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REGULATION

Promoter

The Hippo (Hpo) kinase cascade restricts tissue growth by inactivating the transcriptional coactivator Yorkie (Yki), which regulates the expression of target genes such as the cell death inhibitor diap1 by unknown mechanisms. The TEAD/TEF family protein Scalloped (Sd) is a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th) locus harbors a minimal Sd-binding Hpo Responsive Element (HRE) that mediates transcriptional regulation by the Hpo pathway. Sd binds directly to Yki, and a Yki missense mutation that abrogates Sd-Yki binding also inactivates Yki function in vivo. sd is required for yki-induced tissue overgrowth and target gene expression, and that sd activity is conserved in its mammalian homolog. These results uncover a heretofore missing link in the Hpo signaling pathway and provide a glimpse of the molecular events on a Hpo-responsive enhancer element (Wu, 2008).

The Hpo signaling pathway has emerged as a central and highly conserved mechanism that regulates organ size in animals. At the core of this pathway is a kinase cascade that impinges on the transcriptional coactivator Yki to regulate the transcription of target genes involved in cell growth, proliferation, and survival. Given Yki's pivotal position in the Hpo pathway, understanding the mechanisms by which Yki regulates target gene expression should provide important mechanistic insights that can facilitate therapeutic manipulation of this crucial size-control pathway (Wu, 2008).

A major gap in understanding of the Hpo signaling pathway concerns how Yki regulates target gene transcription. This study shows that Sd represents a crucial missing link between Yki and the regulatory DNA of Hpo pathway target genes. First, an unbiased dissection of diap1 regulatory region revealed a minimal Sd-binding enhancer element (HRE) that confers Hpo-responsive regulation. The HRE not only responded to Yki activity in vivo, but also conferred Sd-dependent and Yki-dependent transcriptional activity in cultured Drosophila cells. In a parallel line of experiments, Sd was identified in an unbiased screen for proteins that bind to a critical N-terminal Yki domain defined by a missense allele, ykiP88L. The fact that this missense mutation disrupted binding of Yki to Sd supports the physiological relevance of a Sd-Yki transcription complex in vivo. The identification of Sd as a cognate Yki partner using two unbiased approaches, combined with the genetic interactions between sd and yki, provide strong evidence that Sd is a critical DNA-binding factor that mediates the transcriptional output of the Hpo signaling pathway. It is worth noting that the requirement for sd in yki-driven overgrowth is highly specific, since the same sd mutation had no effect on overgrowth driven by the activated Ras oncogene. The observation that Yki, but not Sd, can be overexpressed or mutated to elicit tissue overgrowth further suggests that Sd is normally present in excess, and the activity of the Sd-Yki complex is regulated through modulation of Yki activity effected by the Hpo kinase cascade (Wu, 2008).

The founding member of the TEAD family transcription factors, TEAD-1/TEF-1, was initially identified based on its binding to the GTIIC motif of the simian virus 40 (SV40) enhancer. The TEAD family transcription factors have been mostly studied in the context of muscle-specific gene transcription, and their roles in cell proliferation and cell survival are poorly understood. The observation that TEAD-2 and YAP have similar activity to Sd and Yki, respectively, suggests that the growth-regulatory activity of the Sd-Yki complex is likely conserved in the mammalian Hpo pathway. It is also worth noting that besides growth regulation, the Hpo pathway has also been implicated in controlling other biological processes such as rhodopsin gene expression in mature photoreceptors and dendrite morphogenesis in postmitotic neurons. It remains to be determined whether Yki partners with Sd or other (unknown) DNA-binding factors in such nongrowth contexts (Wu, 2008).

Despite their elevated transcription upon inactivation of Hpo pathway tumor suppressors or activation of the Yki oncoprotein, it was previously unknown whether Yki regulates the known Hpo pathway target genes directly or indirectly through intermediary transcriptional regulators. This study has taken an unbiased approach to this question by isolating a HRE for diap1. This DNA element provided several important insights into how Hpo signaling activity is converted into transcriptional output. First, the Sd protein directly binds to the HRE and activates an HRE-luciferase reporter in cell culture in conjunction with Yki, supporting the notion that Yki directly regulates the transcription of the diap1 gene. Second, the minimal diap1 HRE contains non-Sd-binding sequence that is indispensable for HRE activity, suggesting that the HRE likely binds to additional transcription factors besides Sd in vivo. This latter characteristic is not unique to the HRE, but is a general feature that has been observed for many signaling pathways in Drosophila. For example, Notch-regulated enhancers contain not only binding sites for the signal-regulated transcription factor Suppressor of Hairless, but also binding sites for additional cofactors whose activity is Notch independent. It will be important to identify the factors that bind to the non-Sd sequence in the diap1 HRE, and to investigate whether such factors play a general role in mediating the Hpo responsiveness of other target genes (Wu, 2008).

The identification of a minimal HRE makes possible several new avenues of investigation to better understand the Hpo signaling pathway. The minimal HRE revealed in this study should facilitate a comprehensive cataloguing of Hpo pathway target genes, many of which remain to be identified. It also provides a useful tool for constructing reporters that can be used to monitor the specific activity of the Hpo pathway in vivo. Furthermore, this work will facilitate cell-based RNAi screens for components or modulators of the Hpo pathway, as illustrated by the successful use of pathway-specific luciferase reporters for interrogating other signaling pathway (Wu, 2008).

An interesting and somewhat unexpected finding from this study concerns the differential requirement of yki for the basal expression of diap1 and expanded (ex), with the former being yki dependent and the latter being yki independent, respectively. Thus, different Hpo target genes, and by inference different enhancer elements or their combinations, can respond to different threshold levels of Yki activity. It is suggest the basal expression of ex is mediated by non-HRE sequence in the ex locus and is therefore independent of yki. Excessive yki activity, either directly via an HRE in the ex locus or indirectly by turning on another factor, promotes ex transcription above the basal level. In contrast, Yki (through Sd, non-Sd DNA-binding factors, or both), regulates the basal level transcription of diap1. It is noted that the basal transcription of diap1 is not necessarily regulated through the HRE, which was identified by virtue of reporter expression under hyperactive Yki activities. Indeed, it was found that although the HRE is responsive to yki overexpression, it is largely unresponsive to loss of yki. Thus, the diap1 HRE revealed in this study is uniquely sensitive to unrestrained Yki activity (Wu, 2008).

The exquisite sensitivity of yki-induced overgrowth to sd dosage suggests that Sd/TEAD could be specifically targeted to ablate certain unwanted tissue growth, such as that caused by aberrant Hpo signaling, with minimal effect on normal growth. Thus, Sd/TEAD belongs to a growing list of genes that cause 'non-oncogene addition' -- genes that cannot be mutated or overexpressed to an extent that directly promotes tumorigenesis, but are still rate limiting to their specific signaling pathways. The requirement of such non-oncogenes in tumor cells makes them excellent targets for the development of new cancer therapeutics (Wu, 2008).

Transcriptional Regulation

The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP: Yorkie targets cycE and diap1

Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a the Hippo/Warts pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).

Activation of yki leads to increased transcription of diap1 and CycE. The increased cell proliferation and decreased apoptosis resulting from yki overexpression are strikingly similar to those caused by loss of hpo, sav, or wts, suggesting that Yki functions in the Hpo pathway. To further explore this possibility, the transcription of cell-death inhibitor diap1 and cell-cycle regulator cycE, known targets of the Hpo pathway were examined. Elevated DIAP1 protein is detected in yki-overexpressing clones in the eye discs. This regulation is largely mediated at the level of diap1 transcription since the expression of thj5c8, a P[lacZ] enhancer trap reporter inserted into the diap1 locus, is similarly elevated in yki-overexpressing clones in a cell-autonomous manner. A cycE-lacZ reporter containing 16.4 kb of the 5′ regulatory sequence of cycE is also increased in yki-overexpressing clones, especially those close to the MF, although the effect is less profound than that observed with the diap1 reporter. Thus, like loss of hpo, sav, or wts, overexpression of yki results in increased transcription of diap1 and cycE. It is worth noting that previous analyses of hpo mutant clones also revealed a 'tighter' regulation of diap1: while diap1 transcription is elevated in all hpo mutant cells irrespective of their relative position to the MF, cycE transcription is only elevated in hpo mutant cells close to the MF (Wu, 2003). These observations suggest that diap1 might represent a more direct transcriptional target of the Hpo pathway (Huang, 2005).

Mats regulates thread transcription

Appropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Growth inhibitory functions are described for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. Mats and Wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. Since Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans (Lai, 2005).

Apoptosis provides an important mechanism for the control of cell number and organ size. To test if mats plays a role in cell death control, expression of DIAP1 in eye discs was examined. DIAP1 is a caspase inhibitor essential for cell survival. Through immunostaining of mats mosaic eye discs, it was found that the level of DIAP1 protein is increased in mats clones. To examine if mats regulates diap1 at the transcriptional level, an enhancer trap line thj5C8 was used, in which a lacZ reporter gene is inserted in diap1 and expression pattern of diap1-lacZ reflect that of the endogenous diap1 gene. It was found that expression of diap1-lacZ was elevated. Thus, mats is required to negatively regulate DIAP1 expression. To directly test the idea that mats promotes apoptosis, mats mutant clones were induced in larval eye discs that overexpress an apoptosis-promoting gene head involution defective (hid) in all cells behind the MF. As expected, expression of hid in a wild-type background increased apoptosis to cause a reduced eye phenotype. Notably, removal of mats function blocks hid-induced cell death and significantly suppresses the small eye phenotype. In these same tissues, developmental cell death is observed in regions anterior to the MF, where expression of the hid transgene is not induced. In these cases, apoptosis occurs only in wild-type tissues but not in mats mutant clones. Thus, mats is also required for developmentally programmed apoptosis. All together, these findings support a model that mats is required to facilitate cell death, and loss of mats’ apoptosis-promoting activity may contribute to tumor development (Lai, 2005).

A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration

Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, a screen was performed for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppresses the migration defect. Moreover, loss-of-function mutations in th causes migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescues RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility (Geisbrecht, 2004).

The work reported here demonstrates a new function for DIAP1 in promoting cell migration. The strongest evidence for this is that border cells lacking DIAP1 fail to migrate. This finding is surprising since there has been no previous indication that IAP proteins contribute to cell motility. However, in most cells, it would be difficult or impossible to uncover a requirement for DIAP1 in cell migration because loss of the protein typically results in cell death (Geisbrecht, 2004).

The effect of DIAP1 on cell migration appears to be through the small GTPase Rac and its effects on the actin cytoskeleton based on genetic, biochemical, and cell culture experiments. First, overexpression of DIAP1 suppresses RacN17 migration defects specifically and does not rescue border cell migration defects that are due to other causes. Moreover, overexpression of either actin5C or profilin, both of which would be expected to increase the amount of polymerization competent G-actin in the cell, also rescue RacN17 border cell migration defects. The association of DIAP1 protein with Rac and profilin in S2 cells together with the finding that overexpression of DIAP1 can enhance activated Rac's effects on the actin cytoskeleton in cultured cells further support the conclusion that DIAP1 affects cell migration via Rac and the actin cytoskeleton. Additional support is provided by the finding that overexpression of Rac in border cells results in increased accumulation of DIAP1 protein and F-actin in vivo (Geisbrecht, 2004).

The effect of DIAP1 on border cell migration is clearly independent of its role in preventing apoptosis. The lack of apoptosis in th mutant follicle cell clones is striking since at other stages of Drosophila development, cells fail to survive in the absence of DIAP1. However, IAP proteins are thought to play a less critical role in survival of certain mammalian cells as well, where the current view is that the balance between proapoptotic and antiapoptotic BCL-2 family proteins is the deciding factor between life and death. The results presented here suggest that DIAP1 is not required for survival of every cell and tissue of the fly either. It may be that in both flies and mammals, different cell types have distinct requirements for particular classes of survival molecules (Geisbrecht, 2004).

Although the ability of DIAP1 to rescue RacN17 border cell migration defects is independent of its function in preventing cell death, and independent of its inhibition of effector caspases, the effects result from inhibition of the initiator caspase Dronc. The finding that inhibition of Dronc can rescue RacN17 border cell migration defects indicates that Dronc activity has a negative effect on migration. Since Dronc is a protease, the most parsimonious hypothesis would be that Dronc cleaves one or more proteins required for Rac-mediated cell motility. Previous studies have shown that Rac can be cleaved and inactivated by caspase 3 in lymphocytes. Therefore one possibility is that Rac itself is a Dronc substrate in border cells. A number of cytoskeleton-associated proteins are cleaved by caspases, including actin. Since reduced profilin levels was observed in th mutant follicle cells, it is also possible that profilin is a Dronc substrate, though no increased accumulation of actin or profilin was detected in cells overexpressing DIAP1. Further study will be required to pinpoint the physiologically relevant Dronc substrate in border cells (Geisbrecht, 2004).

The observation that two different dark mutant alleles cause mild border cell migration defects suggests that Dronc, which is thought to be constitutively active at a low level in most cells, contributes to normal migration. In fact, caspases have been shown to function in cell proliferation and differentiation in a variety of cell types, in addition to their better known role in promoting apoptosis. In some cases, caspase activity is required for terminal differentiation events that resemble incomplete apoptosis. For example, terminal differentiation of Drosophila sperm requires removal of much of the cytoplasm and requires caspase activity. Similarly, differentiation of mammalian lens cells and erythrocytes requires caspase activity. Other differentiation events, such as those of macrophages and skeletal muscle, do not overtly resemble apoptosis and yet require caspase activity. There must be some mechanism in such cells, and in border cells, to restrict the caspase activity to selected substrates so that apoptosis does not occur (Geisbrecht, 2004).

DIAP1 is a member of an evolutionarily conserved family of proteins that contain BIR domains. BIR domain-containing proteins are found in organisms from yeast to man and seem to have arisen in evolution prior to the apoptotic machinery. For example, in yeast, BIR1p is a protein required for proper chromosome segregation and cytokinesis. Yet the yeast genome does not encode an obvious caspase and yeast are not known to undergo apoptosis. In C. elegans, there is a BIR domain protein that does not suppress apoptosis when overexpressed. Reduction in the expression of this protein by RNA interference leads to defective cytokinesis and a phenotype that is very similar to loss of the worm formin protein. This is interesting since formin homology proteins can bind Rac, stimulate actin polymerization in concert with profilin, and promote cell migration. However it is not known if the C. elegans BIR domain protein interacts with formin or profilin, or whether it functions downstream of Rac. Taken together, these observations suggest that a primitive function of BIR domain proteins may have been regulation of cell division and the cytoskeleton (Geisbrecht, 2004).

It is well known that growth factors promote both survival and proliferation, as well as migration of specific cells. For example, Steel factor acting through the c-kit receptor tyrosine kinase regulates survival and proliferation of primordial germ cells and melanocytes in the mouse embryo. In addition, Steel factor and c-kit may contribute to guiding the embryonic migrations of these two cell populations. Conversely, overexpression of a factor that functions in repulsive guidance of Drosophila primordial germ cells causes excessive germ cell death. This intimate relationship between guidance and survival may exist to ensure that only those cells that migrate to the appropriate location survive and proliferate (Geisbrecht, 2004).

