DRONC of caspase-1 substrate YVAD-afc was observed (Dorstyn, 1999).
Since DRONC interacts with drICE, the ability of active DRONC to cleave drICE CdeltaA, lamin DmO , the DNA fragmentation factor DREP-1 and the baculovirus caspase inhibitor p35 was assayed. Both DRONC and drICE cleave drICE CdeltaA, lamin DmO and DREP-1. The cleavage products generated by DRONC and drICE are clearly different, indicating that DRONC and drICE each cleave lamin DmO and DREP-1 at different sites. Unlike drICE, however, DRONC is 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 (Meier, 2000).
Many caspases induce apoptosis when expressed in mammalian cells. It was therefore asked whether pro-DRONC, DeltaN DRONC or the catalytically inactive mutant of DeltaN DRONC (DeltaN DRONC CdeltaA) kill Rat-1 fibroblasts. Expression of DeltaN DRONC, which lacks its pro-domain, is very effective at inducing cell death, as is expression of either of the positive controls, caspase-8 and the Fas pathway adaptor FADD. However, in complete contrast, expression of full-length DRONC exerts no lethal effect. DRONC therefore resembles caspases-4 and -5 , both of which kill mammalian cells only when expressed without their respective pro-domains. As in S.pombe, the catalytically inactive DeltaN DRONC CdeltaA mutant has 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 DeltaN 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 DeltaN DRONC would then render the caspase no longer inhibitable by such putative factors, resulting in the spontaneous activation of DeltaN 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 (Meier, 2000).
Some caspases autocatalytically cleave and activate themselves. To determine if and where DRONC cleaves itself, a COOH-terminal His6-tagged version of DRONC was expressed and purified from E. coli. The purified protein consisted of two major bands, presumably consisting of the processed large and small subunits. This protein is active as a caspase. To determine the site of cleavage between the large and small subunits, Edman degradation amino-terminal sequencing was performed on the smaller band. The NH2-terminal sequence that was determined occurs COOH-terminal to the sequence TQTE352, suggesting that DRONC cleaves itself following a glutamate rather than an aspartate. To test this hypothesis DRONC TQTE352 was directly mutated to TQTA352. 35S-Labeled wild type DRONC and DRONC TQTAE352A were generated by in vitro translation and incubated with bacterially produced DRONC or DCP-1. DRONC cleaved itself to generate a product corresponding in size to the prodomain and large subunit. This band is not seen when DRONC TQTAE352A is the substrate, consistent with the hypothesis that DRONC processes itself following TQTE352. DCP-1 and drICE cleave DRONC at several sites. This cleavage is unaffected by the presence of the TQTA352 mutation, suggesting that these caspases cleave elsewhere in DRONC, perhaps in the DRONC prodomain. To explore the possibility of cleavage within the DRONC prodomain a form of DRONC, DRONCpD4A, was generated in which the P1 aspartates of four potential caspase target sites within the prodomain, DEKD66, ESVD110, DESD113, and DIVD135, were changed to alanine. 35S-labeled in vitro translated DRONCpD4A was still processed by wild type DRONC, but not by DCP-1. Similar results were obtained with cleavage by drICE. Thus DCP-1 and drICE can process DRONC within the prodomain, but not at the large-small subunit boundary. If this processing occurs in vivo it may serve as a point of regulation of DRONC function (Hawkins, 2000).
Positional scanning synthetic combinatorial libraries (PS-SCL) have been a useful tool to determine cleavage site specificities of other caspases. The PS-SCL is composed of three separate sublibraries of 8,000 compounds each. In each sublibrary, one position is defined with one of 20 amino acids (excluding cysteine), while the remaining two positions contain a mixture of amino acids present in approximately equimolar concentrations. Analysis of the three sublibraries (20 samples each) affords a complete understanding of the amino acid preferences in the P2, P3, and P4 positions. This approach was used to characterize DRONC's preferences for given amino acids at each of these positions. The positional scanning synthetic combinatorial libraries available all contain aspartate at the P1 position. While DRONC cleaves itself after glutamate, it is also able to cleave protein substrates after aspartate. Thus it was reasoned that the existing aspartate-based libraries would yield useful information about DRONCs cleavage specificity. DRONC shows a strong preference for Thr, Ile, or Val at the P2 position. A wider spectrum of amino acids was tolerated at the P3 and P4 positions. This analysis suggests that TATD constitutes an optimal DRONC P1 aspartate tetrapeptide cleavage site (Hawkins, 2000).
The results of the PS-SCL analysis are supported by experiments in which DRONC activity was tested directly with a number of commonly used tetrapeptide activity substrates. DRONC shows highest levels of activity with the tetrapeptides VEID-AMC and IETD-AMC, and somewhat lower levels of activity with DEVD-AMC. However, little if any activity is seen with WEHD-AMC or YVAD-AMC, which are predicted to be poor substrates. DRONC has higher levels of activity with the pentapeptide GIETD-AMC than with the tetrapeptide IETD-AMC. This suggests that a P5 residue is important for optimal DRONC activity (Hawkins, 2000).
To further characterize DRONCs cleavage preferences assays were carried out in which the cleavage activities of DRONC and DCP-1 were measured for two different peptide substrates: Ac-TQTE-AFC and Ac-DEVD-AFC. Ac-TQTE-AFC is derived from the known DRONC autoprocessing site and is also predicted to correspond to a good DRONC cleavage site based on the results obtained from PS-SCL analysis. Ac-DEVD-AFC is a tetrapeptide substrate for caspases generally grouped together as effectors of apoptosis (group II caspases). DRONCs activity is low in absolute terms compared with DCP-1. However, DRONC shows a clear cleavage preference for the Ac-TQTE-AFC substrate over Ac-DEVD-AFC. As expected, DCP-1, which has a common variant of the standard caspase active site pentapeptide (QACQG), has a strong preference for the tetrapeptide substrate with a P1 aspartate, Ac-DEVD-AFC (Hawkins, 2000).
Despite the fact that DRONC shows relatively low levels of activity with tetrapeptide substrates containing a P1 aspartate, DRONC is efficiently inhibited by the broad range tripeptide caspase inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk) (Hawkins, 2000).
It was of interest to determine DRONC's P1 specificity with respect to aspartate and glutamate. To do this a second tetrapeptide substrate, Ac-TQTD-AFC, was synthesized that differs from Ac-TQTE-AFC only by the P1 residue. These substrates were used to measure DRONCs activity. DRONC shows only a slight preference for cleavage of tetrapeptide substrates with a P1 aspartate over those with a P1 glutamate. DRONC is, however, a particularly poor catalyst of tetrapeptide hydrolysis. The calculated activity values for DRONC are roughly 40-180-fold lower than those described for caspase-9, which itself is a very inefficient enzyme in isolation as compared with most other caspases. This may reflect the fact that DRONC has an intrinsically low turnover rate or that optimal in vitro assay conditions have not been identified. However, DRONC activity may also be regulated allosterically through interactions with the Drosophila homolog of Apaf-1, HAC-1, and DARK in a manner similar to that of mammalian caspase-9 by Apaf-1. Alternatively, since DRONC shows similar levels of activity to DCP-1 on the protein substrate drICE, optimal DRONC cleavage may require additional sequences surrounding the target site (Hawkins, 2000).
