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 (Wu, 2003) were examined. Elevated DIAP1 protein is detected in yki-overexpressing clones in the eye discs. This regulation is largely mediated at the level of diap1 transcription since the expression of thj5c8, a P[lacZ] enhancer trap reporter inserted into the diap1 locus, is similarly elevated in yki-overexpressing clones in a cell-autonomous manner. A cycE-lacZ reporter containing 16.4 kb of the 5′ regulatory sequence of cycE is also increased in yki-overexpressing clones, especially those close to the MF, although the effect is less profound than that observed with the diap1 reporter. Thus, like loss of hpo, sav, or wts, overexpression of yki results in increased transcription of diap1 and cycE. It is worth noting that previous analyses of hpo mutant clones also revealed a 'tighter' regulation of diap1: while diap1 transcription is elevated in all hpo mutant cells irrespective of their relative position to the MF, cycE transcription is only elevated in hpo mutant cells close to the MF (Wu, 2003). These observations suggest that diap1 might represent a more direct transcriptional target of the Hpo pathway (Huang, 2005).
The results are consistent with a model wherein Yki acts antagonistically to Hpo, Sav, and Wts in a common signaling pathway that coordinately controls cell proliferation and apoptosis. Based on the physical interactions between Yki and Wts, and given that YAP, the mammalian homolog of Yki, is known to function as a transcriptional coactivator (Yagi, 1999; Strano, 2001; Vassilev, 2001), it was further hypothesized that Yki functions downstream of Wts to regulate transcription of genes such as diap1 and that the Hpo pathway negatively regulates the coactivator activity of Yki (Huang, 2005).
To test the hypothesis that the coactivator activity of Yki is negatively regulated by the Hpo pathway, a transcription assay was established for Yki activity in Drosophila S2 cells. Since the cognate transcription factor(s) that partner with Yki are not yet identified, Yki was fused to the DNA binding domain (DB) of the yeast Gal4 transcription factor. The activity of this fusion construct was then assayed using a Gal4-responsive reporter. Consistent with previous reports of YAP as a transcriptional coactivator in mammalian cells, the Gal4DB-Yki fusion protein exhibited potent transcriptional activation. Strikingly, transcriptional activity of the Gal4DB-Yki fusion was abolished when Hpo, Sav, and Wts plasmids were coexpressed. This effect is specific to Yki since activity of the full-length Gal4 (with its own activation domain) was unaffected by the coexpression of Hpo, Sav, and Wts. These results suggest that the Hpo pathway negatively regulates the coactivator activity of Yki (Huang, 2005).
To further probe a functional link between yki and the Hpo pathway, their genetic interactions were investigated. While expression of hpo or wts directly from the GMR promoter results in viable flies with rough or slightly rough eyes, respectively, cointroduction of GMR-hpo and GMR-wts into the same animals results in 100% lethality at early pupal stage (Wu, 2003). Strikingly, such lethality is completely rescued by coexpression of yki from a GMR-yki transgene. Interestingly, this lethality is also completely rescued by coexpression of the human YAP gene. In another line of experiments, advantage was taken of the complete pupal lethality caused by the overexpression of UAS-hpo driven by the GMR-Gal4 driver. Interestingly, this lethality is also rescued by the coexpression of yki (100% rescue) or YAP (21% rescue). Taken together, these genetic interactions further support the model that Yki acts antagonistically to Hpo, Sav, and Wts in a common signaling pathway. The ability of a human YAP transgene to rescue the lethality of flies caused by Hpo pathway hyperactivation reveals a functional conservation between Yki and YAP, suggesting that YAP might play a similar role in mammalian growth control (Huang, 2005).