Rho family GTPases have also been demonstrated to affect both migration and survival. By activating gene expression through the JNK pathway, Rac1 protects COS 7 cells from apoptosis induced by ultraviolet light. Rac is also required for survival of cerebellar granule neurons. Another pathway required for both cell survival and Rac-mediated cell migration is the phosphatidylinositol 3-kinase pathway (PI3K). Activation of PI3K, and its downstream effector Akt, is capable of promoting neuronal survival in the absence of growth factors. Akt is also essential for Rac-mediated motility in mammalian fibroblasts. Akt is activated by Rac, and phosphorylated Akt colocalizes with Rac at the leading edge of fibroblasts. Therefore, there are several biochemical pathways that control, and possibly coordinate, cell survival and cell motility.

The mammalian formin homology protein FRL functions as a survival signal, in addition to its role in Rac-mediated regulation of the cytoskeleton. Overexpression of a truncated form of FRL, containing only the N-terminal Rac binding site, results in inhibition of cell growth and apoptosis in the macrophage cell line P388D1. These lines of evidence support the view that regulation of the cytoskeleton and cell survival are intertwined. The present study demonstrates that the inhibitor of apoptosis proteins, well known for their role in cell survival, can also promote cell migration, thus demonstrating a new and unexpected molecular link between survival and migration (Geisbrecht, 2004).

Dmp53 activates the Hippo pathway to promote cell death in response to DNA damage

Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size. Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki). This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although some of the events downstream of the Hpo kinase are understood, its mode of activation remains mysterious. This study shows that Hpo can be activated by Ionizing Radiations (IR) in a p53-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or p53 ectopic expression (Colombani, 2006).

Hpo is the ortholog of the Mammalian Sterile Twenty-like (MST) kinases, which belong to the Ste20 family of kinases. MSTs are highly similar to Hippo (Hpo) in their N-terminal serine/threonine kinase domains as well as in the C-terminal Salvador (Sav) binding region (or SARAH domain). MST1 functions both downstream and upstream of caspases to promote chromatin condensation and nuclear fragmentation, as well as activation of the JNK (Jun N-terminal kinase) and p38 pathways. Like most Ste20 family kinases, MST1/2 auto- or trans-phosphorylates at a number of residues. One of these, T183 in the activation loop, has been shown to be required for full kinase activity and has been used as a useful marker of MST1 activation in cultured cells. In order to study events upstream of Hpo, antibodies that have previously been shown to recognize MST1/2 phosphorylated on T183 were tested for their ability to cross-react with Hpo on the equivalent residue (T195). Interestingly, it was found antibodies that specifically recognized the phosphorylated form of Hpo upon treatment with staurosporine (sts), a known activator of MST1/2. This signal is abolished by RNAi-mediated Hpo depletion and disappears upon phosphatase treatment. Moreover, the antibodies recognize overexpressed tagged Hpo before immunoprecipitation. By contrast, the antibodies did not recognize a nonphosphorylable (T195A) Hpo mutant protein. Myc-tagged wild-type and T195A Hpo were immunoprecipitated and their auto-kinase activity and their activity on an exogenous substrate (Histone H2B, not shown) were measured in both the presence and absence of sts. As has been observed for MST1/2, overexpression of Hpo leads to its activation, presumably via trans-phosphorylation. Sts treatment potently stimulates Hpo kinase activity (5-fold). By contrast, the T195A mutant is severely compromised both in its unstimulated and stimulated activities, suggesting that T195 phosphorylation is crucial to normal Hpo kinase activity. Thus, these phospho-specific antibodies can be used as readouts of Hpo pathway activity (Colombani, 2006).

In the course of testing stimuli that would activate Hpo in tissue culture, it was observed that γ-irradiation potently and rapidly induced Hpo activation. The fly p53 ortholog has been shown to mediate cell death upon ionizing radiation (IR)-induced DNA damage. Although the pro-apoptotic genes reaper (rpr), hid, and sickle are p53 transcriptional targets, removal of these three proteins via chromosomal deficiencies only partially suppresses the cell-death effects of IR in embryos, suggesting that additional death signals act downstream of p53. This prompted an examination of whether the Hpo pathway could function downstream of Drosophila p53 in the response to IR (Colombani, 2006).

Initially, wing imaginal discs (the larval precursors of the adult wing) containing clones of hpo, wts, and sav mutant cells were treated with γ-rays and cell death was examined by staining for activated caspases. Interestingly, although caspase activation was efficiently induced in wild-type tissue or control discs, cell death was severely reduced in hpo, wts, and sav mutant clones and in p53 mutant discs. Quantification of the caspase staining indicated that apoptosis was reduced by 2- to 3-fold in hpo, wts, and sav clones compared to wild-type tissue. This was also true in eye imaginal discs (Colombani, 2006).

Overexpression of p53 in the posterior portion of late larval eye imaginal dics was sufficient to induce apoptosis. Loss of function of hpo, wts, and sav decreased cell death in this context, although the effect was less pronounced in sav clones, perhaps as a reflection of the weaker phenotype of the sav mutants. This suggests that the Hpo complex may function as an effector in the p53-mediated response to IR. To test this hypothesis, Hpo activation was measured in cultured cells treated with γ-rays in the presence or absence of dsRNAs directed against p53. Excitingly, depletion of Dmp53 markedly reduced Hpo phosphorylation by IR. The residual level of Hpo activation observed in p53-depleted cells can probably be explained by the fact that the dsRNA-mediated p53 depletion was never complete, as measured by RT-PCR. To check that the increased Hpo phosphorylation observed corresponded to increased activity, IP kinase assays were performed on cells expressing ectopic Hpo. It was observed that IR treatment potently induced Hpo kinase activity. Furthermore, p53 expression alone, in the absence of IR, was sufficient to activate Hpo phosphorylation. Finally, it was determined whether p53-dependent Hpo activation could be observed in vivo by taking advantage of the fact that p53 is not required for viability. Dissected ovaries from p53 mutant and wild-type flies were treated with γ-rays and examin Hpo activity was examined by Western blotting. Interestingly, although γ-rays potently activated Hpo in wild-type flies, this response was abolished in p53 mutant animals. p53 expression in the ovaries was able to induce apoptosis, ovary degeneration, and total loss of fecundity. It is concluded that Hpo is activated as part of a p53-dependent DNA-damage response both in cultured cells and in vivo (Colombani, 2006).

MST1 and 2 are known to be activated by caspase 3 through proteolytic cleavage. Therefore, the possibility exists that the Hpo activation observed is merely a by-product of Rpr-dependent caspase activation. Several lines of evidence suggest that this is not the case. First, reaper overexpression in S2 cells did not increase Hpo activity. Second, depletion of DIAP1 from cultured cells, which potently induces caspase activation, fails to trigger detectable Hpo activation. Third, the phospho-Hpo signal detected corresponds to full-length Hpo rather than a caspase-cleaved fragment. In fact, the caspase cleavage site present in the MSTs is not thought to be conserved in Hpo, and no evidence was seen of Hpo cleavage upon apoptotic stimuli. Fourth, treatment of cultured cells with caspase inhibitors did not affect Hpo activation by IR. Thus, it is unlikely that Hpo is stimulated via p53-dependent caspase activation (Colombani, 2006).

The time course of Hpo activation by IR (2–3 hr for maximal activation) suggests that transcription may be required for this response. Indeed, treatment of cells with IR in the presence of the transcription inhibitor Actinomycin D (ActD) abolishes Hpo activation. Thus, Hpo activation in response to IR requires new gene transcription, which could be mediated, at least in part, by p53. Hpo activity is induced by p53 expression, but Hpo protein itself does not appear to be a target of p53 because Hpo levels are not detectably upregulated when p53 is expressed in the posterior portion of the eye imaginal disc or in Dmp53-expressing clones in the wing disc. Future studies will be aimed at determining the exact mechanism through which Dmp53 promotes Hpo activation (Colombani, 2006).

This study has demonstrate by genetic and biochemical approaches not only that the Hpo pathway is required for the full apoptotic response induced by γ-ray irradiation but also that DNA damage triggers Hpo kinase activity in a p53-dependent manner both in vivo and in vitro. The apoptosis induced by p53 overexpression is strongly affected in hpo, wts, and sav mutant clones and p53 does not modulate Hpo levels. This study constitutes the first description of an upstream activating signal of the Hpo complex in vivo and during organism development (Colombani, 2006).

It is noted that the blockage of p53-induced apoptosis is not complete in hpo clones; this incomplete blockage likely reflects the role of other pro-apoptotic proteins, such as Reaper, Hid, and Sickle, in this process. Thus, it is proposed that, after exposure to ionizing radiations, the ATM, Chk2, p53 signaling pathway is activated and induces apoptosis by targeting expression of pro-apoptotic effectors such as Reaper, as well as by activating the Hpo pathway. This cell-death response to irradiation requires the caspase DRONC and leads to upregulation of JNK activity in a p53-dependent manner. Because Hpo has been shown to induce JNK activation when overexpressed in vivo, it will be interesting to determine whether Hpo is necessary for IR-induced JNK activation (Colombani, 2006).

Several reports have suggested that the mammalian homologs of members of the Hpo pathway might behave as tumor suppressors in humans. In addition, mice lacking the Wts homolog mLats1 are more sensitive to tumor-inducing agents. The current data suggest that one effect of mutations in Hpo-pathway members may be to protect these cells from DNA-damage-induced apoptosis and thus promote tumor progression and the accumulation of additional mutations. Further work on the Hpo pathway should further understanding of the DNA-damage response and its role in the transformation process (Colombani, 2006).

The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control

The Hippo (Hpo) signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes; however, the transcription factor of the pathway has remained elusive. This study provides evidence that the TEAD/TEF family transcription factor Scalloped (Sd) acts together with the coactivator Yorkie (Yki) to regulate Hpo pathway-responsive genes. Sd and Yki form a transcriptional complex whose activity is inhibited by Hpo signaling. Sd overexpression enhances, whereas its inactivation suppresses, tissue overgrowth caused by Yki overexpression or tumor suppressor mutations in the Hpo pathway. Inactivation of Sd diminishes Hpo target gene expression and reduces organ size, whereas a constitutively active Sd promotes tissue overgrowth. Sd promotes Yki nuclear localization, whereas Hpo signaling retains Yki in the cytoplasm by phosphorylating Yki at S168. Finally, Sd recruits Yki to the enhancer of the pathway-responsive gene diap1, suggesting that diap1 is a direct transcriptional target of the Hpo pathway (Zhang, 2008).

The Hpo pathway has emerged as a conserved signaling pathway that plays a critical role in controlling tissue growth and organ size. Despite the growing recognition of the importance of this pathway in development and cancer, the transcription factor that links the cytoplasmic components to the nuclear events has remained elusive and thus represents a major gap in the pathway. This study demonstrates Sd is the missing transcription factor of the Hpo pathway based on several lines of genetic and biochemical evidence. (1) Sd and Yki form a transcriptional complex to activate a reporter gene in S2 cells and this transcriptional activity is inhibited by Hpo signaling. Furthermore, Sd and Yki synergize in vivo to promote Hpo target gene expression and tissue overgrowth. (2) More importantly, loss of Sd function suppresses tissue overgrowth induced by Yki overexpression or loss-of-function mutations in hpo, sav, and wts. In addition, Sd inactivation either by RNAi or a genetic mutation blocks the ectopic expression of Hpo responsive genes induced by excessive Yki activity. (3) RNAi knockdown of Sd phenocopies knockdown of Yki, which is manifested by reduced organ size and diminished expression of Hpo pathway-responsive genes. (4) A constitutively active form of Sd activates multiple Hpo pathway-responsive genes and promotes tissue overgrowth. (5) Sd promotes Yki nuclear translocation and recruited Yki to the diap1 enhancer (Zhang, 2008).

Several sd null alleles were generated to further explore the consequence of loss of Sd. sd null clones located in the wing pouch region were found to exhibit growth deficit such that early-induced clones (48-72 hrs AEL) were eliminated by the end of late third instar. However, late-induced clones (72-96 hrs AEL) survived and exhibited diminished expression of diap1. In contrast, early-induced clones were recovered in the notal region of wing discs and in eye discs without showing discernible change in Diap1 levels. However, a previous study showed that yki mutant clones exhibited reduced diap1 expression in eye discs. It is possible that low levels of residual Sd activity persist in sd mutant clones, which are sufficient to support the basal expression of the Hpo target genes. Alternatively, Yki may act through another transcription factor to maintain the basal expression of Hpo target genes. Nevertheless, sd null mutation suppresses the overgrowth phenotype and ectopic cycE expression induced by excessive Yki activity, suggesting the residual Sd in sd mutant clones is insufficient to support the elevated Yki activity (Zhang, 2008).

The identification of Hpo pathway transcription factor provided an opportunity to assess direct transcriptional targets of the pathway. To this end, the diap1 enhancer was characterized, and a 1.8 kb enhancer element critical for diap1 expression was identified. This region contains a total of seventeen predicted Sd binding sites. Using the ChIP assay, it was demonstrated that both Sd and Yki physically interact with the 1.8 kb diap1 enhancer and the association of Yki with the diap1 enhancer is mediated by Sd. These results suggest that Sd recruits Yki to the diap1 enhancer to activate its transcription (Zhang, 2008).

It has been shown that Sd acts in conjunction with Vg to promote wing development by directly regulating the expression of wing patterning genes. This study has demonstrated that Sd acts in conjunction with Yki to control organ size by regulating the expression of genes involved in cell proliferation, cell growth, and apoptosis. These observations raise an important question of how Yki-Sd and Vg-Sd transcriptional complexes specifically select their targets. One possibility is that Vg-Sd and Yki-Sd prefer to interact with distinct Sd binding sites. Indeed, a previous study showed that binding of Vg to Sd modulates the DNA binding selectivity of Sd. Another possibility is that target selectivity could be influenced by cofactors that bind in the vicinity of Sd binding sites. In support of this notion, previous studies have shown that wing specific enhancers contain both Sd binding sites and binding sites for transcription factors that mediate specific signaling pathways. It is also possible that Vg-Sd and Yki-Sd may share common targets. For example, diap1 could be activated by Vg-Sd in the wing pouch, which might explain why sd mutant clones in this region exhibits diminished diap1 expression (Zhang, 2008).

In principle, the Hpo pathway could regulate the activity of Yki-Sd transcriptional complex at several levels. For example, Hpo signaling could regulate the formation Yki-Sd complex or the recruitment of other factor(s) to the Yki-Sd transcriptional complex. Alternatively, Hpo signaling could regulate the nuclear-cytoplasmic transport of Yki. In support of the latter possibility, Yki exhibits elevated nuclear localization in wts or hpo mutant clones. In addition, coexpression of Hpo with Yki depletes nuclear Yki in S2 cells, suggesting that Hpo signaling impedes nuclear localization of Yki and thereby limits the amount of active Yki-Sd transcriptional complex (Zhang, 2008).