If DRONC is an apical cell death caspase, likely substrates include other Drosophila caspases. drICE is a good candidate to be such a target since immunodepletion experiments show that drICE is required for rpr-dependent apoptotic events in cell extracts, and genetic interactions suggest that DRONC contributes to rpr-, hid-, and grim-dependent cell death. 35S-labeled in vitro translated drICE was generated and it was incubated with bacterially produced DRONC or DCP-1. drICE was efficiently cleaved by DRONC and DCP-1. The generation of a band corresponding in size to that of the mature large subunit Ala29-Asp230 was observed. Several other cleavage products were also generated. These correspond to full-length drICE lacking the prodomain, Ala29-Val339 and a fragment comprising the prodomain and large subunit processed at the COOH terminus of the large-small subunit linker region, 1-Asp230. To show that DRONC and DCP-1 process drICE at TETD230, the proposed natural drICE cleavage site between large and small subunits, this site was changed to TETA230. DRONC and DCP-1 do not cleave 35S-labeled in vitro translated drICE TETA230 between the large and small subunit, implying that they both cleave drICE at TETD230. These results, taken together with the observed site of DRONC autoprocessing in bacteria and in vitro , and the results of tetrapeptide cleavage experiments, argue that DRONC cleaves following glutamate as well as aspartate. DRONC efficiently processesdrICE at QTETD230. However, it processes DCP-1 very poorly at the equivalent site in the large-small subunit linker, VTETD215. These results are consistent with the possibility that the optimal DRONC peptide substrate is a pentapeptide (Hawkins, 2000).
The drICE TETA230 mutant is still cleaved by DCP-1 at one position, perhaps at the prodomain-large subunit boundary. To explore this possibility the P1 aspartate of the proposed prodomain-large subunit boundary caspase target site, DHTD28, was altered to alanine, generating drICED28A. DRONC and DCP-1 both process drICED28A to generate a fragment corresponding in size to the prodomain and large subunit processed at Asp230. A slightly smaller band, probably corresponding to the prodomain and the large subunit processed at the NH2 terminus of the large-small subunit linker, 1-Asp217, was also produced. However, no bands corresponding in size to full-length drICE lacking the prodomain or the fully processed large subunit were observed. These observations demonstrate that DCP-1 processes drICE in the prodomain as well as at TETD230 (Hawkins, 2000).
Addition of DRONC to in vitro translated drICE results in production of a mature drICE large subunit lacking prodomain sequences, but DRONC is unable to process drICE TETA230 within the prodomain. These observations suggested that drICE cleaved by DRONC at TETD230 is autocatalytically removing its own prodomain. To test this possibility DRONC and DCP-1 were incubated with an in vitro translated version of drICE, drICEC211A, in which the active site cysteine was changed to alanine. This caspase should remain inactive following cleavage at TETD230. DRONC cleavage of drICEC211A results in the appearance of only a single band corresponding to the prodomain and large subunit. This observation suggests that drICE autocatalytically removes its own prodomain following cleavage between the large and small subunits. Mature drICEC211A large subunit was generated in the presence of DCP-1. This further supports the argument that DCP-1 cleaves drICE in the prodomain as well as at the large-small subunit boundary (Hawkins, 2000).
What purpose could be served by DRONC having an altered cleavage specificity? One possibility is simply that DRONC has unique targets other than itself, and that a different target site preference is required for cleavage of these substrates. DRONC's novel cleavage site specificity, in conjunction with the sequence of the linker between the large and small subunits, may also provide a mechanism for limiting DRONC's ability to become activated by other caspase cascades. DCP-1 or drICE do not process DRONC to any significant extent at the large-small subunit boundary. This is not surprising because there are only two aspartates in the linker region between the large and small subunits, DEYD324 and KWPD348. Based on positional scanning synthetic combinatorial library analysis of tetrapeptide substrates, these sequences are predicted to be very poor substrates for all known mammalian caspases and DCP-1. The possibility that processing of DRONC by unknown proteases occurs at these sites in vivo cannot be ruled out. However, because DRONC is able to process itself in the linker region at TQTE352, but other tested caspases are not, it seems reasonable that DRONC's altered cleavage specificity, coupled with the lack of good target sites for other caspases in the large-small subunit linker region, may serve at least in part to make activation of DRONC more strictly DRONC-dependent. This may provide a mechanism for limiting cross-talk between other caspase cascades and pathways activated by DRONC (Hawkins, 2000).
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 results in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc< or dark largely suppresses this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibits Ice processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induces DRONC processing, which occurs in two steps. The first step appears to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulates when dark is silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 results in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing is 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 (Muro, 2002).
A number of caspases have been shown to induce apoptosis when overexpressed. DRONC was transiently co-expressed with ß-galactosidase in NIH 3T3 cells. At 48 hr posttransfection approximately 60% of the ß-galactosidase positive cells had undergone apoptosis. DRONC-induced cell death was almost completely abolished by coexpression of baculovirus P35 and inhibited to a lesser extent by MIHA, OpIAP, and Bcl-2. CrmA, an inhibitor of caspase-1, is least effective in inhibiting DRONC-induced apoptosis. A substitution mutation of the catalytic Cys-318 to Gly completely abolishes the cell-killing activity of DRONC, suggesting that cysteine protease activity is responsible for the apoptotic function of DRONC. The localization of DRONC protein was examined in transfected cells by using DRONC-GFP fusion constructs. Fusion of GFP to the carboxyl terminal of DRONC does not affect its cell-killing activity. At 24 hr, when most of the transfected cells appeared morphologically normal, DRONC was mostly localized in the cytoplasmic fraction of cells. In some cells, DRONC protein appeared to be concentrated asymmetrically near the cellular nucleus, possibly associated with some subcellular structures. Staining of transfected cells with mitochondrial markers suggests that DRONC does not localize to mitochondria. At 48 hr after transfection, DRONC-GFP protein is uniformly distributed in apoptotic cells (Dorstyn, 1999).
To determine whether ectopic expression of DRONC can induce cell death in D.melanogaster, the GAL4/UAS system was used to express various forms of DRONC in the developing Drosophila compound eye. Independent transgenic Drosophila lines were generated carrying pro-dronc, DeltaN dronc, pro-dronc CdeltaA, DeltaN dronc CdeltaA 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 in differentiating photoreceptors and pigment cells posterior to the morphogenetic furrow in the eye imaginal disc. The DRONC-induced phenotypes that were observed were of variable severity, depending on the insertion line used, presumably because of insertion site-specific effects on the transgene expression level. 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-DeltaN dronc line (Meier, 2000).