The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Hippo signaling restricts tissue size by promoting apoptosis and cell-cycle arrest, and animals carrying clones of cells mutant for hippo develop severely overgrown adult structures. The Hippo pathway is thought to exert its effects by modulating gene expression through the phosphorylation of the transcriptional coactivator Yorkie. However, how Yorkie regulates growth, and thus the identities of downstream target genes that mediate the effects of Hippo signaling, are largely unknown. This study reports that the bantam microRNA is a downstream target of the Hippo signaling pathway. In common with Hippo signaling, the bantam microRNA controls tissue size by regulating cell proliferation and apoptosis. hippo mutant cells had elevated levels of bantam activity; and bantam is required for Yorkie-driven overgrowth. Additionally, overexpression of bantam is sufficient to rescue growth defects of yorkie mutant cells and to suppress the cell death induced by Hippo hyperactivation. Hippo regulates bantam independently of cyclin E and diap1, two other Hippo targets, and overexpression of bantam mimics overgrowth phenotypes of hippo mutant cells. These data indicate that bantam is an essential target of the Hippo signaling pathway to regulate cell proliferation, cell death, and thus tissue size (Nolo, 2006).
To test whether the activity of the bantam miRNA is regulated by Hpo signaling, use was made of a GFP bantam sensor that reports the spatial activity of bantam. This bantam sensor expresses GFP under the control of a ubiquitously active tubulin promoter and has two perfect bantam target sites in its 3′ UTR. When present, the bantam miRNA reduces GFP expression through its RNAi effect. The expression pattern of GFP is thus a negative image of the activity pattern of the bantam miRNA. In third-instar wing imaginal discs, the bantam sensor is expressed in a complex pattern with higher levels along the presumptive wing margin, in the anterior compartment along the anteroposterior compartment boundary, and in several patches in the thorax region. Overexpression of the bantam miRNA in the developing wing eliminated the GFP expression of the bantam sensor in the corresponding region, demonstrating that the expression of GFP is indeed under the control of bantam. In developing eye discs, the bantam sensor is also broadly expressed, with higher levels in differentiating photoreceptor cells. As in wing discs, overexpression of bantam downregulated GFP expression in eye discs. The bantam sensor thus reflects the activity of the bantam miRNA in eye and wing discs (Nolo, 2006).
To address whether Hpo signaling regulates the activity of the bantam miRNA, GFP expression of the bantam sensor was monitored in imaginal discs that had defects in Hpo signaling. It was found that hpo or wts mutant cells had lower levels of bantam-sensor-driven GFP expression throughout the mutant clones. Significantly, hpo and wts mutant clones showed lower levels of GFP in multiple tissues, including the wing, antenna, and eye imaginal discs. In eye imaginal discs, wts clones affected the bantam sensor anterior to the morphogenetic furrow, where cells are still uncommitted as well as posterior to the furrow in differentiating photoreceptor cells. In all cases, the regulation of the bantam sensor was cell autonomous. In addition, wing imaginal discs that overexpressed Yki had lower levels of bantam sensor expression in the entire region of Yki overexpression. In summary, it is concluded that Hpo signaling generally regulates bantam expression in multiple imaginal discs and cell types (Nolo, 2006).
A model is postulated in which bantam is an essential target of the Hpo signaling pathway to regulate cell proliferation, cell death, and thus tissue size. This model is based on several observations. First, it was found that bantam is regulated by Hpo signaling broadly and in various tissues. This regulation is a specific downstream effect of Hpo signaling and is not simply the consequence of the cell proliferation induced in hpo mutant cells. Second, bantam is required for Yki to drive tissue overgrowth, because removal of bantam suppresses the overgrowth phenotypes caused by overexpression of Yki in the retina. Third, overexpression of bantam rescues the cell death induced by overexpressed Hpo and significantly rescues growth defects of yki mutant cells. And fourth, bantam overexpression mimics the phenotypes of hypomorphic hpo mutations. Taken together, these data support a model in which bantam is an important downstream target of the Hpo pathway (Nolo, 2006).