Mutating Yki S168 to Ala increases nuclear localization and growth promoting activity of Yki. In addition, it has been demonstrated that phosphorylation of Yki S168 was stimulated by Hpo. Phosphorylation of Yki by Hpo signaling increases their association with 14-3-3, which is abolished by mutating Yki S168 to Ala. Since 14-3-3 often regulates nuclear-cytoplasmic shuttling of its interacting proteins, these observations suggest that Hpo signaling inhibits Yki at least in part by phosphorylating Yki S168, which promotes 14-3-3 binding and cytoplasmic sequestration of Yki (Zhang, 2008).

The Hpo pathway appears to restrict cell growth and control organ size in mammals. The finding that Sd is critical for Yki-induced tissue growth has raised the interesting possibility that the effect of YAP in promoting tissue growth may rely on the TEAD/TEF family of transcription factors. Corroborating this hypothesis, TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with YAP (Vassilev, 2001). Furthermore, YAP can bind to and stimulate the trans-activating activity of all four TEAD/TEF family members (Vassilev, 2001). The TEAD/TEF family members exhibit overlapping but distinct spatiotemporal expression patterns and thus may have redundant but unique roles during development . It will be important to determine which TEAD/TEF family members are involved in the mammalian Hpo pathway and whether YAP employs distinct sets of TEAD/TEF transcription factors in different tissues. Since abnormal activation of YAP is associated with multiple types of cancer, disrupting YAP-TEAD/TEF interaction may provide a new strategy for cancer therapeutics (Zhang, 2008).

Enhanced sensitivity of midline glial cells to apoptosis is achieved by HOW(L)-dependent repression of Diap1

The selective sensitivity of cells to programmed cell death (PCD) depends on the positive and negative death-inducing signals that converge into the apoptotic pathway. In Drosophila, the midline glial (MG) cells undergo selective death during development. This study shows that the long isoform of the RNA-binding protein Held Out Wing (HOW(L)) is essential for enhancing the sensitivity of the MG cells to PCD. In how mutant embryos, the number of MG cells was elevated. This phenotype could be rescued by midline expression of the HOW(L) repressor isoform. In how mutant embryos, the levels of the caspase inhibitor of apoptosis, Diap1 were elevated, in parallel to reduction in the levels of activated caspase. Similarly, reducing the levels of HOW in S2 cells led to elevation of Diap1, whereas over expression of HOW(L) promoted reduction of Diap1 protein as well as mRNA levels. Importantly, deletion of the two HOW binding sites from diap1 3'UTR abrogated HOW-dependent repression of Diap1, suggesting that HOW represses diap1 by binding to its 3'UTR. These results suggest that HOW(L) enhances the sensitivity of MG cells to apoptotic signals by reducing the levels of diap1 in these cells in, demonstrating a novel mode of regulation of PCD at the mRNA level (Reuveny, 2009).

The sensitivity of cells to apoptotic signals depends on the balance between the pro-apoptic and anti-apoptotic signals expressed within a cell in a given developmental context. MG cells represent a unique system in which to study apoptosis because only a small subset of the cells (2-3 out of 6) are doomed to die, and the death must be executed in a relative short period of time (around 3 h), during the migration of the AMG pair towards the next segment. HOW functions to enhance the sensitivity of these cells to the pro apoptotic signals by reducing the levels of the anti-apoptotic protein, Diap1 (Reuveny, 2009).

Regulation through pro apoptotic signals, e.g. the activities of Reaper, Grim and Hid (RGH) proteins, or the anti-apoptotic signal, by influencing the activity of Diap1, enables cells to respond to a wide array of signaling pathways. The convergence of these signals in a single cell determines not only whether the cell will undergo PCD, but also the timing during development at which this process will occur. In case of MG cells, the timing is critical, as the cells die prior to their arrival to the commissure, and thus do not receive the survival signal through MAPK activation (Reuveny, 2009).

RGH proteins were shown to affect the levels of Diap1 at several regulatory stages. Reaper and Hid affect Diap1 levels via ubiquitination and proteosomal degradation. In addition, Morgue has been shown to promote Diap1 degradation whereas Reaper was shown to also inhibit translation of diap1. Despite the expression of Hid and Reaper in MG cells, only partial PCD is induced in these cells, possibly due to high levels of Diap1 (Reuveny, 2009).

Previous data has suggested that HOW(L) mediates developmental processes in other tissues (e.g., mesoderm, tendon cells), by a temporal reduction of the levels of key regulatory proteins. For example, in gastrulating embryos, HOW(L) reduces the mRNA levels of string/cdc25 to arrest cell division during mesoderm invagination, and at a later stage, HOW(L) reduces the levels of miple1 to allow mesoderm spreading. Similarly, this study shows that HOW contributes to the timing of MG cell apoptosis by reducing the levels of Diap1, thereby sensitizing these cells to pro-apoptotic signals. An effect of HOW on cell division through regulation of String is not favored by this study, since HOW is detected in the midline cells only at stage 12-13 at which the MG cells do not divide anymore (Reuveny, 2009).

Several lines of evidence support the idea that HOW(L) might affect MG cell apoptosis through its repression of Diap1 levels. First, it was shown by antibody staining that Diap1 levels are elevated in how mutants. Second, reducing HOW levels by introducing HOW-specific dsRNA or elevating HOW(L) levels in S2 cells leads to corresponding opposing effects on Diap1 protein expression, elevation of Diap1 when HOW is reduced and reduction of Diap1 when HOW(L) is elevated. The diap1 3' UTR contains two binding sites for HOW, and is capable of binding to HOW(L) in vitro. Nevertheless, a corresponding elevation of diap1 mRNA could not be detected in the S2 cells in which HOW was knocked down by dsRNA, possibly due to a continuous positive transcriptional input of diap1 in these cells. However, a reduction of diap1 mRNA and protein levels was induced, following over expression of HOW(L) in S2 cells and in embryos. Also, whether the splicing pattern of diap1 was altered in S2 cells depleted of HOW was examined, since HOW was demonstrated to mediate alternative splicing in other tissues. To this end, an RT-PCR was performed with primers specific for each of the three diap1 splice variants; however, no change in the pattern of diap1 splicing was observed (Reuveny, 2009).

Thus, HOW(L) might affect Diap1 protein levels by repressing both its mRNA levels as well as its translation. Alternatively it could affect Diap1 indirectly by influencing the levels of an upstream regulator of Diap1. The results support a direct effect of HOW through its association with the HOW-binding sites in diap1 3' UTR, since deletion of these sites abrogated the reduction of Diap1 detected in the presence of HOW(L) (Reuveny, 2009).

Gld-1 the C. elegans orthologue of HOW affects both translation and stability of its target mRNAs, apparently by affecting the length of the polyA tail of the target mRNA. HOW(L) might act in a similar fashion on diap1 mRNA (Reuveny, 2009).

Whereas HOW(L) does not induce apoptosis in other tissues, where it is highly expressed (e.g. mesoderm, tendon cells etc.), it was shown to have pro apoptotic effects in MG cells and in the adult fly eye. It is suspected that in these tissues, a delicate balance between the levels of the pro apoptotic and anti apoptotic proteins is maintained, so that the cells become highly sensitive to Diap1 levels, and thus are responsive to reduced or elevated levels of HOW(L) (Reuveny, 2009).

Interestingly, one isoform of the mammalian orthologue of HOW, Quaking7 (QKI-7), has been shown to induce apoptosis of fibroblasts and primary rat oligodendrocytes. The molecular mechanism of QKI-7-induced apoptotic activity has yet to be elucidated, but the unique C' terminal tail of QKI-7 appears to be necessary for this apoptotic activity. In contrast, C. elegans GLD-1 was shown to repress the levels of cep1 an orthologue of mammalian P53. In that system, GLD-1 exhibits an anti apoptotic effect. It appears therefore, that STAR proteins are not dedicated to a defined direction of apoptotic regulation. Rather, their basic activity is to elevate or reduce the levels of critical components in the process to enable the execution of PCD or to allow other developmental process to occur (Reuveny, 2009).

Previous studies demonstrated that how transcription is induced in response to the activation of the Ecdyson (Ecd) pathway however, the biological significance of this induction was not clear. During larval stages, a high titer of Ecd acts through the ecdyson receptor EcR/Ultraspiracle nuclear receptor heterodimer to signal puparium formation and destruction of several larval tissues including the midgut and salivary glands. The Ecd pathway triggers a transcriptional cascade that culminates in rpr and hid induction to initiate tissue destruction. Interestingly, the Ecd pathway induces parallel repression of diap1 via the activity of the CREB binding protein, CBP. CBP is both necessary and sufficient to down-regulate Diap1, providing the cells with the competence to die. Whereas the contribution of CBP to MG cell apoptosis has yet to be elucidated, it is possible that in this system, in parallel to the induction of CBP transcription, the Ecd pathway triggers HOW(L) transcription to enhance Diap1 destruction, possibly due to a need to induce rapid death of the MG cells (Reuveny, 2009).

To address whether HOW(L) is sufficient to rescue the excess in MG cells in disembodied (dib) mutant embryos, defective in ecdysone biosynthetic, HOW(L) was overexpressed in dib mutant embryos that carried the MG-specific enhancer trap, AA142, using the sim-gal driver. Embryos over expressing HOW(L) in dib mutant embryos still maintained a high number of MG cells, suggesting that HOW(L) is not sufficient to rescue the dib mutant phenotype. Importantly, the levels of Diap1 in MG cells in these embryos were significantly reduced (Reuveny, 2009).

The inability of HOW(L) to reduce the number of MG cells following its over expression in the dib mutant embryos might be explained by the involvement of the Ecd pathway not only in PCD but also in repression of MG cell division in an earlier developmental stage. Also, since the Ecdysone pathway positively regulates Hid, it is possible that the MG cells did not contain enough pro-apoptotic signals to induce PCD, and therefore it is not surprising that HOW(L) did not provide rescue of the MG cell number (Reuveny, 2009).

In summary, this study have identified the KH-domain RNA-binding protein, HOW, as a novel regulator of PCD in MG cells, likely acting as a regulator of Diap1 translation and/or stability. It is proposed that HOW provides the MG cells with enhanced sensitivity to the pro apoptotic effects of Hid and Reaper, triggering the rapid apoptosis of MG cells during their migration (Reuveny, 2009).

The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex (see Rbf) has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue (Clavier, 2014).

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. This study shows that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. These data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth (Graves, 2012).

This study identifies a novel role of Notch signaling for non-cell autonomous control of apoptosis via induction of Yki activity in neighboring cells. It has previously been shown that Notch signaling controls cell proliferation both autonomously and non-cell autonomously in the developing eye. The non-cell autonomous component of proliferation control was attributed to Notch-dependent activation of Jak/Stat signaling although that recently came into question. Nevertheless, Jak/Stat activation is not sufficient to mediate the effect of Notch on non-cell autonomous control of apoptosis. This study identified the Hpo/Wts/Yki pathway as a target of Notch signaling for the non-cell autonomous control of apoptosis both in eye and wing imaginal discs. Because the Hpo/Wts/Yki pathway also controls proliferation, it is likely that Notch promotes non-cell autonomous proliferation through both Jak/Stat and Hpo/Wts/Yki activities (Graves, 2012).

It is also interesting to note that this non-autonomous control of the Hpo/Wts/Yki pathway by Notch occurs in a position-dependent manner. For example, vps25 mutant clones or NICD-expressing clones located in the hinge and notum of wing discs triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the wing pouch did not. Additionally, vps25 mutant clones located anterior to the morphogenetic furrow triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the posterior of the eye disc did not. The reason for this position-dependence is unknown. However, the regions which do not induce Hpo/Wts/Yki signaling non-autonomously correspond to the zone of non-proliferating (ZNP) cells in the wing disc and post-mitotic, differentiating cells in the eye disc. Therefore, one potential reason for the position-dependence may be that the post-mitotic nature of the cells in the ZNP of the wing pouch and in the posterior of the eye disc render them inert to growth-promoting signals that trigger the Hpo/Wts/Yki pathway. However, while this is one possibility, there may also be additional mechanisms that influence the response to growth-promoting signals (Graves, 2012).

How Notch exerts this non-autonomous effect is an important and interesting question. Based on its function as a transcriptional regulator, it is possible that increased Notch signaling in vps25 mutant cells could lead to transcription of a secreted or transmembrane protein that communicates to surrounding tissue and induces Yki activity. Expression of proteins known to non-cell autonomously activate Yki signaling such as Fat, Dachsous (Ds) and Four-Jointed as well as ds-lacZ, however, are not altered in vps25 mosaic discs. Identification of this non-cell autonomous signaling mechanism may also be critical for understanding tumorigenesis, as mutations in the Notch pathway, the Hippo pathway and in ESCRT components have been implicated in many different types of human cancer. In conclusion, this study provides a mechanism by which neoplastic cells influence the behavior of neighboring wild-type cells, which may be critical for generating a supportive microenvironment for tumor growth by preventing cell death and promoting the proliferation of wild-type cells (Graves, 2012).

JAK/STAT autocontrol of ligand-producing cell number through apoptosis

During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. This study shows that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. These results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila (Borensztejn, 2013).

A role for STAT in cell death and survival has been clearly documented in mammals, and depending on which of the seven mammalian Stat genes is considered and on the cellular context, both pro- and anti-apoptotic functions have been characterised. In the Drosophila developing wing, phosphorylated Stat92E has been shown to be necessary for protection against stress-induced apoptosis, but not for wing developmental apoptosis. This study provides evidence that Upd and the JAK/STAT pathway control developmental apoptosis during Drosophila oogenesis (Borensztejn, 2013).

This study demonstrated that the JAK/STAT pathway ligand, Upd, and all components of the JAK/STAT transduction cascade (the receptor Dome, JAK/Hop and Stat92E) are involved in promoting apoptosis of supernumerary PCs produced during early oogenesis. It is argued that The JAK/STAT pathway is essential for this event for several reasons. Indeed, in the strongest mutant context tested, follicle poles containing large TFC and PC clones homozygous for Stat92E amorphic alleles, almost all of these (95%) maintained more than two PCs through oogenesis. Also, RNAi-mediated reduction of upd, dome and hop blocked PC number reduction and deregulated several apoptosis markers, inhibiting Hid accumulation, Diap1 downregulation and caspase activation in supernumerary PCs. Altogether, these data, along with what has already been shown for JAK/STAT signalling in this system, fit the following model. Upd is secreted from PCs and diffuses in the local environment. Signal transduction via Dome/Hop/Stat92E occurs in nearby TFCs, interfollicular stalks and PCs themselves, leading to specific target gene transcription in these cells, as revealed by a number of pathway reporters. An as-yet-unidentified Stat92E-dependent pro-apoptotic relay signal (X) is produced in TFCs, interfollicular stalks and possibly PCs, which promotes supernumerary PC elimination via specific expression of hid in these cells, consequent downregulation of Diap1 and finally caspase activation. An additional cell-autonomous role for JAK/STAT signal transduction in supernumerary PC apoptosis of these cells is also consistent with, though not demonstrated by, the results (Borensztejn, 2013).

Relay signalling allows for spatial and temporal positioning of multiple signals in a tissue and thus exquisite control of differentiation and morphogenetic programmes. In the Drosophila developing eye, the role of Upd and the JAK/STAT pathway in instructing planar polarity has been shown to require an as-yet-uncharacterised secondary signal. In the ovary, the fact that JAK/STAT-mediated PC apoptosis depends on a relay signal may provide a mechanism by which PC apoptosis and earlier JAK/STAT-dependent stalk-cell specification can be separated temporally (Borensztejn, 2013).