Pro-droncW flies carrying one copy of the transgene exhibit 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. Such eyes have an essentially normal external morphology and size, in contrast to eyes expressing Rpr under the control of GMR, which are severely reduced in size. By comparison, pro-droncS and DeltaN dronc transgenic flies exhibit dramatically 'roughened eyes' that are severely reduced in size. Scanning electron microscopy (SEM) analysis of pro-droncS and DeltaN dronc eyes confirms that surface morphology is severely distorted, erupted and rough. As with pro-droncW flies, eyes from pro-droncS and DeltaN dronc flies are 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 color. The phenotypes induced by DRONC expression are a consequence of DRONC caspase activity since overexpression of catalytically inactive CdeltaA mutants of DRONC exerts no detectable effect on eye development (Meier, 2000).
To investigate in detail the consequences of DRONC expression on the survival of photoreceptor and pigment cells underlying the eye surface, transverse sections of adult transgenic eyes were examined. 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. These remnant pigment cells, containing the red pigment pteridine, were responsible for the red 'spots' observed in the pro-droncW fly eyes. It was therefore concluded that GMR-driven DRONC expression kills both pigment and photoreceptor cells (Meier, 2000).
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. Third instar larval eye discs were examined for the appearance of apoptotic cells using acridine orange, which stains apoptotic cells. Compared with controls, third larval instar eye discs expressing DeltaN DRONC exhibit dramatic and super-numerary apoptosis posterior to the morphogenetic furrow. In contrast, no such sign of excessive apoptosis is evident in eye discs from third instar larvae expressing full-length pro-droncW. However, during later development (60 h after puparium formation), eye discs of pro-droncW pupae exhibit a dramatic increase in numbers of apoptotic cells. 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, DeltaN dronc or GMR-rpr transgenic flies is consistent with the observed induction of cell death much earlier during larval eye development (Meier, 2000).
The pro-domain-less DeltaN DRONC generates a consistently more severe eye ablation phenotype than does pro-DRONC. Indeed, all DeltaN dronc transgenic lines die when crossed with GMR-gal4 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 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 DeltaN 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, it was found that flies with severely deformed and black eyes can indeed be rescued by manually opening the puparium at the end of their development (Meier, 2000).
To examine the function of Dronc in a whole animal, transgenic flies were generated containing Dronc tagged with GFP or the inactive dronc mutant, droncC318G, also tagged with GFP, under the control of the yeast UAS(GAL4) in pUAST. Expression of these constructs was then achieved by crossing flies to various GAL4 drivers. To show that the constructs were expressed, UAS-dronc and UAS-droncC318G flies were crossed to flies containing the GMR-GAL4 driver, allowing expression in the posterior region of third instar larval eye imaginal discs. Eye imaginal discs from these third instar larvae stained specifically in the posterior region with antiGFP and antiDronc antibodies, demonstrating that high levels of specific expression were achieved and that the antiDronc antibody was specifically detecting Dronc protein. To determine whether ectopic overexpression of dronc could induce cell death, acridine orange staining of these eye imaginal discs was carried out to detect dying cells. Expression of the droncC318G construct has little effect on the normal pattern of dying cells in the eye imaginal disc, whereas wild type dronc expression results in a massive induction of cell death in the posterior part of the eye disc. Expression of dronc during embryogenesis or in different tissues during larval development using the heat shock-inducible hsp70-GAL4 driver also results in ectopic cell death (Quinn, 2000).
To examine the phenotypic consequence of expression of dronc in the eye disc, progeny of the cross of GMR-GAL4 to the UAS-dronc construct were allowed to develop into adults. Many died as pupae, which has been observed previously and attributed to the poor ability of the adults to break through the pupal case. The few adults from this cross that survive exhibit severely ablated eyes. By contrast, no death during the pupal stage was observed with flies from the cross of GMR-GAL4 to UAS- droncC318G, and adult flies showed normal eyes. Thus, the expression of dronc results in an almost complete ablation of the eye, similar to that obtained with expression of the apoptosis inducers rpr, hid, or grim from the GMR enhancer (Quinn, 2000).
Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).
Analysis of the Drosophila genome sequence reveals a single predicted transcript that encodes a type II membrane protein with structural similarities to members of the TNF superfamily. This protein is referred to as Eiger, in memory of the numerous mountaineers that have been killed by the Eiger Nordwand, the 'wall of death.' The Eiger protein contains a cytoplasmic domain, a transmembrane region between amino acid residues 36 and 62, and an extracellular domain of 353 amino acids. The C-terminal TNF homology domain (THD) of Eiger shows comparable homology to several human TNF family members (20%–25% identity). In situ hybridization has revealed a weak expression in imaginal discs with a pronounced pattern in the eye (Moreno, 2002).
Like a subset of human TNF ligands, Eiger can induce caspase-dependent apoptosis. Targeted expression of Eiger in the eyes and wings of Drosophila causes a severe ablation of these organs, and Eiger-expressing cell clones are rapidly eliminated. Both of these effects can be suppressed by coexpression of the pan-caspase inhibitor p35 (Moreno, 2002).
Caspase-8 is the key initiator caspase of death ligand-induced apoptosis in mammals. Upon stimulation by TNF, the adaptor protein FADD recruits and aggregates several molecules of procaspase-8 that mutually cleave and activate each other. Due to the involvement of an extracellular ligand, this pathway has been referred to as the 'extrinsic death pathway'. DREDD is the Drosophila caspase most similar to caspase-8 and has been shown to physically interact with Drosophila FADD. Surprisingly, complete removal of DREDD function fails to block Eiger-induced apoptosis, indicating that Eiger triggers cell death by a DREDD/caspase-8-independent pathway (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
To examine genetic interactions between Dronc and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).
Whether Dronc is able to induce cell death in the hemocyte-derived SL2 cells was also examined. Surprisingly, transfection of these cells with full-length Dronc only resulted in 25% cell death. Because previous studies have shown that Diap1 binds to the prodomain of Dronc and may inhibit Dronc function, a truncated version of Dronc lacking the prodomain (MPD-Dronc) was transfected into SL2 cells. This resulted in a significant increase in cell death (50%). Because previous studies had failed to observe an effect of the caspase inhibitor P35 on dronc-induced cell death, a test was performed to see whether co-transfection of P35 could suppress MPD-Dronc-induced cell death. In contrast to previous results, P35 was able to significantly suppress MPD-Dronc-induced cell death in SL2 cells. This result is consistent with a previous observation showing that P35 inhibits Dronc-induced cell death in a mammalian overexpression system. However, it should be noted that co-expression of P35 does not rescue MPD-Dronc-induced cell death as well as Diap1, z-VAD-fluoromethylketone, or the dominant negative Dronc mutant, DroncC318G, although rescue is significantly better than that observed with Diap2 (Quinn, 2000).
Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).
Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).