The finding that Hpo signaling regulates the expression of bantam raises the question of how important this effect is for Hpo signaling to control tissue size. Removal of bantam suppresses the induction of extra interommatidial cells in the retina by Yki overexpression but does not cause a general elimination of retinal cells in a wild-type background. These data indicate that the regulation of bantam is an essential downstream effect of Hpo signaling to regulate tissue size. However, loss of bantam only partially suppresses the effects of Yki overexpression, indicating that Yki regulates other targets in addition to bantam. Hpo was found to regulate bantam independently of cyclin E and diap1, two other genes known to be regulated by Hpo signaling. bantam is thus not a component of the Hpo signal transduction pathway itself, but is one of several downstream target genes. Yki must have targets in addition to bantam, cyclin E, and diap1, because overexpression of bantam, Cyclin E, and DIAP1 together did not induce the amount of overgrowth caused by Yki overexpression in wing discs. Nevertheless, overexpression of bantam alone caused phenotypes resembling hypomorphic situations for Hpo signaling, indicating that bantam is a critical mediator of Hpo function. Whether the regulation of bantam by a Yki-containing transcription factor complex is direct remains to be determined. However, the fact that Hpo regulates bantam cell autonomously and in multiple tissues is consistent with such a model (Nolo, 2006).
bantam expression is spatially modulated, and patterning signals such as Wg and Dpp also regulate the expression of bantam to generate its expression pattern. These patterning signals regulate specific aspects of the bantam expression pattern, and they have different effects on cell proliferation as well as bantam activity in different regions in various imaginal discs. In contrast, hpo mutant cells upregulate bantam activity independently of cell type and in multiple imaginal discs, indicating an intimate relationship. Hpo is thus a more general and ubiquitous regulator of bantam expression in imaginal discs. An important question that remains to be answered is how these patterning signals regulate tissue growth and bantam expression and whether they regulate bantam expression directly and independently of Hpo signaling or through the regulation of Hpo activity (Nolo, 2006).
Surprisingly, just the opposite of hpo mutant cells, TSC1 mutant cells had lower levels of bantam activity although these cells overgrow, indicating that TSC1 mutant cells induce growth independently of bantam. Neither Myc, Ras, nor Cyclin D-Cdk4 expression induced bantam, although they induce cell growth and proliferation. bantam is thus not simply a part of the cell-intrinsic machinery that executes cell growth and division but rather acts as an upstream component to instruct cells to proliferate. In summary, although Hpo is a key regulator of bantam expression, bantam is also regulated by other pathways potentially integrating the effects of several growth-regulatory and patterning pathways (Nolo, 2006).
miRNAs and their target genes often show mutually exclusive expression patterns, and miRNAs induced during differentiation tend to target messages that were abundant in the previous developmental stage. miRNAs may thus provide a rapid and effective means to suppress expression of residual, unwanted mRNAs while the transcriptional program in a cell is changing. Hpo signaling is involved in regulating cell proliferation and apoptosis in developing imaginal discs. Cell lineages and cell proliferation show significant plasticity in growing imaginal discs, which can rapidly respond to surgical ablation or genetic insults by regenerating missing (eliminated) cells or by ablating unwanted (extra) cells. This adjustment of cell proliferation and apoptosis requires a mechanism that can rapidly change the growth properties of a cell. Yki appears to regulate cell number on the one hand by inducing the expression of positive regulators of cell proliferation and cell survival and on the other hand by inducing the expression of bantam, which posttranscriptionally suppresses the expression of proteins that inhibit cell proliferation and induce apoptosis. An example of such cooperative action of Yki and bantam is the regulation of Hid: Yki suppresses the expression of hid, but also induces bantam, which then suppresses the translation of hid mRNAs that may still be present in a cell. The induction of bantam by Yki may also accelerate the repression of negative growth regulators, thereby enabling a cell to more quickly and robustly adjust its rate of cell proliferation. It will be interesting to elucidate how bantam regulates growth and how its growth targets are integrated with other targets of Hpo signaling (Nolo, 2006).