Although neither the identity, nor the nature, of the relay signal are known, it is possible to propose that the signal is not likely to be contact-dependent, and could be diffusible at only a short range. Indeed, Stat92E homozygous mutant TFC clones in contact with PCs, as well as those positioned up to three cell diameters away from PCs, are both associated with prolonged survival of supernumerary PCs, whereas clones further than three cell diameters away from PCs are not. In addition, fully efficient apoptosis of supernumerary PCs may require participation of all surrounding TFCs, stalk cells and possibly PCs, for production of a threshold level of relay signal. In support of this, large stat mutant TFC clones are more frequently associated with prolonged survival of supernumerary PCs, and the effects of removing JAK/STAT signal transduction in several cell populations at the same time are additive. Interestingly, the characterisation of two other Drosophila models of developmental apoptosis, interommatidial cells of the eye and glial cells at the midline of the embryonic central nervous system, also indicates that the level and relative position of signals (EGFR and Notch pathways) is determinant in selection of specific cells to be eliminated by apoptosis (Borensztejn, 2013).

The results indicate that only the supernumerary PCs respond to the JAK/STAT-mediated pro-apoptotic relay signal, whereas two PCs per pole are always protected. Indeed, this study found that overexpression of Upd did not lead to apoptosis of the mature PC pairs and delayed rather than accelerated elimination of supernumerary PCs. Recently, it was reported that selection of the two surviving PCs requires high Notch activation in one of the two cells and an as-yet-unknown Notch-independent mechanism for the second cell. Intriguingly, expression of both Notch and Stat reporters is dynamic in PC clusters and PC survival and death fates are associated with respective activation of the Notch and JAK/STAT pathways. However, this study found that RNAi-mediated downregulation of upd did not affect either expression of Notch or that of two Notch activity reporters. Therefore, JAK/STAT does not promote supernumerary PC apoptosis by downregulating Notch activity in these cells. Identification of the relay signal and/or of Stat target genes should help further elucidate the mechanism underlying the induction of apoptosis in selected PCs (Borensztejn, 2013).

Interfollicular stalk formation during early oogenesis has been shown to depend on activation of the JAK/STAT pathway. The presence of more than two PCs during these stages may be important to produce the appropriate level of Upd ligand to induce specification of the correct number of stalk cells. Later, at stages 7-8 of oogenesis, correct specification of anterior follicle cell fates (border, stretch and centripetal cells) depends on a decreasing gradient of Upd signal emanating from two PCs positioned centrally in this field of cells. Attaining the correct number of PCs per follicle pole has been shown to be relevant to this process and border cells (BC) specification seems to be particularly sensitive to the number of PCs present. Previously work has shown apoptosis of supernumerary PCs is physiological necessary for PC organiser function, as blocking caspase activity in PCs such that more than two PCs are present from stage 7 leads to defects in PC/BC migration and stretch cell morphogenesis. This study now shows that the excess PCs produced by blocking apoptosis lead to increased levels of secreted Upd and induce specification of excess BCs compared with the control, and these exhibit inefficient migration. These results indicate that reduction of PC number to two is necessary to limit the amount of Upd signal such that the correct numbers of BCs are specified for efficient migration to occur. Taken together with the role shown for Upd and JAK/STAT signalling in promoting PC apoptosis, it is possible to propose a model whereby Upd itself controls the size of the Upd-producing organising centre composed of PCs by inducing apoptosis of supernumerary PCs. Interestingly, in the polarising region in the vertebrate limb bud, which secretes the morphogen Sonic Hedgehog (Shh), Shh-induced apoptosis counteracts Fgf4-stimulated proliferation to maintain the size of the polarising region and thus stabilise levels of Shh. It is likely that signal autocontrol via apoptosis of signal-producing cells will prove to be a more widespread mechanism as knowledge of apoptosis control during development advances (Borensztejn, 2013).

Huang, X., Shi, L., Cao, J., He, F., Li, R., Zhang, Y., Miao, S., Jin, L., Qu, J., Li, Z. and Lin, X. (2014). The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut. J Genet Genomics 41: 429-438. PubMed ID: 25160975

The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut

The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. This study shows that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell (ISC) proliferation and midgut homeostasis loss. Meanwhile, it was find that the STAT signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diap1 and bantam, is dramatically increased in Tao knockdown intestines. Consistently, it was shown that the Yorkie (Yki) acts downstream of Tao to regulate ISC proliferation. Together, these results provide insights into understanding of the mechanisms of stem cell proliferation and tissue homeostasis control (Huang, 2014).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

Protein Interactions

Many members of the inhibitor of apoptosis (IAP) family of proteins suppress programmed cell death, at least in part, by physically interacting with and inhibiting the catalytic activity of caspases. An important functional unit in all death-inhibiting IAP proteins is the so-called baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. The Drosophila genome contains four genes that encode proteins with BIR domains. The overexpression of two of these, DIAP1 and DIAP2, inhibit both normal developmental cell death and apoptosis induced by expression of proapoptotic genes. In addition, DIAP1 is required for cell survival in the embryo and in a number of adult tissues. These observations, in conjunction with others showing that DIAP1 binds and inactivates several Drosophila caspases and that loss of DIAP1 results in an increase in caspase activity in vivo, argue that DIAP1's function as a caspase inhibitor is required for cell survival. DIAP1 contains two N-terminal BIR repeats and a C-terminal RING domain. DIAP1 fragments containing the BIR2 domain are sufficient to prevent cell death in a number of contexts. Interestingly, fragments consisting of the BIR2 and surrounding linker sequences also bind multiple proapoptotic proteins, including the apical caspase DRONC, and Hid, Grim, and Reaper (Wu, 2001 and references therein).

One mechanism by which Hid, Grim, and Reaper promote cell death is by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor. Although Hid, Grim, and Reaper perform a similar function in promoting cell death, they only share homology in the N-terminal 14 residues of their primary sequences. These N-terminal sequences are sufficient to mediate interactions with DIAP1 and with several mammalian IAPs. In the case of Hid in insects, and Hid and Reaper in mammalian cells, these N-terminal sequences are essential for proapoptotic function (Wu, 2001 and references therein).

In mammalian cells, caspase inhibition by IAPs is negatively regulated by a mitochondrial protein Smac/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol upon apoptotic stimuli. Smac/DIABLO physically interacts with multiple IAPs and relieves their inhibitory effect on both initiator and effector caspases. Thus, Smac/DIABLO represents the mammalian functional homolog of the Drosophila Hid, Grim, and Reaper proteins. Recent structural studies reveal that the N-terminal tetrapeptide of Smac/DIABLO binds a surface groove on XIAP-BIR3, thus competitively removing the inhibition of caspase-9 by XIAP. Smac/DIABLO shares sequence homology with Hid, Grim, and Reaper only in the N-terminal 4 residues, prompting the hypothesis that Hid, Grim, and Reaper interact with DIAP1 using similar tetrapeptides and binding to a similar surface groove on DIAP1 (Wu, 2001 and references therein).

There is currently no structural information on DIAP1 or Hid, Grim, or Reaper. To investigate the structural mechanisms of DIAP1 recognition by the Drosophila Hid, Grim, and Reaper proteins, the DIAP1-BIR2 domain was crystalized by itself and in complex with the N-terminal peptides from both Hid and Grim (these structures were determined at 2.7, 2.7, and 1.9 Angstrom resolution, respectively). By analogy to the Smac-XIAP interactions, the first four amino acids of Hid and Grim bind an evolutionarily conserved surface groove on DIAP1-BIR2. The next 3 conserved residues of Hid and Grim also contribute to the interactions with DIAP1 through extensive van der Waals contacts. Interestingly, peptide binding to DIAP1-BIR2 appears to induce the formation of an additional alpha helix, which appears to stabilize peptide binding. In conjunction with biochemical analysis, this structural study reveals a molecular basis for the conservation and diversity necessary for the recognition of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins. These results have important ramifications for the design of IAP inhibitors toward therapeutic applications (Wu, 2001).

The recently published genome sequence of Drosophila predicts seven caspases in the fly. Five of these caspases have been previously characterised. STRICA, the object of this study, is a caspase with a long amino-terminal prodomain that lacks any caspase recruitment domain or death effector domain. Instead, the prodomain of STRICA consists of unique serine/threonine stretches. Low levels of strica expression are detected in embryos, larvae, pupae and adult animals. STRICA is a cytoplasmic protein that, upon overexpression, causes apoptosis in cultured Drosophila SL2 cells that is partially suppressed by DIAP1. Interestingly, unlike other fly caspases, STRICA shows physical association with DIAP2, in cotransfection experiments. These results suggest that STRICA may have a unique cellular function (Doumanis, 2001).

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation requires both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibits protein translation. Since the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis (Holley, 2002).

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: (1) increased ubiquitination promotes degradation directly; (2) a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 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 three currently known IAP antagonists in Drosophila map to the H99 genomic interval required for all programmed cell death. A fourth member of this genetic group, sickle (skl), is described that maps just outside of the H99 deletion. At its N terminus, Skl shares residues in common with other IAP antagonists in flies (Rpr, Grim, and Hid) and in mammals (Smac/DIABLO and Omi/Htra2). Like other activators of apoptosis mapping in the Reaper region, full-length skl induces apoptosis when overexpressed, and the N terminus of this protein specifically binds to the BIR2 domain of DIAP1. However, unlike the N termini of Grim, Hid, and Rpr, the N terminus of Skl does not induce apoptosis. skl transcripts accumulate in cells that are fated to die in some but not all regions of the embryo. Genotoxic stimuli induce skl expression, but skl is not responsive to all signals that trigger premature apoptosis. skl is potentially a fourth IAP antagonist in the 'Reaper region' and a new candidate transducer of apoptotic damage signaling in Drosophila (Christich, 2002).

Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain (E2). Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce (dBruce) can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The dBruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. dBruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1 BIR2, be intact. dBruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that dBruce can regulate cell death at a novel point (Vernooy, 2002).

Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. A physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. The N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila eye. These data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. It is suggested that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases (Ditzel, 2003).

morgue enhances the actions of the Grim-Reaper proteins and negatively regulates the levels of DIAP1 protein. This gene encodes a novel protein that contains both an F box and a ubiquitin conjugase domain. Interestingly, the Morgue conjugase domain lacks the active site cysteine required for covalent linkage to ubiquitin. Morgue could target IAPs and other proteins for ubiquitination and proteasome-dependent turnover by acting either in an SCF ubiquitin E3 ligase complex, or as a ubiquitin E2 conjugase enzyme variant (UEV) in conjunction with a catalytically active E2 conjugase. Morgue is evolutionarily conserved; a Morgue ortholog was identified from the mosquito, Anopheles gambiae. Elucidation of morgue function should provide novel insights into the mechanisms of ubiquitination and programmed cell death (Schreader, 2003).

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. The Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, regulation of Reaper by ubiquitination has been shown to be a significant factor in determining Reaper biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance (Olson, 2003).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions

Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. This study characterized the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. Reaper concentrates are found on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper is able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation (Freel, 2008).

Degradation of Thread

In melanogaster, apoptosis is controlled by the integrated actions of the Grim-Reaper (Grim-Rpr) and Drosophila Inhibitor of Apoptosis (DIAP) proteins. The anti-apoptotic DIAPs bind to caspases and inhibit their proteolytic activities. DIAPs also bind to Grim-Rpr proteins, an interaction that promotes caspase activity and the initiation of apoptosis. Using a genetic modifier screen, four enhancers of grim-reaper-induced apoptosis were identified that all regulate ubiquitination processes: uba-1, skpA, fat facets (faf), and morgue (modifier of rpr and grim, ubiquitously expressed). Strikingly, morgue encodes a unique protein that contains both an F box and a ubiquitin E2 conjugase domain that lacks the active site Cys required for ubiquitin linkage. A reduction of morgue activity suppresses grim-reaper-induced cell death in Drosophila. In cultured cells, Morgue induces apoptosis that is suppressed by DIAP1. Targeted morgue expression downregulates DIAP1 levels in Drosophila tissue, and Morgue and Rpr together downregulate DIAP1 levels in cultured cells. Consistent with potential substrate binding functions in an SCF ubiquitin E3 ligase complex, Morgue exhibits F box-dependent association with SkpA and F box-independent association with DIAP1. Morgue may thus have a key function in apoptosis by targeting DIAP1 for ubiquitination and turnover (Wing, 2002).

Inhibitor of apoptosis proteins (IAPs) provide a critical barrier to inappropriate apoptotic cell death through direct binding and inhibition of caspases. Degradation of IAPs is an important mechanism for the initiation of apoptosis in vivo. Drosophila Morgue, a ubiquitin conjugase-related protein, promotes DIAP1 down-regulation in the developing retina to permit selective programmed cell death. Morgue complexes with DIAP1 in vitro and mediates DIAP1 degradation in a manner dependent on the Morgue UBC domain. Reaper (Rpr) and Grim, but not Hid, also promote the degradation of DIAP1 in vivo, suggesting that these proteins promote cell death through different mechanisms (Hays, 2002).

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

A homozygous loss of function mutation in DIAP1 results in widespread apoptosis during Drosophila embryogenesis. To determine the effect of depleting IAP proteins from cultured insect cells, two iap genes from different insect species, Sf-iap from the lepidopteran insect S. frugiperda and diap1 from Drosophila melanogaster, were silenced using RNAi. Within 4 h after addition of Sf-iap dsRNA to Sf21 cells or diap1 dsRNA to S2 cells, membrane blebbing was observed in both cell lines consistent with apoptosis. This morphology was indistinguishable from that observed after treatment with actinomycin D, ultraviolet light, or cycloheximide, known inducers of apoptosis in Sf21 and S2 cells. The blebbing intensified over time, and by 24 h more than 99% of the cells had undergone apoptosis. Only very low background levels of apoptosis were observed in mock-treated cells or cells treated with control dsRNAs (Muro, 2002).

To determine whether caspases were involved in the death induced by iap silencing, the effect was examined of caspase inhibitors on death induced by IAP depletion. Sf21 cells were transfected with a plasmid vector expressing the baculovirus caspase inhibitor P35, and then the cells were treated with Sf-iap dsRNA. Transient expression of P35 inhibited the apoptosis induced by the addition of Sf-iap dsRNA. In addition, expression of the baculovirus iap gene Op-iap also inhibited this apoptotic signal. In S2 cells, apoptosis induced by loss of DIAP1 was inhibited by the chemical caspase inhibitor Z-VAD-FMK, indicating that loss of DIAP1 also resulted in caspase activation and caspase-dependent apoptosis (Muro, 2002).

The levels of DIAP1 protein decrease rapidly after addition of diap1 dsRNA to S2 cells, with the protein becoming undetectable by immunoblotting within 7.5 h. These results are consistent with a short half-life for DIAP1 protein, which has been shown to be ~30-45 min in S2 cells following cycloheximide treatment (Muro, 2002 and references therein).