Flies were generated that expressed full-length wild type DRONC under GMR control. While phenotypes displayed by individuals within a line were similar, different lines displayed eyes with various degrees of eye disruption, presumably owing to genomic position effects on the expression level of the transgene. By manipulating the number of copies of the GMR-DRONC transgene in animals, a phenotypic series was inferred in which low levels of DRONC expression (GMR-DRONCW flies) resulted in no outward phenotype, while higher levels of expression (GMR-DRONCM flies) resulted in cell death late in retinal development. These flies had eyes that were normal in size and shape, but that were largely white due to a loss of retinal pigment. Tangential sections through the eyes of GMR-DRONCM flies showed that all retinal cells, including photoreceptors, were missing. Increasing DRONC expression levels still further (GMR-DRONCS flies) resulted in flies with small eyes, similar to those seen in animals overexpressing rpr, hid, or grim. These observations show that DRONC expression in the eye induces cell death in a dose-dependent manner. Consistent with this interpretation, third instar eye imaginal discs from animals expressing GMR-DRONCS show high levels of staining with the vital dye acridine orange, which is taken up and retained by dying cells (Hawkins, 2000).
DIAP1, a Drosophila member of the IAP family of caspase inhibitors, suppresses rpr-, hid-, and grim-dependent cell death in the fly. It was reasoned that if expression of DRONC was activating the same pathway, then the GMR-DRONC eye phenotype might be sensitive to the levels of DIAP1. To test this hypothesis the amount of DIAP1 in the eye was decreased by crossing a strong loss-of-function DIAP1 point mutant, thread 5 (th5), to GMR-DRONCM flies. th5 heterozygotes are phenotypically wild type. However, flies that are heterozygous for th5, and that express GMR-DRONCM, show an enhancement of the GMR-DRONC-dependent small eye phenotype. In contrast, small eyed GMR-DRONCS flies that overexpress DIAP1 because they carry a GMR-DIAP1 transgene show a strong suppression of the small eye and pigment loss phenotypes. These observations, are consistent with the idea that DRONC activity is negatively regulated by DIAP1. However, they do not exclude the possibility that DIAP1's effects on the DRONC overexpression phenotypes are due, at least in part, to DIAP1's ability to suppress the activity of caspases such as drICE, that are activated by DRONC (Hawkins, 2000).
Genetic and biochemical evidence suggests that one mechanism by which RPR, HID, and GRIM promote apoptosis is by blocking DIAP1's ability to inhibit caspase activation or activity, thereby promoting caspase-dependent cell death. To determine if DRONCs activity could be regulated in a similar manner tests were performed to see whether RPR, HID, or GRIM could interfere with DIAP1-dependent inhibition of DRONC-dependent yeast cell death. Yeast were generated in which DRONC was expressed under GAL1 control and DIAP1 was expressed under the control of the copper-inducible CUP1 promoter. A third GAL1 vector was then introduced that was either empty or that expressed RPR, HID, or GRIM. Cells expressing GAL1-DRONC and empty vectors died when plated on medium containing galactose and 100 µM copper, but cells expressing GAL1-DRONC and CUP1-DIAP1 survived. Coexpression of GAL1-RPR had no effect on the survival of yeast expressing GAL1-DRONC and CUP1-DIAP1. However, coexpression of GAL1-HID or GAL1-GRIM completely blocked the survival of these cells. Thus, while these experiments do not exclude the possibility that HID and GRIM might alter DRONC activity directly, they are consistent with other observations arguing that these proteins mediate their effects on caspase activity, and thus presumably caspase-dependent yeast cell killing, by virtue of their interactions with DIAP1 (Watkins, 2000).
Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001).
Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through Dronc, since GrimC-induced death is partially suppressed by a dominant/negative DroncC318S protein. However, the persistence of some eye cell death in the presence of DroncC318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001 and references therein).
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. Cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and the initiator caspase Dronc and effector caspase Ice 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 Ice 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 is 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 (Dorstyn, 2002).
In mammalian cell extracts, addition of cytochrome c and dATP results in the formation of an ~700-kD complex, commonly known as an apoptosome. Studies using purified components have demonstrated that the apoptosome, consisting of Apaf-1, cytochrome c, and procaspase-9, is necessary for caspase-9 activation. Since formation of an apoptosome in Drosophila has not been demonstrated and because cytochrome c is not released from mitochondria during apoptosis, whether a cytochrome c-dependent apoptosome containing Dronc is formed in Drosophila cells was tested. Cell extracts prepared from BG2 cells were fractionated by gel filtration chromatography and individual fractions were analyzed by immunoblotting using specific antibodies. In cell extracts kept at 4°C, the majority of Dronc was eluted in its monomeric form (50 kD) in fractions 20-22. Extracts that were incubated at 27°C with or without cytochrome c and dATP showed a shift of some of the Dronc protein to fractions 3-5, which correspond to a molecular mass of >670 kD. The shift in the absence of added cytochrome c may suggest that endogenous cytochrome c present in cell extracts could be sufficient to allow the formation of the large complex containing Dronc. Similar results have been seen using mammalian cell extracts. Drosophila cells grow at 27°C, however, when the cell extracts are incubated at 37°C, the majority of the Dronc is recruited to the >700-kD complex and there is increased processing of proDronc and proIce. The reason for this is not clear, however recombinant Dronc and Ice and extracts prepared from apoptotic BG2 cells show considerably more caspase activities at 37°C than at 27°C (Dorstyn, 2002).
Does the large complex contains Ice? In cell extracts incubated at 4°C, the majority of the Ice precursor remains in its monomeric form, although some appears to be dimeric. Incubation of cell extracts at 27°C or 37°C, with or without cytochrome c/dATP, results in the recruitment of a fraction of Ice to the high molecular mass complex. Interestingly, in extracts incubated at 37°C, most of the Ice in the high molecular mass complex is processed, whereas most of the monomeric Ice is in the precursor form. These results suggest the formation of an apoptosome containing Dronc and Ice in Drosophila cell extracts (Dorstyn, 2002).
To further explore the role of cytochrome c in the formation of the Dronc-containing complex, cytochrome c was immunodepleted from S100 fractions. These fractions were then subjected to gel filtration experiments. When incubated at 27°C, a small fraction of Dronc is found in the high molecular mass complex. Addition of cytochrome c and dATP causes a significant increase in the recruitment of Dronc to the >700-kD complex. Immunoblotting the fractions with the cytochrome c antibody shows that incubation of S100 at 27°C results in the recruitment of a significant proportion of cytochrome c to the >700-kD complex. Interestingly, only dimeric (26 kD) cytochrome c is detected in the >700-kD complex. These results suggest that cytochrome c and dATP, at least in part, are responsible for the formation of the complex (Dorstyn, 2002).
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. Silencing expression of either or both DC3 and DC4 has no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. Loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 fail to activate caspases in Drosophila cell lysates, but, remarkably, induce caspase activation in extracts from human cells. Overall, these results argue that DARK-mediated DRONC activation occurs independently of cytochrome c (Dorstyn, 2004).