The Hippo signaling pathway acts upon the Yorkie transcriptional activator to control tissue growth in Drosophila. Activated Yorkie drives growth by stimulating cell proliferation and inhibiting apoptosis, but how it achieves this is not understood. Yorkie is known to activate Cyclin E (CycE) and the apoptosis inhibitor DIAP1. However, overexpression of these targets is not sufficient to cause tissue overgrowth. This study shows that Yorkie also activates expression of the bantam microRNA, a known regulator of both proliferation and apoptosis. bantam overexpression mimics Yorkie activation while loss of bantam function slows the rate of cell proliferation. bantam is necessary for Yorkie-induced overproliferation and bantam overexpression is sufficient to rescue survival and proliferation of yorkie mutant cells. Finally, bantam levels are shown to be regulated during both developmentally programmed proliferation arrest and apoptosis. In summary, the results show that the Hippo pathway regulates expression of bantam to control tissue growth in Drosophila (Thompson, 2006).
The Hippo pathway is unique in its direct and dedicated role in the intrinsic program of growth in proliferating tissues. The potency of the Hippo pathway in driving tissue growth appears to reside in its ability to coordinately stimulate cell proliferation and suppress apoptosis. A key goal is to understand how this coordinate control is achieved. The results show that the bantam microRNA, a known regulator of both cell proliferation and apoptosis, is a critical target of the Hippo pathway. Activated Yki is necessary and sufficient to induce bantam expression and to stimulate cell survival and proliferation. bantam appears to be a key target of Yki because loss of Yki can be rescued by overexpression of bantam. Finally, bantam clearly has an important role in both normal growth and Yki-driven overgrowth because loss of bantam strongly reduces the rate of cell proliferation in either case. Although the bantam microRNA appears not to be conserved in vertebrates, it is possible that other microRNAs play a functionally equivalent role as effectors of the Hippo pathway. Recent work has identified human microRNAs involved in this pathway (Thompson, 2006).
Two lines of evidence indicate that bantam is not the only relevant target of the Hippo pathway. Firstly, loss of bantam does not completely mimic loss of Yki in every respect, because bantam mutant cells do not undergo apoptosis. This difference is likely to reflect the contribution of the Yki target DIAP1, whose absence is known to trigger apoptosis. Secondly, Yki retains some ability to stimulate cell proliferation even in the absence of bantam. Again, this activity may reflect the role of other Yki targets, including CycE, in driving cell proliferation. Thus, the results favor the view that bantam acts in a highly cooperative way with other Yki target genes to mediate the effects of the Hippo pathway on cell proliferation and apoptosis (Thompson, 2006).
The expression of bantam during normal development shows a striking pattern of regulation; it is expressed in proliferating cells but not in quiescent cells or, as has been shown in this work, in certain cells destined for apoptosis. These findings indicate that regulation of bantam is a key feature of the normal program of tissue growth. Previous work has shown that high levels of the Wingless (Wnt) morphogen represses bantam as cells arrest proliferation at the presumptive wing margin. Since the results show that the Hippo pathway regulates bantam, the pattern of bantam expression may reflect regulation of Hippo pathway activity by positional signals. Thus, positional signals could determine the behavior of cells along the spectrum from rapid proliferation to apoptosis simply by controlling the Hippo pathway. Alternatively, positional signals and the Hippo pathway may act independently, with the bantam locus being a regulatory nexus that integrates information from a number of different signaling pathways (Thompson, 2006).
The final finding is that the Hippo pathway also influences epithelial morphogenesis. This function appears to be independent of its role in controlling cell survival and proliferation and does not involve bantam. Interestingly, expression of Yki and Hippo have reciprocal effects on the epithelium, with overexpressed Yki driving apical bulging and overexpressed Hippo causing basal outfolding. In both cases, the cells remain epithelial, indicating that the Hippo pathway controls cell shape without affecting epithelial polarity or integrity. These observations are consistent with previous reports that clones of cells with elevated pathway activity (i.e. mutant for Hippo or other negatively acting components) have a rounded appearance, indicating altered cell affinities, and that mutation of warts also causes apical bulging of epithelia that is attributed to an expanded apical membrane domain. Why cells use the same pathway to control survival, proliferation, and shape remains an intriguing question (Thompson, 2006).