The fact that depletion of DIAP1 stimulates caspase-dependent apoptosis suggests that DIAP1 normally promotes cell viability at least in part by inhibiting the activity of one or more caspases. However, the caspase(s) inhibited by DIAP1 in vivo have not been identified. An obvious candidate for a caspase targeted by DIAP1 is the caspase DRONC, which is widely expressed in the developing Drosophila embryo and is required for developmentally programmed embryonic cell death. In addition, DIAP1 binds to both the pro-domain and core subunits of DRONC (Meier, 2000), and death induced by ectopic DRONC expression in the fly and in yeast has been shown to be inhibited by DIAP1 (Meier, 2000). S2 cells were treated with dronc dsRNA for 24 h, which reduced the amount of full-length DRONC protein to a non-detectable level. This treatment was followed by addition of diap1 dsRNA to induce apoptosis. Remarkably, in cells that had been treated with dronc dsRNA, death induced by silencing of diap1 is largely suppressed, whereas cells that had been pre-treated with control dsRNA undergo almost complete apoptosis. After 12 h, the surviving cells that were pre-treated with dronc dsRNA began to divide, resulting in an apparent increase in viability. Thus, death in these surviving cells appears to be completely inhibited, not just delayed, by depletion of DRONC (Muro, 2002).

In mammals, activation of caspase-9 requires dATP, cytochrome c, and Apaf-1. The homolog of Apaf-1 in Drosophila is DARK. In vitro activation of DRONC is decreased in extracts made from mutant fly embryos lacking DARK, indicating that DARK may play a role in DRONC activation similar to that of Apaf-1 in caspase-9 activation. In order to determine whether DARK is also required for apoptosis stimulated by depletion of DIAP1, S2 cells were treated with dark dsRNA, and 24 h later diap1 dsRNA was added to induce apoptosis. Co-silencing of dark and diap1 also suppresses apoptosis induced by loss of DIAP1 and results in even higher cell viability than cells co-silenced for dronc and diap1. Also, similar to dronc dsRNA-treated cells, dark dsRNA-treated cells surviving after 12 h of diap1 dsRNA treatment begin to divide. Together, these data indicate that an important function of DIAP1 in S2 cells is to inhibit the activity of DRONC, and that DRONC activity is in turn dependent on DARK (Muro, 2002).

S2 cells treated with cycloheximide undergo caspase-dependent apoptosis within 3-4 h. Similar to diap1 RNAi, silencing dronc or dark prior to cycloheximide treatment dramatically delays apoptosis. Silencing dronc also strongly inhibits apoptosis stimulated by UV light. Likewise, depletion of DARK by RNAi protects S2 cells from stress-related apoptotic stimuli, including ultraviolet light and cycloheximide (Muro, 2002).

The observation that depletion of dronc or dark does not completely suppress apoptosis induced by a reduction in DIAP1 may have been due to small amounts of DRONC or DARK protein remaining after 24 h of dsRNA treatment. Alternatively, there may be other apical caspases, such as DREDD or STRICA/Dream that can become activated following loss of DIAP1. This latter possibility is supported by the greater protection seen with co-silencing of dark than with dronc. Nevertheless, these results indicate that DRONC appears to be the major apical caspase that is activated following depletion of DIAP1, because the majority of cells are protected and continue dividing following diap1 and dronc RNAi (Muro, 2002).

In addition to DRONC, another caspase, Ice, is also known to be activated in Drosophila embryos lacking DIAP1. Furthermore, immunodepletion of Ice from apoptotic S2 cell lysates removes all of the detectable chromatin condensing activity from the lysates, suggesting that Ice plays a vital role in apoptosis. These findings and the data showing the requirement for DRONC and DARK in apoptosis stimulated by depletion of DIAP1 or UV light led to examine an examination of the activation of DRONC and Ice following treatment with these stimuli. Lysates from S2 cells treated with diap1 dsRNA or UV light were immunoblotted for DRONC and Ice. Within 3 h after treatment with either stimulus a processed form of DRONC, hereafter referred to as Pr1, is detected. By 6 h, a second, smaller processed form of DRONC, hereafter referred to as Pr2, is also seen. Pr1 disappears as Pr2 accumulates over time, suggesting but not proving that Pr1 is being further processed into Pr2. Full-length DRONC also disappears over time, although the decrease in full-length DRONC is sometimes difficult to detect because it runs as a tight doublet with a nonspecific background band. Yet Ice processing is not detected until 6 h after treatment with either stimulus. The appearance of the Pr2 form of DRONC coincides with the onset of Ice processing, and thus may be a result of cleavage of Pr1 by Ice or another effector caspase. Both Pr1 and Pr2, as well as full-length DRONC, were affinity-labeled with biotinylated Z-VAD-fmk indicating that they are enzymatically active processed forms of DRONC and not merely inactive degradation products (Muro, 2002).

Because of the length of their prodomains, it is widely assumed that DRONC and Ice are apical and effector caspases, respectively. The observation that DRONC is processed earlier than Ice following stimulation of apoptosis supports this hypothesis. In order to determine whether processing of Ice is dependent on DRONC, Ice processing was examined following depletion of DRONC. S2 cells treated with dronc dsRNA for 24 h and then treated with diap1 dsRNA were immunoblotted for Ice and showed almost no Ice processing. Ice processing was also almost completely inhibited when S2 cells were treated with UV light after dronc RNAi. In both cases, the cells remained viable throughout the experiment. These results are consistent with DRONC being an apical caspase that is required for processing of the effector caspase Ice following an apoptotic stimulus. In addition, these results also indicate that in normal, living cells, DIAP1 promotes cell viability by inhibiting DRONC activity and not that of Ice, because Ice processing does not occur in the absence of active DRONC (Muro, 2002).

Fly embryos lacking DIAP1 spontaneously undergo massive apoptosis, as do S2 cells depleted of DIAP1 by RNAi. This suggests that not only does DIAP1 inhibit DRONC activity, but also that this activity is constitutively present in cells because DIAP1 is required to prevent spontaneous apoptosis. Because DIAP1 can bind to DRONC, this inhibition may be direct and/or it may be due to DIAP1 acting as an E3 and causing the degradation of DRONC by the proteasome. To determine whether DRONC is targeted for proteasome degradation, S2 cells were treated with the proteasome inhibitor MG132 and immunoblotted for DRONC. Interestingly, MG132-treated cells consistently exhibit an accumulation of the Pr1 form of processed DRONC, even though MG132 treatment itself has no effect on cell viability. However, MG132 treatment does not result in an increase in the levels of full-length DRONC. In addition, there is no further processing of DRONC to the Pr2 form seen in cells undergoing apoptosis induced by either UV treatment or diap1 dsRNA. Thus, even in the absence of an apoptotic signal, DRONC is continuously processed to the Pr1 form, and the Pr1 form is subject to proteasome-mediated degradation. Given the fact that either diap1 RNAi or UV light cause a rapid decrease in DIAP1 levels, and the observation that DRONC is a target for ubiquitination by DIAP1, it appears that DIAP1 is required to prevent accumulation of the Pr1 processed form of DRONC, probably by directing its ubiquitination (Muro, 2002).

These results also indicate that there are least two steps involved in DRONC processing in vivo. It is suggested that the first cleavage, resulting in the Pr1 form, may be due to autocatalytic processing, whereas the second cleavage resulting in Pr2 is specifically seen in dying cells and may be due to cleavage by an effector caspase such as Ice. The apparent molecular weight of Pr1 is consistent with an in vitro autocatalytic cleavage event for DRONC. DRONC autoprocesses itself in vitro after a glutamate residue, Glu-352, and the apparent molecular weight of Pr1 is similar to that expected if cleavage occurred at Glu-352 (40.3 kDa). Furthermore, the size of Pr2 is consistent with Pr1 being further cleaved at the canonical caspase cleavage site (DEYD) located at the boundary between the large and small subunits at position 324 (expected size of 37.0 kDa). The timing of the appearance and disappearance of Pr1 and Pr2 suggests (but does not prove) a precursor-product relationship between these two forms of processed DRONC. Because DRONC is believed to be an apical caspase, it would be expected that the initial cleavage event is autocatalytic. Pr1 is the first cleavage product of DRONC observed following an apoptotic stimulus and occurs before Ice activation, whereas the appearance of Pr2 correlates with Ice activation (Muro, 2002).

DARK is required for efficient processing of DRONC in vitro and in the absence of DARK, apoptosis induced by either diap1 dsRNA addition or cycloheximide treatment is largely suppressed. DARK may therefore play a role in the continuous autoprocessing of DRONC. S2 cells were treated with dark dsRNA and after 24 h immunoblotted for DRONC. Remarkably, these cells show an accumulation of full-length DRONC, suggesting that DARK is indeed required for the continuous autoprocessing of DRONC. These cells also contain some of the Pr1 form of DRONC, which may have been due to low levels of DARK remaining after RNAi or to spontaneous DRONC dimerization and autoactivation that may occur without the need for DARK when DRONC accumulates to high levels, similar to the Apaf-1-independent activation observed when caspase-9 is present at higher than normal concentrations. The presence of Pr1 DRONC in cells treated with dronc dsRNA for 24 h suggests that the half-life of Pr1 in nonapoptotic cells may be relatively long compared with the time required for autoprocessing of full-length DRONC. If, as is thought, DRONC first autoprocesses itself to Pr1 and only then is subject to proteasome degradation, then it would be expected that full-length DRONC would disappear faster than Pr1 following silencing of dronc by RNAi. In addition, cells treated with dronc dsRNA are not apoptotic, and thus Pr1 is not being further processed to Pr2, possibly further lengthening Pr1 half-life (Muro, 2002).

Together these results provide evidence for a model in which DRONC continuously undergoes processing to the Pr1 form in normal living cells, and this processed form, which is suggested to be due to autoprocessing, is continuously degraded via the E3 activity of DIAP1. This initial processing step proceeds through a mechanism that requires DARK, perhaps involving apoptosome formation. In this model, DIAP1 is required to inhibit the over-accumulation of Pr1 DRONC through its ability to act as an E3. However, once a death signal is received and DIAP1 is removed, either through binding to apoptotic inducers such as Hid, Reaper or Grim, or by degradation, this suppression is released and the Pr1 form of DRONC accumulates, activating effector caspases such as Ice, which can further cleave Pr1 DRONC to the Pr2 form as well as cleave other apoptotic substrates, leading to apoptosis. This model does not rule out the possibility that DIAP1 may also directly inhibit the enzymatic activity of full-length and/or partially processed DRONC. In fact, this possibility is suggested by the fact that MG132 treatment results in an over-accumulation of Pr1 DRONC, but these cells did not die (Muro, 2002).

Prior to this work, the identity of the caspase(s) that are normally inhibited by IAP proteins in any living cells had not been determined. These results indicate that continuous expression of the short lived IAP protein DIAP1 is required to inhibit the activity of the caspase DRONC and that DRONC acts as an apical caspase in Drosophila S2 cells. Importantly, the results showing induction of apoptosis in Sf21 cells following Sf-iap RNAi demonstrate that this pathway is probably conserved in other insects as well (Muro, 2002).

Although it is widely assumed that DIAP1 inhibits caspase activity in Drosophila, this is the first identification of a specific caspase that must be inhibited by DIAP1 to promote cell survival. The activation of mammalian caspase-9 is known to require Apaf-1, cytochrome c, and dATP. The current results suggest that the Apaf-1 homolog DARK is also required for activation of DRONC, since depletion of DARK causes an excess accumulation of full-length DRONC, and silencing of dark prior to reducing DIAP1 levels protects cells from apoptosis. However, unlike caspase-9, DRONC appears to undergo continuous autoprocessing, even in normal living S2 cells. It is possible that in insect cells, there is some level of constitutive apoptosome formation that may not require cytochrome c but may involve other factors. Recent data suggest that cytochrome c is not required for apoptosome formation in Drosophila cells, although addition of cytochrome c further stimulates formation of an apoptosome-like complex (Muro, 2002 and references therein).

Treatment of cells with the proteasome inhibitor MG132 results in over-accumulation of the larger Pr1 processed form of DRONC, even though these cells remained viable throughout the experiment. This result argues that this processed form, which may result from autoprocessing at Glu-352, is continuously produced in cells but is normally targeted for degradation by the proteasome. In contrast, the data do not indicate that full-length DRONC is a proteasome substrate in vivo; there was no detectable excess accumulation of full-length DRONC following treatment with MG132. The involvement of DIAP1 in this degradation process is supported by the increased levels of processed DRONC following silencing of diap1, and the report that DIAP1 is capable of directing ubiquitination of DRONC (Muro, 2002 and references therein).

In conclusion, the results of this study show that the apical caspase DRONC is continuously processed in living Drosophila cells, probably by an autocatalytic mechanism, and that DIAP1 is required to prevent accumulation of this processed form of DRONC. This initial processing step is dependent on the Apaf-1 homolog DARK and may occur by DARK promoting DRONC dimerization, similar to the mechanism by which Apaf-1 activates caspase-9. Removal of DIAP1 results in excess accumulation of processed DRONC, activation of the downstream effector caspase Ice, and apoptosis. The results thus have implications for therapeutic strategies aimed at disrupting IAP function in mammalian cells, because they suggest that targeting interactions between IAP proteins and apical caspases are likely to be more effective at inducing cell death than targeting interactions between IAP proteins and downstream effector caspases (Muro, 2002).

Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1

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).

Elevated Thread levels are found in salvador and hippo mutants; Hippo phosphorylates Thread

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, the gene salvador (sav) that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and Thread/DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

In sav1 clones in the adult retina, almost all the ommatidia contain the normal complement of eight photoreceptor cells. However, there is increased spacing between adjacent ommatidia. In contrast to wild-type retinas from late pupae that contain a single layer of interommatidial cells, mutant clones contain many additional interommatidial cells. Generation of sav1 mutant clones in a white+ background indicates that most of these additional interommatidial cells contain pigment. Thus, these cells can undergo terminal differentiation. The more disorganized retinas of the sav3 allele display all of these phenotypic abnormalities. In addition, almost half of the ommatidia in sav3 clones lack one or more photoreceptor cells (Tapon, 2002).

In wild-type imaginal discs, S phases, as visualized by BrdU incorporation, are observed anterior to the morphogenetic furrow (MF) and as a single stripe of incorporation posterior to the furrow referred to as the second mitotic wave (SMW). In sav clones, many BrdU-incorporating nuclei are observed posterior to the SMW. Clones spanning the MF have some BrdU-incorporating nuclei in the anterior half of the MF, a region that is normally composed of cells arrested in G1. Using the anti-phosphohistone H3 antibody, additional cells in mitosis are also visualized in sav mutant clones posterior to the MF, suggesting that at least some of these cells are completing additional cell cycles. BrdU incorporation persists in mutant clones during the first 12 hr after puparium formation (APF) but has ceased by 24 hr APF. Thus, sav mutant cells continue to proliferate for 12–24 hr after wild-type cells stop dividing but are eventually able to exit from the cell cycle and undergo terminal differentiation (Tapon, 2002).