The data clearly show that neither of the two cytochrome c species in Drosophila are required for caspase activation or apoptosis. Previous studies reported that a P-element insertion in the dc3 gene (bln1) results in loss of DRICE activity in testis (Arama, 2003). However, a recent report indicates that the bln1 P-element insertion also disrupts a number of other genes (Huh, 2004), thus questioning whether DC3 is responsible for DRICE activity. Additionally, DRICE activation during spermatogenesis appears to be independent of DARK and DRONC (Huh, 2004). If DC3 is required for caspase activation in Drosophila, a loss of function mutation in dc3 should lead to severe developmental defects and lethality. Furthermore, although a tissue-specific function has been suggested for DC3, it is unlikely that DC3 functions only during spermatogenesis, given its ubiquitous expression. Although disruption of the dc4 gene is embryonic lethal, DC4 cannot induce caspase activation and apoptosis in Drosophila cells (Dorstyn, 2004).
The question remains: how does DARK mediates DRONC activation? One possibility is that other factors can substitute for cytochrome c function during apoptosis. Alternatively, removal of DIAP1 from DRONC may be sufficient to allow an interaction with DARK and activation. Given that transcription plays a major role in developmental PCD in Drosophila, changes in the concentration of DIAP1, DRONC, and DARK proteins could facilitate caspase activation in the fly. These studies, combined with published work, demonstrate that Drosophila and mammalian cytochrome c proteins are functionally similar since they can both mediate respiration and Apaf-1 activation in mammalian cell lysates. Therefore, the requirement for cytochrome c in caspase activation in mammals is likely to have evolved late in evolution (Dorstyn, 2004).
The physical interaction between DRONC and drICE was assessed by testing for the ability of the two proteins to co-immunoprecipitate from cell extracts. FLAG-tagged, full-length, catalytically inactive DRONC (pro-DRONC CdeltaA, 1-451) was co-expressed in 293T cells together with Myc-tagged catalytically inactive pro-drICE CdeltaA (1-339), DeltaN drICE CdeltaA (29-339) or Bcl-10. 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-immunoprecipitates both pro-drICE and DeltaN drICE, but not Bcl-10, indicating that DRONC and drICE form a stable complex in cell extracts (Meier, 2000).
The observed difference between the pro-apoptotic activity of pro-DRONC and pro-domain-lacking DeltaN 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, Drosophila proteins were sought 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, 56 DRONC-interacting clones were recovered, of which 17 encoded DIAP1. The second BIR domain of DIAP1 is necessary and sufficient for the interaction with the pro-domain of DRONC. 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 (Meier, 2000).
To verify the observed interaction between DIAP1 and DRONC, co-immunoprecipitation experiments were performed on cellular extracts obtained from 293T cells. FLAG-tagged pro-DRONC CdeltaA, DeltaN DRONC CdeltaA 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). As expected, full-length DRONC and the isolated pro-domain of DRONC (DRONC-CARD) both co-immunoprecipitate with BIR1/2 and BIR2 but not with BIR1, consistent with yeast two-hybrid data showing that the BIR2 domain of DIAP1 is required for the interaction with DRONC. Somewhat surprisingly, however, DeltaN 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 is required for this interaction between DeltaN DRONC and DIAP1, since DeltaN DRONC forms 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 (Meier, 2000).
Several lines of evidence suggest that pro-DRONC activation is negatively regulated via its pro-domain. This is best illustrated by the biology underlying the relatively weak eye phenotype in flies expressing full length pro-droncW: (1) most UAS-pro-dronc lines are viable when crossed to a strong GMR-gal4 line and kept at 25°C: in contrast, virtually all GMR-driven DeltaN dronc transgenic lines tested die under such conditions; (2) most pro-dronc lines exhibit essentially normal outer eye structure, whereas rare surviving DeltaN dronc transgenic flies never display this 'weak' eye phenotype and have severely deformed eyes; (3) ectopic expression of pro-DRONC induces no significant increase of cell death in the eye discs of third instar larvae, whereas excessive cell death is evident posterior to the morphogenetic furrow in the eye discs of third instar larvae expressing DeltaN DRONC; (4) ectopic expression of DRONC in mammalian Rat-1 cells induces apoptosis only when its pro-domain had been removed, suggesting the existence of an innate inhibitor of DRONC activation acting through the DRONC-CARD domain. All of these observations implicate the DRONC pro-domain in repressing activation of the caspase and suggest that DRONC activation is kept in abeyance in metazoan cells through the action of some CARD-binding innate inhibitor. In contrast, studies of DRONC in S.pombe unambiguously show that isolated pro-DRONC is, by itself, perfectly capable of undergoing catalytic autoprocessing resulting in its activation. Indeed, in yeast, pro-DRONC proved more toxic than DeltaN DRONC, suggesting that the presence of the pro-domain may actually enhance DRONC activation in the absence of other modulating influences (Meier, 2000).
In insect cells, a candidate for such an innate DRONC repressor is the inhibitor of apoptosis, DIAP1, which interacts with the DRONC pro-domain: co-expression of DIAP1 completely reverts the eye ablation phenotype of pro-droncW flies, whereas the eye ablation phenotype induced by DeltaN DRONC is largely unaffected. If endogenous DIAP1, or an analog, were not expressed in the Drosophila eye until very late in its development, this would provide the requisite mechanism for holding the activity of DRONC in abeyance until very late, so generating the 'spotted eye' phenotype observed. Indeed, heterozygosity at the diap1 locus greatly enhances the eye phenotype induced by pro-DRONC overexpression, indicating that endogenous DIAP1 negatively regulates DRONC activation in vivo. This is analogous to the way in which c-IAP1, c-IAP2 and XIAP bind to, and inhibit activation of, the pro-form of the apical caspase-9 in mammalian cells. The notion that it is DIAP1, in particular, that most likely fulfils the role of in vivo suppressor of DRONC is reinforced by studies in yeast that show that while DIAP1 both interacts with, and protects from the lethal effects of, pro-DRONC, Drosophila DIAP2 and the mammalian IAP homologs MIHA, MIHB, MIHC, MIHD and XIAP offer no such protection (Meier, 2000).
Currently, very little is known about how IAPs suppress apoptosis, although the most convincing biological evidence for the ability of IAPs to regulate cell death comes from genetic studies in D.melanogaster. Deletion of the chromosomal region encoding DIAP1 enhances cell death induced by ectopic expression of Rpr, and genetic loss of DIAP1 function leads to early and widespread apoptosis, indicating that DIAP1 is essential for survival of many cell types. Furthermore, overexpression of DIAP1 suppresses cell death induced by either Rpr, Grim or Hid through direct interaction between these various pro-apoptotic proteins and the second BIR domain of DIAP1, the same BIR domain that is sufficient for its interaction with pro-DRONC. It is noteworthy that it is also the second BIR repeat of the mammalian IAP family members c-IAP1, c-IAP2 and XIAP that appears sufficient for their anti-apoptotic activity (Meier, 2000 and references therein).