A fuller understanding of the network connecting the Hippo pathway with the basic machinery controlling survival, proliferation, and morphology will be needed to understand how size regulation is connected to pattern formation during normal development. This work allows sketching of the outline of one facet of this network, with the Yki targets bantam, CycE, and DIAP1 cooperating to control survival and proliferation (Thompson, 2006).
Studies of the Hpo signaling pathway placed Wts as the most downstream component among Hpo, Sav and Wts. In an effort to extend this pathway further downstream, a yeast two-hybrid screen was carried out for Wts binding proteins. Using the noncatalytic N-terminal portion of Wts (1-608) as bait and from 1 million cDNA clones, three independent clones were isolated representing partial sequences of a gene annotated as CG4005 by the Berkeley Drosophila Genome Project. This gene was named yorkie (yki) after Yorkshire Terriers, one of the world’s smallest breeds of pet dogs, according to its loss-of-function phenotype. Consistent with the yeast two-hybrid results, Wts and Yki coimmunoprecipitate with each other in Drosophila S2 cells (Huang, 2005).
The three independent Wts-interacting clones isolated from the yeast two-hybrid screen define the C-terminal half of Yki (residues 229-418) as a Wts binding region. This region contains the two predicted WW domains, suggesting that the WW domains are required for Yki-Wts binding. Consistent with this hypothesis, mutating two critical residues of the WW domains abolishes the binding between Yki and Wts. Likewise, the N-terminal half of the Yki protein, which does not contain the WW domains, did not bind to Wts in the same assay. Thus, the WW domains of Yki are required for its interaction with Wts (Huang, 2005).
Given the direct interaction between Yki and Wts and that Wts encodes a protein kinase, it was hypothesized that Yki is regulated by the Hpo pathway through Wts-mediated phosphorylation. To test this possibility, phosphorylation of Yki by the Hpo pathway was tested using an S2 cell-based assay. Coexpression of Wts and Yki results in a small mobility retardation of Yki. Coexpression of Hpo-Sav with Yki also results in a mobility shift of Yki, and coexpression of Hpo-Sav-Wts results in an even greater mobility shift of Yki. The mobility shift of Yki induced by Hpo-Sav-Wts expression was abrogated by phosphatase treatment, demonstrating that this shift is due to protein phosphorylation. It is worth noting that the increasing phosphosphorylation of Yki induced by Wts, Hpo-Sav, and Hpo-Sav-Wts in the S2 cell assay correlates with the severity of the overexpression phenotype caused by the respective transgenes in vivo: expression of Wts by the GMR promoter results in slightly rough eyes; expression of Hpo-Sav results in strong rough eyes with reduced size, and expression of Hpo-Sav-Wts results in complete animal lethality. These results suggest that Yki phosphorylation is a relevant output of the Hpo signaling pathway (Huang, 2005).
To determine whether Yki is a direct substrate of Wts, in vitro kinase assays were performed. When expressed alone, Wts shows little kinase activity on Yki. When coexpressed with Hpo-Sav, however, Wts displays specific kinase activity on Yki but not a control substrate. Moreover, a kinase-dead mutation of Wts completely abolishes the in vitro kinase activity of Wts toward Yki. These data confirm that Yki is a kinase substrate of Wts. Furthermore, the observation that Hpo-Sav coexpression stimulates the kinase activity of Wts on Yki is consistent with the activation of Wts by Hpo-Sav as measured by the phosphorylation status of Wts (Huang, 2005).