In cycling cells in the anterior portion of the eye imaginal disc, the distribution of mutant cells in the cell cycle, as assessed by flow cytometry, is extremely similar to that of wild-type cells. The mutant cells are very slightly smaller than their wild-type counterparts. Posterior to the MF, mutant populations have an increased proportion of cells in S and G2, indicating that mutant cells continue to cycle in this portion of the disc. Mutant cells are of normal size. The population doubling times of clones of mutant cells and wild-type cells generated in the wing imaginal disc during the proliferative phase of development did not differ significantly. Thus, when they are proliferating, mutant cells behave like wild-type cells. However, exit from the cell cycle is delayed in sav cells (Tapon, 2002).

Elevated levels of Cyclin E protein are found in the basal nuclei of sav clones posterior to the MF. These are the nuclei of the undifferentiated cells that continue to proliferate in sav clones. Such discs were examined for levels of cyclin E RNA. When sav clones are generated using eyFLP, a large proportion of cells in third instar discs are mutant, and these discs contain large patches of mutant tissue. In wild-type discs, cyclin E RNA is expressed in a narrow stripe immediately posterior to the morphogenetic furrow. In discs containing sav clones, the stripe of expression is broader and more intense, indicating that cyclin E RNA levels are elevated in these discs. Thus, the increased level of Cyclin E protein is likely to result, at least in part, from an increase in cyclin E RNA levels (Tapon, 2002).

In wild-type eyes, excessive interommatidial cells are eliminated by a wave of apoptosis that is evident in 38 hr pupal retinas. Even in sav mutant clones, cell proliferation, as assessed by BrdU incorporation, has ceased within 24 hr APF. When mosaic retinas were examined 38 hr APF, cell death is mostly confined to the wild-type portions of the retina. Thus, the apoptotic cell deaths that are part of normal retinal development appear to require sav function (Tapon, 2002).

Apoptosis in the pupal retina requires hid function, since hid mutants display additional interommatidial cells. Hid is thought to induce caspase activation by binding to the DIAP1 protein and preventing it from inhibiting caspase function. Overexpression of hid using the eye-specific GMR promoter generates a small eye. The induction of cell death by hid is severely impaired in sav mutant clones. As a consequence, eyes derived from GMR-hid-expressing discs that contain sav mutant clones are larger than those derived from wild-type discs that express GMR-hid. Since sav function is required for hid-induced cell death, sav is likely to function either downstream of hid or in a parallel pathway (Tapon, 2002).

Several studies have shown that Hid and Rpr activate caspases by another mechanism in which they induce the autoubiquitination of DIAP1 and target it for degradation by the proteasome. DIAP1 levels are markedly elevated in sav clones in the larval eye disc and remain elevated in the interommatidial cells in mutant clones in the pupal eye disc. Thus, increased levels of DIAP1 in sav cells may be able to overcome the effect of many proapoptotic signals (Tapon, 2002).

To examine DIAP1 RNA levels, in situ hybridization was used to examine 20 wild-type discs and 20 mutant discs. The presence of sav (GFP-) clones in the mutant discs was confirmed by examining the discs by fluorescence microscopy prior to hybridization. There is a modest level of DIAP1 RNA expression posterior to the furrow in both populations of discs and no evidence of increased DIAP1 RNA in the discs containing sav clones. Thus, at least at this level of detection, the increased DIAP1 expression in sav cells does not appear to result from increased transcription (Tapon, 2002).

In wild-type eye discs, DIAP1 protein is expressed at higher levels posterior to the morphogenetic furrow. DIAP1 protein levels are downregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression. In sav mutant clones expressing GMR-rpr, DIAP1 protein levels remain elevated. Similar results are observed with GMR-hid. Thus, neither GMR-rpr nor GMR-hid appears capable of downregulating the elevated levels of DIAP1 sufficiently in sav clones to activate caspases (Tapon, 2002).

Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice. An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).

A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAP kinase phosphorylation. GMR-hid-Ala5 is a more potent inducer of cell death than is GMR-hid, as assessed by the extent of Drice activation in the eye disc. Cell death induced by GMR-hid-Ala5 is only partially blocked in sav clones, indicating that the increased potency of Hid-Ala-5 may be able to overcome increased DIAP1 levels (Tapon, 2002).

Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).

Proteins such as Hid, Rpr, and Grim are thought to downregulate DIAP1 levels by stimulating its autoubiquitination or by repressing general protein translation, which has the greatest effect on short-lived proteins such as DIAP1. To investigate whether hpo can modify the function of such proteins, hpo clones were generated in flies overexpressing the grim gene under the control of the GMR promoter. When overexpressed in the Drosophila eye, grim induces extensive cell death as visualized by TUNEL, which results in a small, rough eye. When hpo clones were generated in eyes overexpressing Grim, eye size was significantly restored and Grim-induced cell death was greatly reduced in hpo mutant clones. sav and wts clones are relatively resistant to cell death induced by Rpr or Hid. The increased basal level of DIAP1 found in sav, wts, or hpo clones may make it more difficult for proteins such as Rpr, Hid, or Grim to reduce DIAP1 levels sufficiently to activate caspases in these cells (Harvey, 2003).

Since DIAP1 protein levels are elevated in hpo clones and in S2 cells treated with hpo RNAi, the possibility that hpo might regulate DIAP1 stability was tested. When hpo is overexpressed in Drosophila S2 cells, endogenous DIAP1 protein is consistently reduced to approximately 60%-70% of normal levels, when normalized to loading controls. Since Wts and Hpo are both predicted to have kinase activity, it is possible that a complex consisting of Sav, Hpo, and Wts regulates the phosphorylation state of DIAP1 and hence regulates its turnover. Indeed, both Sav-associated and Hpo-associated complexes are capable of phosphorylating DIAP1 in vitro, and DIAP1 is destabilized in the presence of Hpo, presumably as a result of Hpo-dependent phosphorylation of DIAP1 (Harvey, 2003).

Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death remains largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila tumor-necrosis factor receptor-associated factor 1 (Traf1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates Traf1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of Traf1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of Traf1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of Traf1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).

Three Drosophila genes, reaper, hid, and grim, have been identified as key regulators of apoptosis during Drosophila embryogenesis. Products of all three genes induce apoptosis through a pathway that requires activation of caspase. Through interactions mediated by the N terminus, each of these proteins binds to DIAP1. Genetic and biochemical data indicate that one way in which these proteins promote apoptosis is by inhibiting the ability of DIAP1 to prevent death-inducing caspase activity. Smac (also known as DIABLO) and HtrA2 (also known as Omi), mammalian mitochondrial proteins whose truncated N termini share similarity with Rpr, Hid and Grim, also inhibit the antiapoptotic function of XIAP and enhance caspase activation (Kuranaga, 2002).

To study the genetic regulation of this conserved cell death mechanism, a dominant modifier screen of Rpr was carried out that covered more than 70% of the Drosophila genome. Overexpression of Rpr using an eye-specific promoter (GMR) gave rise to dose-dependent cell death through caspase activation, resulting in flies with small eyes. Df(2L)sc19-8 was identified as a suppressor of Rpr through deletions covering the region 24C2-25C8. Because this suppressor line had a large deletion, lines with smaller deletions around the 24C2-25C8 region were subsequently screened, including Df(2L)ed-dp, Df(2L)dp-h25, Df(2L)M24F-B, Df(2L)tkv3 and Df(2L)ed1. Of all the strains examined, three overlapping deletions, Df(2L)ed-dp, Df(2L)dp-h25 and Df(2L)M24F-B, suppressed the Rpr-induced small eye phenotype: this information allowed a narrowing down of the suppressor region to 24E4-25A2. The ability of mutants covering the 24E4-25A2 region to improve the Rpr-induced small eye phenotype was tested: a P-element insertion, EP(2)578, in the first exon in the noncoding region of the Drosophila homolog of TRAF1 (Traf1) substantially suppresses the reduced eye size caused by Rpr (Kuranaga, 2002).

To assess the reduced expression of the Traf1 transcript in this homozygous mutant, polymerase chain reaction was carried out with reverse transcription (RT-PCR) using specific primers that detected Traf1 messenger RNA from wild-type and mutant third-instar larvae. The expression of Traf1 was markedly reduced in the homozygous mutant. Thus, Traf1 mutant was identified as a putative suppressor allele on Rpr (Kuranaga, 2002).

Df(3R)H-B79 was also identified using deletions covering the 92B3-F13 region. A search of the translated nucleotide databases, using the TBLASTN program, identified an expressed sequence tag (EST) clone, LD40486, that maps to this region (Berkeley Drosophila Genome Project) and has similarity to the kinase domain of the mitogen-activated protein kinasae kinase kinase (MAPKKK) family of serine/threonine kinases. Previously, the gene from the genomic region that encoded the MAPKKK had been isolated as PK92B, from an eye-antennal imaginal disc complementary DNA library using a PCR-based approach. It has an open reading frame (ORF) of 650 amino acids. The EST clone (LD40486) encoding the PK92B cDNA was analyzed and it can potentially encode a longer form of the protein (1,367 amino acids). Notably, the longest ORF of the cDNA showed substantial overall homology to the human MAPKKK ASK1, with the kinase domain of this cDNA showing 73% identity and 83% similarity with the amino acid sequences of ASK1 (Kuranaga, 2002).

To determine whether PK92B has a role in JNK activation in Drosophila, Basket/JNK and PK92B were coexpressed in Drosophila S2 cells along with the kinase-dead K618M mutant of PK92B. JNK activation by PK92B was suppressed by PK92BK618M in a dose-dependent manner, which suggests that PK92BK618M is a dominant-negative form of PK92B and that PK92B is involved in JNK signalling. The kinase-dead PK92BK618M mutant was used to test whether the kinase activity of PK92B is important in mediating Rpr-induced cell death; the Rpr-induced reduced eye size is visibly suppressed by coexpression of PK92BK618M (Kuranga, 2002).

To test whether the Drosophila Basket/JNK pathway is involved in Rpr-induced cell death, the genetic interactions of GMR-Rpr with several mutants or transgenic lines of the JNK signalling pathway were examined. Two mutants in the JNK pathway were tested -- basket2 (bsk encodes JNK) and hemipterous1 (hep1, hep encodes DJNK kinase) -- as well as two transgenic lines, UAS-DJNK DN (expressing a dominant-negative form of Basket) and UAS-Dp38 DN (expressing a dominant-negative form of Drosophila p38; Dp38). In flies with reduced JNK signals, but not in those with reduced p38 signals, the reduced eye size of the GMR-Rpr flies is visibly improved. The transient expression of Rpr strongly activates Basket in third-instar larvae. These results suggest that Rpr can activate the JNK pathway, probably through Traf1 and PK92B (Kuranga, 2002).

In mammals, ASK1 interacts with members of the TRAF family and is sufficient and necessary for the activation of JNK induced by TNF-alpha and TRAF2. Traf1 has been reported to be involved in JNK activation. Therefore it was asked whether Traf1 and PK92B could activate DJNK in Drosophila S2 cells. These cells were transfected with PK92B or Traf1, and phosphorylation of Basket was detected by immunoblotting using an antibody against phosphorylated Basket. Both PK92B and Traf1 strongly induce Basket phosphorylation. Whether Traf1 could activate PK92B was examined. Traf1 and Flag-PK92B were cotransfected into S2 cells, and Flag-PK92B was immunoprecipitated with an antibody against Flag. The activation status of PK92B was then assessed using immunoblotting analysis with an antibody specific for phosphorylated ASK1. Consistent with the ability of Traf1 to activate Basket, PK92B is strongly activated by the coexpression of Traf1. A Basket-PK92B interaction was observed in S2 cells. Thus, it is possible that PK92B is a downstream target of Traf1 and that Basket is activated through a direct interaction with PK92B (Kuranga, 2002).

How Rpr affects the Traf1/PK92B/Basket signalling pathway was investigated. Because DIAP1 is a molecular target of Rpr, whether DIAP1 might affect the DJNK activation pathway was investigated. Traf1 was transfected into S2 cells with or without DIAP1 and the viability of cells was examined. Traf1-induced cell death was markedly suppressed by DIAP1. To confirm that Traf1-induced cell death is suppressed by DIAP1, a strain of UAS-Traf1 transgenic flies was generated. The overexpression of Traf1 driven by GMR-GAL4 causes a rough and small eye phenotype and increases the number of dying cells in the third-instar larval eye disc. The massive cell death caused by expression of Traf1 was mediated by the JNK pathway in the fly eye, because the Traf1-induced small eye phenotype is markedly improved in a heterozygous hep1 mutant background. The coexpression of DIAP1 substantially suppresses the Traf1-induced rough eye phenotype (Kuranga, 2002).

Next, the phosphorylation of Basket was examined by immunoblotting with an antibody against phosphorylated JNK in S2 cells. Activation of Basket by Traf1 is suppressed by DIAP1 in a dose-dependent manner, but DIAP1 does not affect the activation of Basket by PK92B. Notably, increased amounts of DIAP1 lower the amounts of Traf1. DIAP1 can directly interact with Traf1, which suggests that the downstream target of DIAP1 is Traf1, and not PK92B or Basket. DIAP1 and DIAP2, mammalian cIAP1 and cIAP2, and X-linked IAP (XIAP) all possess RING-finger and baculovirus IAP repeat (BIR) domains. The BIR domain is required for caspase binding and inhibition, whereas ubiquitination by several E3 ubiquitin ligases is dependent on the RING-finger motif. Notably, cIAP1 and XIAP catalyse their own ubiquitination in a manner dependent on their RING domain. It was therefore thought that the Traf1-DIAP1 interaction might represent ubiquitination of Traf1 by DIAP1. This possibility was tested by transfecting a fusion protein of Traf1 and hemagglutinin A (Traf1-HA), HA-ubiquitin and Flag-DIAP1 into S2 cells, and then carrying out immunoprecipitations and immunoblotting with an antibody against HA to examine the amount of ubiquitinated Traf1. Traf1 was heavily ubiquitinated in the presence of DIAP1 in a dose-dependent manner. Taken together, these results suggest that DIAP1 stimulates Traf1 degradation through ubiquitination. This regulation of Traf1 would therefore prevent Traf1-induced JNK activation as well as cell death (Kuranga, 2002).

Although Rpr could interact with DIAP1 through its N-terminal region, no direct interaction between Rpr and components of the JNK pathway (Traf1, PK92B and DJNK) was detected, which suggests that Rpr may be activating the JNK pathway through DIAP1. In agreement with this, expression of a Rpr mutant truncated at its N terminus (UAS-RprN) using the hs-GAL4 driver in third-instar larvae failed to activate DJNK, suggesting the importance of the Rpr and DIAP1 interaction for both caspase and DJNK activation. It has been shown that Rpr not only inhibits IAP function but also promotes the degradation of DIAP119-23. On the basis of these observations, it was reasoned that the degradation of DIAP1 by Rpr might be able to promote JNK activation. To assess this possibility, whether Rpr could prevent the degradation of Traf1 mediated by DIAP1 was examined. Not only did the expression of Rpr substantially inhibit the DIAP1-mediated degradation of Traf1, but it also activated Basket/JNK. These results suggest that expression of Rpr can stimulate activation of JNK by degrading DIAP1 and subsequently stabilizing Traf1 (Kuranga, 2002).