The finding that DIAP1 directly binds to and inhibits cell death caused by ectopic expression of DRONC, as well as by Rpr, Grim and Hid, underscores the key role played by DIAP1 in the regulation of apoptosis in D.melanogaster and raises the possibility that Rpr, Hid or Grim may exert some, or all, of their pro-apoptotic action through displacement of DIAP1 from the pro-domain of DRONC, so allowing activation of the caspase and consequent cell death. The isolation of DIAP1 mutants that display greatly reduced binding for Rpr, Hid and Grim and significantly suppress Rpr, Hid and Grim cell killing strongly supports this idea. According to this model, IAPs function as 'guardians' of the apoptotic machinery: these guardians act to suppress the chance of spontaneous activation of the intrinsic cell death machinery by neutralizing pro-apoptotic caspases, so establishing a buffered threshold that must be either exceeded or neutralized in order to initiate the destruction of a cell (Meier, 2000).
The biochemical interaction of Dronc with various apoptosis regulators was examined by co-precipitation methods. When 293T cells were transfected with Dronc-GFP and Diap1-Myc or Diap2-Myc, it was found that Diap1, but not Diap2, was present in the Dronc immunoprecipitated complex, showing that Dronc and Diap1 can interact. Dronc can form a complex with Diap1, but not with Diap2, in SL2 cells. The H99 proteins, Rpr, Hid, and Grim, were examined after transfecting 293T cells with Dronc-GFP and with Rpr-FLAG, Hid-FLAG, or Grim-FLAG constructs. Proteins associating with antiGFP-immunoprecipitated Dronc were immunoblotted with the antiFLAG antibody. Dronc co-immunoprecipitates with Grim, but not with Rpr or Hid, suggesting that Dronc forms a complex with Grim. Whether Dronc can form a complex with P35 was examined by transfecting SL2 cells with Dronc tagged with the HA and 6xHis epitopes and with HA-tagged P35. P35 is present in the Dronc complex, indicating that these proteins can associate (Quinn, 2000).
To determine whether the interactions of Dronc with Grim or P35 are direct, whether these proteins can interact in vitro was tested. Whereas Grim can bind to Diap1 in vitro, Dronc is unable to bind to either Grim or P35. Thus, under the conditions where Grim and Diap1 can interact, Dronc and P35 or Grim do not interact, suggesting that the interaction observed between Dronc and Grim or P35 in cells is indirect. Furthermore, the addition of Diap1 allows Grim to be co-immunoprecipitated with Dronc, suggesting that the complex formed in vivo between Dronc and Grim may be mediated by Diap1 (Quinn, 2000).
The amino-terminal region of Apaf-1-related-killer (Ark) containing the CARD and CED-4/Apaf-1 homology domains but lacking the WD40 repeats has been shown to bind to Dredd and Drice. Furthermore, in mammalian tissue culture cells, Dark has been shown to bind to Dronc. To investigate the region of Dark required for the interaction of Dark with Dronc, SL2 cells were transfected with a construct containing a Myc-tagged Dark amino-terminal region [Ark(1-411)], containing the CARD and CED-4/Apaf-1 homology domains, alone or with a FLAG-tagged Dronc construct. Dark-Myc was immunoprecipitated with antiMyc antibodies; pelleted complexes, and cell lysates were analyzed by immunoblotting with antiFLAG antibodies. Dronc-FLAG protein (50% of the protein present in the lysate) was detected in the antiMyc-Arc immunoprecipitate. Thus, the amino-terminal region of Dark containing the CARD domain and the CED-4/Apaf-1 homology region is sufficient for Dronc association in vivo (Quinn, 2000).
To test the requirement of Ark for Dronc activation, extracts prepared from darkCD8 homozygous flies were examined for caspase activity and for their ability to cleave Dronc in vitro. Ark mutant flies have reduced caspase activity compared with wild type for several caspase substrates. VEID is the preferred substrate for Dronc, although VDVAD is also cleaved well by Dronc. DEVD is a caspase-3 substrate that is cleaved poorly by Dronc but preferred by the downstream caspases Dcp1, Decay, and Drice. Thus, Ark mutant extracts contain lower cleavage activity toward both preferred Dronc substrates and preferred downstream caspase substrates. Lower caspase activity has also been observed in extracts from Ark mutant embryos. Furthermore, Ark mutant extracts showed considerably reduced ability to cleave Dronc to its active form, showing that Ark is important for Dronc processing. Because darkCD8 (and darkCD4) are hypomorphic mutants, and it is not known whether they are completely null because a deficiency of the Ark region is not available, the residual Dronc processing observed may be due to residual Ark activity or to an alternative mechanism (Quinn, 2000).
Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when 'Reaper-like' proteins bind to IAPs and liberate caspases from IAPs. The thioredoxin peroxidase Jafrac2 has been identified as an IAP-interacting protein in Drosophila cells that harbors a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition (Tenev, 2002).
Jafrac2 was recovered as a DIAP1-interacting protein in the cell using the tandem affinity purification (TAP) system. Like Rpr, Grim, Hid, Sickle, Smac/DIABLO and HtrA2/Omi, Jafrac2 bears a conserved N-terminal IAP-binding motif (IBM) essential for IAP interaction. Jafrac2 is synthesized as a precursor protein with an N-terminal signal peptide that targets it to the ER. Upon import into the ER, the signal peptide of Jafrac2 is cleaved off, thereby exposing the IAP interacting domain that allows this mature Jafrac2 isoform to interact with DIAP1, DIAP2 and XIAP (Tenev, 2002).
In living cells Jafrac2 is compartmentalized and sequestered in the ER away from IAPs, where it exists exclusively in the processed from. This is evident because mature Jafrac2, like cytochrome c, which is compartmentalized in mitochondria, remains associated with the membrane fraction in healthy cells. Following stimulation of apoptosis by UV irradiation or ER stress-inducing agents, mature Jafrac2 is released from the membrane fraction and is present in the cytosol where it can interact with DIAP1 and DIAP2. Because the pro-apoptotic, IAP-interacting form of Jafrac2 is released only upon cell death insult, the major regulatory step for Jafrac2 appears to be its release from the ER lumen. The release of Jafrac2 from the ER of UV-irradiated cells occurs early in UV-mediated apoptosis. This is evident because Jafrac2 expression becomes diffuse in otherwise morphologically normal cells within 3-4 h following UV exposure. In similar experiments, the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi that occurs, early in apoptosis, also became apparent within 3-4 h following UV treatment. Thus, Jafrac2 resembles Smac/DIABLO and HtrA2/Omi that are similarly compartmentalized in healthy cells and that promote caspase activation after their release from mitochondria following the cell death trigger. Furthermore, analogous to Smac/DIABLO and HtrA2/Omi, Jafrac2 also requires N-terminal processing to generate its pro-apoptotic form. Hence, Jafrac2, Smac/DIABLO and HtrA2/Omi all undergo a maturation process through cleaving off their signal peptide following import into their respective organelles. This organelle-specific maturation ensures that newly synthesized Jafrac2, Smac/DIABLO and HtrA2/Omi will not promote apoptosis prior to their sequestration into organelles (Tenev, 2002).
