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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 th^j5c8, 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 th^j5C8 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).

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).

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 selD^ptuf) can serve as a tool to understand the link between ROS accumulation and cell death. To this aim, the mechanism by which selD^ptuf mutant cells become apoptotic was analyzed in Drosophila imaginal discs. The apoptotic effect of selD^ptuf 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 selD^ptuf mutant cells to death. Moreover, the ectopic expression of the inhibitor of apoptosis DIAP1 rescues the cellular viability of selD^ptuf mutant cells. These observations indicate that selD^ptuf 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 selD^ptuf, is a recessive null mutation and homozygous individuals have extremely reduced and abnormal imaginal discs and die as third instar larvae. Heterozygous selD^ptuf flies are apparently wild type, however, a downregulation of the Ras/MAPK pathway has been observed in transheterozygous combinations of the selD^ptuf 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 selD^ptuf 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 selD^ptuf mutant cells and that ROS increase plays an important role in selD^ptuf 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 selD^ptuf 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 selD^ptuf 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 selD^ptuf 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 DmIKKε^G19R, DmIKKε^D160N, DmIKKε^G250D, 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, NAK^K41M, 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).

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