To determine the endogenous function of Traf1, the phenotype of adult flies that were homozygous for the Traf1 mutant, Traf1EP(2)578, was examined. The only marked characteristic of these flies was additional numbers of adult dorsal bristles. The external sensory organ on the notum is a typical structure of the Drosophila peripheral nervous system, where four large bristles (macrochaetes) are always observed in the wild-type scutellum. The ectopic bristles seen in Traf1EP(2)578 flies are probably the result of altered caspase activation, because they are also found in Drosophila Apaf-1 mutants, in flies that ectopically express caspase inhibitory proteins p35 and DIAP1, and in a mutant for a ubiquitin-conjugating enzyme that promotes the degradation of DIAP1. These results suggest that Traf1 and DIAP1 affect the activation of caspase in order to regulate the number of macrochaetes in adults; thus, Traf1 probably affects processes involving caspase-mediated events. The possibility that Traf1 has a role in the canonical JNK pathway cannot be ruled out, because the Traf1EP(2)578 mutant allele that was used in this study may not be a null allele (Kuranga, 2002).

In summary, it is proposed that Basket/JNK activity can be negatively regulated by DIAP1. Overexpression of DIAP1 prevents Traf1-induced Basket/JNK activation through degradation of Traf1. Rpr facilitates the degradation of DIAP1 through the ubiquitin-proteasome system, thereby inducing activation of Basket/JNK mediated by Traf1 and PK92B. These findings suggest that the degradation of IAPs in cells that have been instructed to undergo cell death may represent an evolutionarily conserved mechanism to facilitate cell death (Kuranga, 2002).

DIAP1 suppresses ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

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).

It is not yet well understood how certain processes such as oxidative stress can trigger specific pathways of apoptosis. Aerobic metabolism uses molecular oxygen as a terminal electron acceptor for mitochondrial respiratory energy production. Reactive oxygen species (ROS) are generated as by-products of this process. These are a variety of oxygen metabolites that have either unpaired electrons (i.e., OH·) or the ability to abstract electrons from other molecules (i.e., hydrogen peroxide). Transient fluctuations in ROS serve important regulatory functions, but when present at high and/or sustained levels, they can cause severe damage to DNA, proteins and lipids, which may finally lead to apoptosis (Forey, 2003).

A number of defense systems have evolved to counteract the persistent state of oxidative siege associated with aerobic life conditions and among them enzymatic intracellular scavengers play an essential role. In this category, mammalian selenoproteins, which contain selenium in the form of selenocysteine, are important for the control of unwanted ROS as most of them catalyze oxidation-reduction reactions or act as antioxidants. The machinery of selenocysteine incorporation is conserved in bacteria, archaea and eukarya and depends on reading the inframe UGA stop codon as the amino acid selenocysteine and on the recognition of a downstream stem loop structure. Four genes (selA-D) of Escherichia coli are essential for selenocysteine synthesis, codon recognition and polypeptide elongation. One of them, the selD gene, encodes for selenophosphate synthetase, which catalyses a selenide-dependent ATP hydrolysis reaction to generate monoselenophosphate, the selenium intermediate necessary for the synthesis of selenocysteine. In flies and mammals two highly conserved selD genes have been identified, sps1 (selD in flies) and sps2. Two effects have been described for a mutation in the Drosophila selD/sps1 gene, perturbation of selenoprotein biosynthesis and accumulation of ROS (Alsina, 1999). This mutation, hereafter called selDptuf, is a recessive null mutation and homozygous individuals have extremely reduced and abnormal imaginal discs and die as third instar larvae. Heterozygous selDptuf flies are apparently wild type, however, a downregulation of the Ras/MAPK pathway has been observed in transheterozygous combinations of the selDptuf mutation and activated members of this signaling pathway. Both loss of survival signals and perturbations in the redox balance, among others, are intracellular stimuli that have been shown to trigger apoptosis in mammals. In Drosophila, the cell death genes reaper (rpr), head involution defective (hid) and grim are potent activators of caspase-dependent apoptosis. Extensive research has been carried out to uncover how distinct death-inducing stimuli converge to activate a common apoptotic program. Both rpr and grim are expressed in cells doomed to die. In contrast, hid is expressed not only in cells that die, but also in living cells. Thus, these different expression patterns imply that the cell death genes integrate different signals regulating apoptosis. Survival signals regulate Drosophila apoptosis and several studies have shown the need for Ras/MAPK activity for cell survival in flies. In the subset of hid-expressing cells prone to die, downregulation of the Ras/MAPK pathway increases hid expression and activity. In the case of rpr, besides inducing developmentally programmed cell death, it also triggers apoptosis in response to other stimuli such as aberrant development, steroid hormone signaling and X-irradiation. rpr is also a transcriptional target of the Drosophila p53 protein, making its expression responsive to genotoxic stress caused by X-irradiation. Because X-irradiation, in addition to direct DNA damage, generates ROS in the aqueous cytoplasm that can also damage DNA, the p53/Rpr pathway is a candidate pathway to be activated in a situation of increased ROS levels such as in the selDptuf mutant (Forey, 2003).

A genetic approach has been taken to study how an increase in ROS levels due to impairment of the selD/sps1 function triggers apoptosis in Drosophila imaginal discs. The results indicate that hid-induced apoptosis may not be the major contributor to the apoptosis observed in selDptuf mutant cells and that ROS increase plays an important role in selDptuf apoptosis through the activation of the p53/Rpr pathway. This apoptotic pathway is mediated by DRONC and DRICE caspases and the inhibitor of apoptosis DIAP1 is able to rescue the viability of selDptuf cells. This work supports the importance of selD/sps1 in the maintenance of cellular viability and demonstrates that ROS-induced apoptosis triggered by the loss-of-function of selD/sps1 is caspase dependent and activated by p53/Rpr function (Forey, 2003).

The results could be explained by an activation of the apoptotic machinery resulting from oxidative stress triggered by the absence of selenoproteins in the selDptuf cells. However, there are some observations that suggest that the fly and mammalian selD/sps1 could have derived a novel function. There are only three selenoprotein genes described so far in the fly genome: selenophosphate synthetase type 2 (sps2), and dselM and dselG, both of unknown function. The presence of non-selenoprotein paralogs of dselM and dselG, makes it hard to account for the lethality of selDptuf by simply removing the selenoproteins. Every known selenoprotein in vertebrates that acts as a detoxifyer enzyme (carrying a UGA-coded selenocysteine in the active center) is not a selenoprotein in the fly (i.e., the selenocysteine is substituted by another amino acid). Taken together, it is unclear whether Drosophila selenoproteins are part of the oxidative stress defense system. In addition, the enzymatic activity of the two highly conserved eukarya selD gene products differs. The selenocysteine amino acid residue of Sps2 is essential for its selenophosphate synthetase activity in mammals, whereas the mammalian Sps1 only weakly complements an E. coli selD mutation. Similarly, the fly Sps2 contains a selenocysteine in its catalitic domain, whereas the Drosophila selD/sps1 does not complement a bacterial selD mutation. It is, therefore, possible that selD/sps1 has a dual function: one mediated by selenoproteins, or involved in their biosynthesis as basal selenophosphate synthetase activity, and a second ROS-related function, independent of selenoproteins, conserved in flies and mammals (Forey, 2003).

Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It has not been clear how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. This study found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in a reconstitution assay. However Grim does not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2 (Yoo, 2005).

Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs

Caspase activation has been extensively studied in the context of apoptosis. However, caspases also control other cellular functions, although the mechanisms regulating caspases in nonapoptotic contexts remain obscure. Drosophila IAP1 (DIAP1) is an endogenous caspase inhibitor that is crucial for regulating cell death during development. Drosophila IKK-related kinase (DmIKKε; FlyBase name, Ik2) as a regulator of caspase activation in a nonapoptotic context. DmIKKε promotes degradation of DIAP1 through direct phosphorylation. Knockdown of DmIKKε in the proneural clusters of the wing imaginal disc, in which nonapoptotic caspase activity is required for proper sensory organ precursor (SOP) development, stabilizes endogenous DIAP1 and affects Drosophila SOP development. These results demonstrate that DmIKKε is a determinant of DIAP1 protein levels and that it establishes the threshold of activity required for the execution of nonapoptotic caspase functions (Kuranaga, 2006).

Whether physical binding occurs between endogenous DmIKKε and DIAP1 was tested. An immunoprecipitation experiment was performed against the endogenous DmIKKε protein using an anti-DmIKKε monoclonal antibody. The specificity of this antibody was confirmed by immunoblotting to detect endogenous DmIKKε protein in normal and DmIKKε knockdown cells. These results indicated that physical binding occurs between endogenous DmIKKε and endogenous DIAP1 in S2 cells. When the S2 cells were treated with DmIKKε dsRNA, endogenous DIAP1 protein was not immunoprecipitated by the DmIKKε antibody. To elucidate the function of the ubiquitin-like (Ubl) domain of DmIKKε, the binding of a deletion mutant for this domain was tested. DmIKKεΔUbl interacted weakly with DIAP1, whereas wild-type or kinase-dead mutants of DmIKKε significantly bound to DIAP1. Consistent with the binding assay, DmIKKεΔUbl did not cause cell death or DIAP1 degradation in S2 cells. The binding of purified DIAP1 and DmIKKε was not detected by a GST pull-down assay in vitro, indicating that this physical binding may not be direct. It is likely that DmIKKε phosphorylates DIAP1 in a protein complex in cells and that the Ubl domain of DmIKKε is required for efficient formation of the DmIKKε/DIAP1 complex (Kuranaga, 2006).

Whether DIAP1 degradation might be regulated by DmIKKε-induced phosphorylation was tested. Wild-type DmIKKε was autophosphorylated and induced the phosphorylation of DIAP1 in S2 cells, whereas none of the four mutant DmIKKεs generated from the alleles DmIKKεG19R, DmIKKεD160N, DmIKKεG250D, or DmIKKεΔUbl did so. Moreover, the kinase-dead DmIKKε mutants did not reduce DIAP1 levels or cause cell death in S2 cells, and they did not cause eye ablation in adult flies (Kuranaga, 2006).

Whether the human IKK-related kinase NAK could also elicit DIAP1 degradation was tested. The expression of human NAK in S2 cells strongly induced cell death as well as DIAP1 degradation, whereas a kinase-dead form of NAK, NAKK41M, did not. These data imply that DIAP1 degradation mechanisms are conserved between mammalian IKK-related kinase and DmIKKε. To investigate whether mammalian IAP could be phosphorylated by mammalian IKK-related kinase, XIAP phosphorylation and degradation by NAK were tested. When 293T cells were cultured in media with 10% serum, a band shift was observed but not reduction of XIAP protein. XIAP band shift was cancelled by phosphatase treatment. The same experiments were performed under low-serum conditions (1%), and it was found that NAK expression induced both a band shift of XIAP and the reduction of XIAP protein. it was also observed that NAK phosphorylated XIAP in vitro, suggesting that the function of IKK-related kinase in the phosphorylation and degradation of IAP is conserved in both Drosophila and mammalian cells (Kuranaga, 2006).

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKKε is a member of the IκB kinase family, and its developmental role has not been clear. Drosophila IKKε localizes to the ruffling membrane of cultured cells and is required for F actin turnover at the cell margin. In IKKε mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibit aberrantly branched morphology. These phenotypes are sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKKε destabilizes F actin-based structures. Expression of a dominant-negative form of IKKε increases the amount of DIAP1. The results suggest that at the physiological level, IKKε acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKKε function involves the negative regulation of the nonapoptotic activity of DIAP1 (Oshima, 2006).

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease

Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. This study demonstrates that Drosophila Omi (dOmi; FlyBase term: HtrA2), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, this study demonstratesin flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein (Challa, 2007).

The role of mitochondria in fly apoptosis remains highly controversial, due in large part to disagreement over whether mitochondria undergo losses in Δψm (mitochondrial membrane potential) and MOMP (mitochondrial outer membrane permeabilization) following stress. Moreover, although mitochondrial release of cytochrome c in mammalian cells initiates formation of the Apaf-1 apoptosome complex and activation of caspases, there is disagreement over the importance of cytochrome c for promoting cell death in flies. The cytochrome c debate notwithstanding, there are additional mitochondrial proteins in mammals that play a role in promoting apoptosis, including the dual IAP antagonist and serine protease, Omi/HtrA2 (Hegde, 2001; Martins, 2001; Suzuki, 2001; Verhagen, 2001). In these studies, attempts were made to determine if the Drosophila homologue of Omi might likewise participate in cell death. It was found that dOmi was highly homologous to hOmi, particularly within the serine protease domain, and that its expression was developmentally regulated. dOmi was imported into fly mitochondria and processed in situ, resulting in the removal of its mitochondrial targeting sequence (MTS) and exposure of two distinct IAP binding motif (IBMs). The mature forms of dOmi were then released into the cytoplasm following stress, through both caspase-dependent and -independent processes. However, once in the cytosol, dOmi induced cell death in S2 cells and in the developing fly eye, primarily through proteolytic degradation of DIAP1 and likely other substrates (Challa, 2007).

Indeed, catalytically inactive Δ79-dOmiS266A and Δ92-dOmiS266A failed to induce significant apoptosis, which was somewhat surprising, given that both forms of dOmi selectively bound to the BIR2 domain in DIAP1 and displaced the initiator caspase DRONC. In particular, the affinity of Δ79-dOmi for BIR2 was lower than that observed for Rpr-IBM, but was slightly higher than that observed for mature Smac with XIAP-BIR3. So why did dOmi require its proteolytic activity to induce cell death, rather than inducing rapid IBM-dependent apoptosis? Notably, unlike other fly IAP antagonists, which exhibit partial preference for either the BIR1 or BIR2 domains, dOmi completely failed to bind the BIR1 domain in DIAP1 and did not displace the active effector caspase DrICE. Thus, it is possible that the continued inhibition of DrICE by DIAP1 was sufficient to inhibit cell death. There is precedence for such a scenario in mammals, since it has been shown that XIAP mutants that fail to bind and inhibit caspase-9 can still prevent apoptosis through inhibition of caspase-3 alone (Challa, 2007).

One of the primary differences between fly and mammalian IAP antagonists relates to their abilities to independently induce apoptosis. Indeed, Rpr, Hid, and Grim induce robust cell death in both cultured cells and, whereas overexpression of mature Smac in the cytoplasm of mammalian cells generally fails to induce apoptosis in the absence of an accompanying prodeath stimulus. A potential explanation for these results may involve their relative capacities to induce RING-dependent autoubiquitinylation upon binding to IAPs. Indeed, while many IAP antagonists in the fly induce DIAP1 autoubiquitinylation, Smac appears to suppress XIAP autoubiquitinylation. In these studies, dOmi failed to induce or suppress DIAP1 autoubiquitinylation upon binding to its BIR2 domain. Thus, in the absence of dOmi's proteolytic activity, DIAP1 may again be free to maintain its inhibition of DrICE via its BIR1 domain. By contrast, given that DIAP1 can protect cells by targeting active DRONC for proteosomal degradation, it is also plausible that DIAP1 might regulate cell death, in part by, promoting the turnover of dOmi. It has been reported that the DIAP1 binding mutant, DRONC (F118E), induces significantly more cell death than wild-type DRONC, when expressed in the developing fly eye, and correspondingly, this study found that Δ92-dOmi consistently produced a more severe phenotype than Δ79-dOmi, in accordance with their relative affinities for DIAP1 (Challa, 2007).