In common with Rpr, Grim and Hid, Jafrac2 interacts genetically and biochemically with DIAP1 and is able to promote cell death. In the Drosophila eye and tissue culture cells, mature Jafrac2, like Rpr, efficiently induces cell death in a DIAP1-binding dependent manner. Recent studies have suggested that Rpr and Grim antagonize the anti-apoptotic activity of IAPs by two distinct mechanisms -- (1) by a mechanism that requires DIAP1 binding, Rpr promotes DIAP1 self ubiquitylation and proteasomal degradation and (2) Rpr and Grim were also found to repress global protein translation by a mechanism that does not rely on IAP binding. The Ub fusion technique has been used to examine whether Jafrac2 and Rpr possess apoptosis-promoting activities that are independent of IAP binding. In vivo Rpr and Jafrac2 promote cell death exclusively in an IAP-binding dependent manner because mutations that impair the binding between DIAP1 and Rpr or Jafrac2 completely abolish their ability to induce cell death in the developing eye and tissue culture cells. Thus, Rpr and Jafrac2 that fail to bind to DIAP1 also fail to induce cell death. Mutations in endogenous diap1, which greatly impair the binding of DIAP1 to Rpr or Jafrac2, suppress Rpr and Jafrac2-mediated cell killing. Together, these data argue that in common with Rpr, mature Jafrac2 promotes cell death, and this activity is contingent upon their binding to DIAP1 (Tenev, 2002).
The interaction between Jafrac2 and the DIAP1 BIR2 domain is indispensable for its pro-apoptotic function. Interestingly, Jafrac2 and Dronc share a common binding site in the BIR2 domain that is distinct from the site of interaction between the DIAP1 BIR2 domain and Rpr and Hid. The th4 mutation of DIAP1's BIR2 domain greatly diminishes binding to Jafrac2 and Dronc, whereas the same mutation does not affect its binding to Rpr and Hid. In addition, the th23-4 DIAP1 mutation that greatly impairs the binding of DIAP1 to Rpr and Hid does not affect the DIAP1-Jafrac2 interaction. Consistent with the biochemical data, flies carrying the th4 mutation, which abolishes Jafrac2 binding, display strongly suppressed Jafrac2-induced eye ablation but enhanced Rpr-induced cell death in the eye (Tenev, 2002).
Several lines of evidence show that the IBM of Jafrac2 is essential for IAP binding and induction of apoptosis. Mutations that delete or obstruct the N-terminus of mature Jafrac2 abrogate the ability of Jafrac2 to bind to DIAP1 and trigger cell death. The view that Jafrac2 harbors a bona fide IAP-binding motif is strongly supported by crystal structure analyses that have identified Ala1 of IBMs as the critical residue to anchor this motif to the BIR surface of IAPs. In addition to the requirement of Ala1, there is a strong preference for Pro3. In accordance with other IBMs, the putative IBM of mature Jafrac2 bears Ala1 and Pro3. Furthermore, the IBM of Rpr is functionally interchangeable with the IBM of Jafrac2. A chimeric Rpr mutant (AKP-Rpr) in which the IBM of Rpr was replaced with the IBM of Jafrac2, displayed the same phenotype and cell death promoting efficacy as wild-type Rpr (AVA-Rpr) in both the Drosophila developing eye and tissue culture cells. Together, these results reveal that whereas Jafrac2 and Rpr share a common IAP-binding motif, they also have some distinct DIAP1-binding requirements that presumably give these interactions their specificity (Tenev, 2002).
Physical interaction between DIAP1 and caspases is essential to regulate apoptosis in vivo because embryos with a homozygous mutation that abolishes Dronc binding die early during embryogenesis due to widespread apoptosis. Unrestrained cell death caused by loss of DIAP1 function requires the Drosophila Apaf-1 homolog DARK because a mutation in dark rescues DIAP1-dependent defects. Thus, loss of DIAP1 function allows DARK-dependent caspase activation. Although activation of downstream, effector caspases is required for normal cell death, the activation of initiator caspases, such as Dronc, is rate limiting for the activation of this cascade. The observed unrestrained cell death caused by loss of DIAP1 function is likely to be triggered by the initiator caspase Dronc because DIAP1 normally suppresses Dronc activation, which in turn is mediated by DARK. In line with the current model on caspase activation, it is argued that the DIAP1-mediated inhibition of Dronc is the key regulatory step in controlling cell death. This view is supported by the observation that flies with diap1 mutations that either abolish binding or ubiquitylation of Dronc completely fail to suppress Dronc- mediated cell death in vivo. Thus, DIAP1 suppresses Dronc activation by binding to and targeting Dronc for ubiquitylation. However, when Rpr-like molecules displace DIAP1 from Dronc, Dronc is recruited into a 700 kDa size apoptosome protein complex that results in Dronc activation. Consequently, cell death is triggered when Dronc is liberated from DIAP1. Thus, the key event in regulating the caspase cascade appears to be inhibition of Dronc by DIAP1 (Tenev, 2002).
Several lines of evidence support the notion that Jafrac2 promotes cell death by interfering with the Dronc-DIAP1 interaction, thereby displacing and liberating Dronc from DIAP1. (1) Jafrac2 and Dronc bind to the same site of the BIR2 domain of DIAP1, since the BIR2 th4 mutation of DIAP1 equally abolished Dronc and Jafrac2 binding. In contrast, Rpr and Hid binding to the th4 DIAP1 mutant remains unaffected. (2) Jafrac2 competes with Dronc for the binding of DIAP1, and Jafrac2 possesses a significantly higher DIAP1-binding affinity compared with that of Dronc to DIAP1, as would be expected of a protein that displaces Dronc from DIAP1. (3) Ectopic expression of Jafrac2 in the developing Drosophila eye causes a phenotype that is highly reminiscent of the phenotype observed in flies ectopically expressing Dronc. (4) Heterozygosity at the dronc locus rescued the eye-ablation phenotype induced by Jafrac2, indicates that apoptotic signal transduction initiated by Jafrac2 is mediated through Dronc. Taken together, these results indicate that Jafrac2 promotes cell death by liberating Dronc from the anti-apoptotic activity of DIAP1 (Tenev, 2002).