If Hpo-Sav activates Wts, which in turn phosphorylates Yki, one would predict that the mobility shift of Yki induced by transfected Hpo-Sav or Wts in the S2 cell assay might require the endogenous Wts or Hpo, respectively. Indeed, RNAi of wts completely reverses the mobility shift of Yki induced by Hpo-Sav expression, and RNAi of hpo completely reverses the mobility shift of Yki induced by Wts expression. These data further support the model that Yki is phosphorylated by Wts upon activation of the Hpo pathway (Huang, 2005).
yki is genetically epistatic to hpo, sav, and wts. The genetic evidence presented so far suggests that yki acts antagonistically to hpo, sav, and wts. Biochemical studies further refined this model and demonstrate that Yki is phosphorylated and inactivated by the Hpo pathway via Wts-mediated phosphorylation. A prediction of this model is that loss-of-function mutations of yki should be genetically epistatic to those of hpo, sav, or wts. To test this hypothesis, clones of cells were generated that were doubly mutant for hpo-yki, sav-yki, or wts-yki. While loss of hpo, sav, or wts results in increased diap1 transcription and overgrowth (Wu, 2003), hpo-yki, sav-yki, or wts-yki double mutant clones display phenotypes indistinguishable from those of yki mutant clones, including retarded growth, decreased DIAP1 protein levels, and decreased diap1 transcription. These genetic observations further strengthen the molecular model implicating Yki as a target of Wts in the Hpo pathway (Huang, 2005).
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).
Coordination of cell proliferation and cell death is essential to attain proper organ size during development and for maintaining tissue homeostasis throughout postnatal life. In Drosophila, these two processes are orchestrated by the Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yorkie (Yki). This study demonstrates that a single phosphorylation site in Yki mediates the growth-suppressive output of the Hippo pathway. Hippo-mediated phosphorylation inactivates Yki by excluding it from the nucleus, whereas loss of Hippo signaling leads to nuclear accumulation and therefore increased Yki activity. A mammalian Hippo signaling pathway has been delimited that culminates in the phosphorylation of YAP, the mammalian homolog of Yki. Using a conditional YAP transgenic mouse model, it has been demonstrated that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results uncover a universal size-control mechanism in metazoan (Dong, 2007).
This study provides several lines of evidence demonstrating that Hippo signaling antagonizes Yki function by changing its subcellular localization. Hippo signaling promotes Yki cytoplasmic localization in cultured Drosophila cells, and accordingly, loss of Hippo signaling promotes nuclear accumulation of Yki in imaginal discs. This is further supported by the ability of phosphorylated Yki (but not unphosphorylated Yki) to bind to 14-3-3 proteins, which are known to promote the cytoplasmic shuttling of other transcription factors in a phosphorylation-dependent manner. Importantly, S168 was identified as a primary Hippo-responsive phosphorylation site on Yki both in vitro and in vivo: the S168A mutation not only abrogates Hippo-induced Yki phosphorylation and cytoplasmic shuttling in S2 cells but, more significantly, causes constitutive Yki activation in developing tissues. These results demonstrate that S168 mediates the growth-suppressive output of the Hippo signaling pathway (Dong, 2007).
Despite the presence of mammalian homologs for all the known components of the Drosophila Hippo pathway (Mst1/2 for Hpo, WW45 for Sav, Lats1/2 for Wts, and YAP for Yki), previous studies in mammals have failed to unite these proteins in a physiologically relevant signaling cascade. The conservation of of the S168 phosphorylation site in mammalian YAP provides the first opportunity to functionally link Mst1/2, WW45, and Lats1/2 in a single kinase cascade that culminates in YAP S127 phosphorylation. The mammalian Hippo signaling pathway antagonizes YAP function by promoting its cytoplasmic localization in a S127 phosphorylation-dependent manner. The identification of S168/S127 as Wts/Lats-mediated phosphorylation site in Yki/YAP is rather unexpected given that previous studies have implicated this residue as an Akt phosphorylation site. The observation that both YkiS168A and YAPS127A result in a loss-of-Wts rather than a loss-of-Akt phenotype in Drosophila strongly suggests that this site is regulated by the Hippo pathway rather than Akt under normal physiological conditions (Dong, 2007).