Others have reconciled such differences between the mammalian and fly IAP antagonists by arguing that, in contrast to the Apaf-1·caspase-9 apoptosome complex, the DARK·DRONC apoptosome complex is constitutively active. Consequently, DIAP1 is required to continuously ubiquitinylate DRONC and mediate its turnover in order to prevent cell death. In this model, Rpr, Hid, or Grim need only displace this active DRONC, in order to promote the activation of effector caspases and induce apoptosis. However, recent studies suggest that, at least for Rpr and Grim, the C-terminus of these IAP antagonists play important roles in promoting both mitochondrial injury and/or inhibition of protein translation. These alternative functions for Rpr and Grim may be necessary to first initiate caspase activation, after which the IBMs serve to displace these active caspases from DIAP1. Therefore, it could be that binding of dOmi to DIAP1-BIR2 per se does not induce apoptosis, because in the absence of another stimulus, there may be very little active DRONC to displace. In any event, regardless of whether dOmi induces cell killing solely through its proteolytic activity, or functions as a pure IAP antagonist in certain contexts, these studies suggest that mitochondria may play a far more important role in apoptosis in the fly than previously thought (Challa, 2007).

Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE

The Drosophila inhibitor of apoptosis protein DIAP1 exists in an auto-inhibited conformation, unable to suppress the effector caspase drICE. Auto-inhibition is disabled by caspase-mediated cleavage of DIAP1 after Asp20. The cleaved DIAP1 binds to mature drICE, inhibits its protease activity, and, presumably, also targets drICE for ubiquitylation. DIAP1-mediated suppression of drICE is effectively antagonized by the pro-apoptotic proteins Reaper, Hid, and Grim (RHG). Despite rigorous effort, the molecular mechanisms behind these observations are enigmatic. This study reports a 2.4 Å crystal structure of uncleaved DIAP1-BIR1, which reveals how the amino-terminal sequences recognize a conserved surface groove in BIR1 to achieve auto-inhibition, and a 3.5 Å crystal structure of active drICE bound to cleaved DIAP1-BIR1, which provides a structural explanation to DIAP1-mediated inhibition of drICE. These structures and associated biochemical analyses, together with published reports, define the molecular determinants that govern the interplay among DIAP1, drICE and the RHG proteins (Li, 2011).

The structural and biochemical information presented in this study gives rise to a model on the interplay of Dronc, drICE, DIAP1, and the RHG proteins. During homeostasis, DIAP1 targets the Dronc zymogen for ubiquitylation and presumably proteasome-mediated degradation. This regulation depends on the interaction between a peptide fragment of Dronc and the conserved groove on DIAP1-BIR2. DIAP1 exists in an auto-inhibited conformation. Caspase-mediated cleavage of DIAP1 after Asp20 disables auto-inhibition, allowing the resulting DIAP1 fragment to bind to and inhibit active drICE. During apoptosis, the RHG proteins use their N-terminal peptides to compete with drICE and Dronc for binding to the conserved peptide-binding grooves on the BIR1 and BIR2 domains, respectively. Such competition results in the release of drICE and Dronc from DIAP1. The freed Dronc zymogen is activated by the Dark apoptosome and the mature Dronc cleaves and activates drICE. In this regard, drICE, DIAP1 and the RHG proteins together provide a fail-safe mechanism to ensure appropriate drICE activation only under bona fide apoptotic conditions (Li, 2011).

The underpinning of this regulatory network is competition among multiple protein-protein interactions mediated by the conserved grooves of the BIR1 and BIR2 domains of DIAP1. Auto-inhibition of DIAP1-BIR1 is achieved by occupation of this groove by its own N-terminal sequence ASVV. The free peptide ASVV does not stably associate with BIR1; the covalent linkage facilitates the binding by increasing the local concentration of ASVV. This binding arrangement allows disabling of auto-inhibition upon cleavage of DIAP1 after Asp20. Inhibition of drICE by BIR1 requires occupation of this groove by the N-terminal sequences ALGS of drICE. Similar to ASVV, the free ALGS peptide exhibited no detectable binding to the BIR1 fragment. Three weak interfaces between BIR1 and drICE cooperate to yield a stable hetero-tetramer with a KD of approximately 1-2 microM. Removal of drICE inhibition by BIR1 depends on the interactions between RHG and the peptide-binding groove, with KD values of 0.12-0.76 microM. Endowing RHG with the strongest interactions ensures an apoptotic phenotype once the RHG proteins are activated in cells. It is acknowledged that the proposed model may be simplistic, as the network of protein-protein interactions and regulation is likely to be more complex in vivo. Nevertheless, the biophysical underpinnings described in this model are likely to have a role in various stages of apoptosis regulation (Li, 2011).

An IAP-binding motif, though not ostensibly abbreviated as IBM, was originally defined to contain four contiguous amino acids that resemble the Smac tetrapeptide AVPI. This structurally defined motif, with binding affinities of 0.1-1 microM, has stringent requirement for the first (P1), third (P3) and fourth (P4) amino acids. The P1 residue must be Ala, which binds to a small hydrophobic pocket on one end of the conserved groove on BIR domain. The P4 residue must be hydrophobic, preferably bulky, to occupy a greasy pocket on the other end of the groove. Deletion of P1 or P4 in a tetrapeptide results in abrogation of stable interaction with the BIR domain. The P3 residue is either Pro or Ala. Pro as P3, with its unique backbone configuration, optimizes simultaneous binding by both P1 and P4 residues for DIAP1-BIR2 or XIAP-BIR3. Ala as P3 can be better accommodated by DIAP1-BIR1 and XIAP-BIR2. In recent studies1, IBM was redefined to contain three contiguous amino acids, with P3 no longer restricted to Pro or Ala. Such tripeptides, ALG/AKG for drICE/Dcp1, or their longer variants, ALGS/AKGC for drICE/Dcp1, do not meet the structural criteria for IBM. Importantly, these peptides in isolation do not form a stable complex with any BIR domain. The definition of such motifs as IBMs insinuates the incorrect assumption that such free peptide motifs may stably interact with the BIR domain. This assumption, in turn, has engendered ample confusion in data interpretation in recent years (Li, 2011).

An IBM at the N-terminus of the caspase-9 small subunit recognizes a conserved surface groove on XIAP-BIR3; this interaction locks caspase-9 in the inhibited state. During apoptosis, Smac/Diablo uses a similar tetrapeptide motif to occupy the BIR3 groove, hence releasing caspase-9 and relieving XIAP-mediated inhibition. Caspase-3 or -7 is inhibited by an 18-residue peptide segment preceding the BIR2 domain of XIAP. Because both caspase-3 and drICE are inhibited directly at the active sites by XIAP and DIAP1, respectively, the overall appearance of the two BIR-caspase complexes is similar. It should be noted, however, that the essential interactions and key features are quite different. For example, caspase-3 or -7 can be inhibited by an isolated 18-residue peptide fused to GST; but the intact BIR1 domain of DIAP1 is absolutely required for drICE inhibition. The orientation of the BIR domain relative to the caspase is different by approximately 90 degrees between caspase-3/BIR2 and drICE/BIR1. Importantly, the peptide-binding groove of XIAP-BIR2 does not have an apparent role in the inhibition of caspase-3 and -7 (Li, 2011).

Using purified, recombinant proteins, this study showed that the BIR1 domain of DIAP1 only forms a stable complex with active drICE following caspase-mediated cleavage of DIAP1 after Asp20. The uncleaved DIAP1-BIR1 exhibited very weak binding to drICE. These observations contrast the report that both uncleaved and cleaved DIAP1-BIR1 bound to drICE similarly using coimmunoprecipitation. The cleaved DIAP1, but not the uncleaved DIAP1, potently inhibits the proteolytic activity of drICE towards both peptide and protein substrates. These observations unambiguously demonstrate that drICE sequestered by cleaved DIAP1 remains catalytically inactive. In fact, the conclusion that DIAP1-sequestered drICE was catalytically active, contradicted the biochemical observation that no protease activity was detectable towards peptide or protein substrate21. It is not uncommon for a substrate to be converted into a protease inhibitor upon cleavage, as exemplified by the pan-caspase inhibitor p35 (Li, 2011).

The key question is not whether BIR1 inhibits drICE, but why BIR1-sequestered drICE continues to exhibit proteolytic activity towards the uncleaved DIAP1. A time course analysis of DIAP1 cleavage shows that the protease activity of drICE was slowed down considerably over time, as the concentration of inhibitor -- cleaved DIAP1 -- increased. The level of drICE activity at the 15-minute time point was at least 6-fold higher than that at 90-minute point. This observation again illustrates that the cleaved DIAP1 is a bona fide inhibitor of drICE (Li, 2011).

Some of the contrasting claims about the regulation of drICE by DIAP1 might be attributable to the limitations of the investigative methods. Biochemical and biophysical investigations, employing homogeneous, recombinant proteins, usually provide mechanistic answers to questions that pertain to protein-protein interactions and enzyme activities. The caveat, however, is whether such observations are biologically relevant, and if yes, to what extent these findings are important. By contrast, investigation by cellular biochemistry, exemplified by coimmunoprecipitation, provides important clues to molecular mechanisms. For both approaches, caution must be exercised for the interpretation of results. Notably, in some cases, the contrasting claims can be reconciled by a complex system. For example, despite structural data demonstrating that the auto-inhibition of DIAP1 involves the binding of its N-terminal sequences to the BIR1 domain, it remains theoretically possible that additional interactions between the N- and C-terminal domains of DIAP1 may contribute to its auto-inhibition1. The best example is Apaf-1, whose auto-inhibition entails two elements: one by the C-terminal WD40 repeats and the other within the N-terminal half. Binding to cytochrome c relieves the auto-inhibition by the WD40 repeats31, and exchange of ADP for ATP defeats the auto-inhibition imposed by intra-domain interactions within the N-terminal-half (Li, 2011).

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. This study reports the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which was named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation (Yang, 2013).

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. This study demonstrates that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. Loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, these data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway (Huang, 2014).

Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis

The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts (Santhanam, 2014).

This study shows that ecdysone-induced expression of PTP52F and the subsequent tyrosine dephosphorylation of TER94 coordinate to construct upstream signaling determinants for a precise time-dependent degradation of larval midgut. The transient expression of Ptp52F gene at the PF stage is regulated by the functional EcR. Immediately after the level of endogenous PTP52F protein is detectable in larval midgut, TER94 becomes dephosphorylated on its Y800 residue. This modification may be critical to the rapid degradation of ubiquitylated proteins through a TER94-dependent regulation of ubiquitin proteasome system (UPS). Although the exact mechanism remains elusive, recent studies have suggested that only the tyrosine-dephosphorylated form and not the tyrosine-phosphorylated form of VCP interacts with cofactors for processing ubiquitylated substrates of UPS. Because VCP and TER94 share some evolutionarily conserved features, it is proposed that the same phosphorylation- and dephosphorylation-dependent mechanism may be adopted by TER94. Ubiquitylated DIAP1, a potential substrate of UPS, was found to be targeted by the Y800 dephosphorylated form of TER94. DIAP1 was rapidly degraded in cells in which levels of PTP52F were increased, as illustrated by in vivo observations in Drosophila midgut during metamorphosis. Consequently, the proteolysis of DIAP1 in response to inducible expression of PTP52F terminates the inhibitory effect on autophagy, allowing the initiation of autophagic cell death accompanied by apoptotic cell death for the destruction of the larval midgut tissues. Since the regulatory role of all Drosophila homologs of caspases have been ruled out in the process of larval midgut histolysis, it is likely that DIAP1 degradation-induced autophagic signaling may activate a yet-unknown pathway leading to the onset of apoptotic cell death in dying midgut. Additional experiments are needed to identify downstream effectors of PTP52F that modulate the cross talk between autophagy and apoptosis in the context of midgut maturation (Santhanam, 2014).

Identification of TER94 as a substrate dephosphorylated by PTP52F in larval midgut is interesting and important. From the time of their original cloning and identification, Drosophila TER94 and its vertebrate ortholog VCP have been characterized as key mediators involved in ER-associated degradation (ERAD), a major quality control process in the protein secretary pathway. Additional investigations have demonstrated degradation of proteins with no obvious relationship to ERAD by a VCP-mediated process, suggesting that TER94 and VCP may perform general functions in the proteolysis of ubiquitylated proteins. However, it remains unknown how this process is regulated under physiological conditions. The current study presents evidence that TER94-dependent degradation of ubiquitylated proteins is enhanced by PTP52F-mediated dephosphorylation of the penultimate Y800 residue. It has been suggested that the penultimate tyrosine (Y805 in VCP and Y800 in TER94) must be in a dephosphorylated form in order to interact with substrate-processing cofactors, such as the peptides N-glycanase (PNGase) and Ufd3, during UPS-mediated proteolysis. In addition, tyrosine phosphorylation levels of VCP/TER94 determine how fast ubiquitylated proteins are degraded by the USP pathway. Clearly, the finding that PTP52F is responsible for dephosphorylation of the penultimate tyrosine residue is critical for uncovering the functional role of VCP/TER94 in the regulation of protein degradation under physiologically relevant conditions (Santhanam, 2014).

This study has demonstrated that the timely degradation of DIAP1 in doomed larval midgut of developing flies is regulated by ecdysone-induced PTP52F. DIAP1 was identified to ubiquitylate proapoptotic proteins in living cells, thereby suppressing cell death signaling. Interestingly, DIAP1 can be ubiquitylated for degradation itself. The proteolytic process of ubiquitylated DIAP1 remained unclear until a recent report suggesting that TER94-mediated UPS pathway is involved in this process. This study has further shown that it is the dephosphorylated form of TER94 that is responsible for rapid DIAP1 degradation. In addition, although a previous study suggested that DIAP1 might suppress Atg1-mediated PCD, it was not known whether degradation of ubiquitylated DIAP1 could promote autophagy in vivo. This study has explored the underlying mechanism through which autophagic cell death is initiated by degradation of DIAP1. The data show that the constitutively tyrosine-phosphorylated form of TER94 acts as a gatekeeper ensuring the death signaling downstream of DIAP1 in'switch-off' mode. Developmental stage-dependent dephosphorylation of TER94 by inducible expression of PTP52F converts the autophagic death signaling into 'switch-on' mode through degradation of DIAP1. These findings thus explain, at least in part, how the massive destruction of larval midgut is precisely controlled by autophagic cell death. In conclusion, this study shows a novel function of PTP52F involved in the onset of autophagy and apoptosis essential for the removal of obsolete midgut tissues. Reversible tyrosine phosphorylation signaling controlled by PTP52F plays an indispensable role in the process of cell death-directed midgut maturation. Therefore, these findings open a new avenue for understanding the previously unexplored function of R-PTPs linked to regulation of autophagic and apoptotic cell death (Santhanam, 2014).

Insect Inhibitor of Apoptosis IAPs are negatively regulated by signal-induced N-terminal degrons absent within viral IAPs

Inhibitor of apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. This study reports that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the lifespan of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independently of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, these findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs, not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover (Vandergaast, 2015).


thread: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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