The observation that Jafrac2, like the apoptotic inducers Rpr, Grim and Hid, induces apoptosis through binding to DIAP1 places Jafrac2 in a potentially pivotal position to regulate apoptosis. The findings are consistent with a model whereby Jafrac2 promotes apoptosis by displacing DIAP1 from Dronc, so allowing activation of the caspase cascade and consequent cell death. The idea is favored whereby Jafrac2 function is additive to, but independent of, Rpr. The early release of Jafrac2 from the ER of UV-irradiated cells is consistent with the view that Jafrac2 is involved in the initiation of apoptosis. Thus, Jafrac2 is released from the ER at a time when other early apoptotic events occur, such as the mitochondrial release of cytochrome c, Smac/DIABLO and HtrA2/Omi in mammalian cells. Once released, Jafrac2 interacts with DIAP1 and thereby liberates Dronc, which in turn is activated by DARK. In line with the notion that Jafrac2 functions in a complementary but distinct cell death pathway to Rpr, Grim and Hid, it is found that a chromosomal deletion that includes the jafrac2 locus does not suppress the eye phenotypes caused by ectopic expression of Rpr, Grim and Hid. However, it is possible that Jafrac2 may also be part of a positive feedback mechanism, which cooperates with Rpr-like proteins to promote apoptosis in response to cellular damage. These two alternatives cannot be distinguished because no jafrac2 mutant flies are available and Jafrac2 is refractory to the effect of dsRNA interference (Tenev, 2002).
The data are consistent with the idea that Jafrac2, with its thioredoxin peroxidase activity and IAP-binding ability, contains two distinct functions. In healthy cells, Jafrac2 may fulfil a 'housekeeping' role through its peroxidase activity by protecting the cell from oxidative damage. Consistent with this view, members of the peroxiredoxin protein family play an important role in protecting cells against oxidative damage by scavenging intracellularly generated reactive oxygen species, such as H2O2. However, upon UV irradiation, mature Jafrac2 is released from the ER and competes with Dronc for the binding of DIAP1 that is independent of its peroxidase activity. Consequently, Jafrac2 liberates Dronc from DIAP1 inhibition and allows activation of the proteolytic caspase cascade, resulting in cell death (Tenev, 2002).
Members of the 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 all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. In vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, 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. These data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination (Wilson, 2002).
The cellular antioxidant defense systems neutralize the cytotoxic by-products referred to as reactive oxygen species (ROS). Among them, selenoproteins have important antioxidant and detoxification functions. The interference in selenoprotein biosynthesis results in accumulation of ROS and consequently in a toxic intracellular environment. The resulting ROS imbalance can trigger apoptosis to eliminate the deleterious cells. In Drosophila, a null mutation in the selD gene (homologous to the human selenophosphate synthetase type 1) causes an impairment of selenoprotein biosynthesis, a ROS burst and lethality. This mutation (known as selDptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selDptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selDptuf does not require the activity of the Ras/MAPK-dependent proapoptotic gene hid, but results in stabilization of the tumor suppressor protein p53 and transcription of the Drosophila pro-apoptotic gene reaper (rpr). Genetic evidence supports the idea that the initiator caspase DRONC is activated and that the effector caspase DRICE is processed to commit selDptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selDptuf mutant cells. These observations indicate that selDptuf ROS-induced apoptosis in Drosophila is mainly driven by the caspase-dependent p53/Rpr pathway (Morey, 2003).
Caspases are well known for their role in the execution of apoptotic programs, in which they cleave specific target proteins, leading to the elimination of cells, and for their role in cytokine maturation. In this study, a novel substrate was identified, that, through cleavage by caspases, can regulate Drosophila neural precursor development. Shaggy (Sgg)46 protein, an isoform encoded by the sgg gene and essential for the negative regulation of Wingless signaling, is cleaved by the Dark-dependent caspase. This cleavage converts it to an active kinase, which contributes to the formation of neural precursor [sensory organ precursor (SOP)] cells. This evidence suggests that caspase regulation of the wingless pathway is not associated with apoptotic cell death. These results imply a novel role for caspases in modulating cell signaling pathways through substrate cleavage in neural precursor development (Kanuka, 2005; full text of article).
Previous genetic studies of sgg mutant flies showed the interesting observation that some phenotypes of sgg mutants can be rescued by the expression of sgg10 or sgg39 (the other sgg isoform similar to sgg10), but not sgg46, suggesting that Sgg46 might be an inactive form. The Sgg10 kinase phosphorylates the Arm protein and induces its degradation. Various forms of Sgg protein were tested for this activity. Expression of Sgg10 induces Arm phosphorylation and degradation in a kinase-dependent manner. In contrast, full-length Sgg46 did not produce the same effects on the Arm protein. Interestingly, expression of a putative cleaved form of Sgg46, containing the kinase domain (myc-Sgg46 DeltaN235 and myc-Sgg46 DeltaN300), led to Arm phosphorylation and degradation in a manner similar to that of Sgg10. These results suggest that full-length Sgg46 is an inactive form that can be converted into an active kinase via caspase-dependent cleavage (Kanuka, 2005).
Whether these findings would be applicable to macrochaete and SOP cell development in vivo was tested by using transgenic flies expressing Sgg proteins. The ectopic expression of Sgg10 by sca-GAL4 caused the loss of macrochaetes and SOP cells. No apoptotic cells in the myc-Sgg10 protein-expressing region of the wing disc could be detected, indicating that this disappearance did not result from the death of SOP cells. Consistent with the immunoblotting results, full-length Sgg46 did not influence macrochaete and SOP cell formation, whereas the cleaved form of Sgg46 (Sgg46 DeltaN300) worked in a manner similar to that of Sgg10. After crossing sca-GAL4+UAS-DRONC DN to UAS-sgg10, most F1 progeny showed a clear loss of macrochaetes in the scutellum, indicating that Sgg kinase activation might be downstream of caspases. These observations suggest that the processing of Sgg46 by caspases leads to the formation of an active kinase that can negatively regulate SOP cell development (Kanuka, 2005).
Finally, whether Sgg46 contributes significantly to macrochaete and SOP cell formation in vivo was investigated. The ectopic expression of Sgg46 D235G/D300G by sca-GAL4 significantly induced extra macrochaetes and SOP cells. Since Sgg46 D235G/D300G could not be cleaved by caspases, this noncleaved Sgg46 might act as dominant-negative form against endogenous Sgg function. Furthermore, an ectopic knockdown of Sgg protein expression by dsRNA-expressing constructs revealed that the specific reduction of the Sgg46 protein induced extra macrochaetes. However, inhibition of Sgg46 is less effective at producing extra macrochaetes than inhibiting Dark or DRONC, suggesting that modulation of Sgg kinase activity may not be the only mechanism contributing to SOP formation. It still remains to be examined whether or not Sgg46 is actually cleaved and converted into an active form in proneural clusters, and will require further examination in vivo. Based on the findings that loss of Sgg function or inhibition of caspase activity resulted in extra macrochaetes mainly in the scutellum of the adult notum (pSC and aSC), where Wingless is highly expressed, and that caspases are activated in scabrous-expressing cluster, it could be considered that scabrous-expressing SOP cells that will produce pSC and aSC macrochaetes are located in specific region, where precise formation of each set of macrochaetes might require both (1) Wg expression (to increase bristle) and (2) caspase activation (to decrease bristle). Thus, it appears that Dark-dependent caspase signaling mediates the total Sgg kinase activity by processing Sgg46 into an active form, thereby negatively regulating Wingless-sensitive macrochaete development (Kanuka, 2005).
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