The identification of a single phosphorylation site as the functional output of the Hippo pathway, and the constitutive active Yki/YAP mutants described in this study, will greatly facilitate future investigation of this important size-control pathway in multiple species. For example, the constitutive active Yki/YAP mutants can be conveniently used to modulate the Hippo pathway in animal models and in genetic epistasis studies to characterize new components of the pathway; the phospho-Yki/YAP antibodies should provide a sensitive assay to link a specific protein to the Hippo pathway. These tools are especially important for the mammalian system, where a functional readout of the Hippo pathway has so far been unavailable. Indeed, this study placed hWW45 in the mammalian Hippo pathway using phospho-YAP as a convenient readout (Dong, 2007).
Despite the conservation of many Hippo pathway components between flies to mammals, previous studies have not revealed a direct role for this pathway in mammalian organ size control. Several recent studies have focused on their involvement in tumorigenesis. For example, YAP was recently shown to transform immortalized mammary epithelial cells in vitro and to accelerate tumorigenesis in conjunction with p53 loss and c-myc overexpression. While suggestive of an involvement of the Hippo pathway in mammalian tumorigenesis, these observations alone do not necessarily prove a direct requirement for the Hippo pathway in the control of organ size, since perturbations of many cellular processes in addition to growth control can contribute to tumorigenesis. It is worth noting that knockout mice have been generated for several components of the mammalian Hippo pathway. However, these mice are either viable, lacking any overt overgrowth characteristic of the respective Drosophila mutants (e.g., Lats1), or embryonic lethal, thus preventing a critical assessment of their involvement in organ size regulation (e.g., Lats2 and YAP (Dong, 2007).
The identification of YAP as the nuclear effector of the mammalian Hippo pathway provides a powerful tool to manipulate this pathway in mammals, in much the same way that Yki overexpression recapitulates loss of Hippo signaling in Drosophila. By manipulating YAP activity in a conditional and tissue-specific manner, this study demonstrates that modulating Hippo pathway activity is sufficient to cause a rapid and reversible change of organ size (up to 500%), therefore offering the first direct evidence implicating the Hippo pathway in mammalian organ size control. It is further demonstrated that, like its Drosophila counterpart, the mammalian Hippo pathway coordinately regulates both cell proliferation and apoptosis. The dual function of YAP in promoting cell proliferation and suppressing apoptosis distinguishes it from a conventional oncogene such as c-myc, whose mitogenic activity is coupled with a proapoptotic activity. It is suggested that this dual activity in promoting cell proliferation and suppressing apoptosis underlies the rapid and uniform expansion of liver mass in the ApoE/rtTA-YAP mice. The ability of YAP to induce organomegaly in postnatal mice is consistent with the notion that the Hippo pathway not only controls organ size during development as demonstrated in Drosophila but also regulates tissue homeostasis in postnatal life (Dong, 2007).
Initially isolated as a yes-associated protein, YAP has since been reported to bind to a large number of proteins in cultured mammalian cells, including EBP50, Smad7, ErbB4, p53BP-2, p73, and hnRNAP U, as well as Runt and TEAD transcription factors. However, it has been difficult to ascertain whether any of these binding partners mediate YAP function in vivo. The antiapoptotic activity observed in the transgenic mouse liver is clearly distinct from the reported ability of YAP to potentiate p73-mediated apoptosis in response to DNA damage in cultured mammalian cells. Given that p73-deficient mice are viable while YAP-deficient mice die at embryonic day 8.5, p73 is unlikely to be a critical partner for YAP in mouse development (Dong, 2007).
Studies from both insects and mammals support the existence of an intrinsic size checkpoint that monitors organ size at the tissue, rather than the cellular, level. For example, while constitutive activity of the myc oncogene drives the growth of individual Drosophila cells, it has little effect on the size of imaginal disc compartments. Therefore, increased cell growth or cell proliferation does not automatically lead to a corresponding increase in tissue size, unless the size checkpoint is simultaneously perturbed. It follows that such intrinsic size-control mechanism must be overridden to permit the sustained overgrowth of tumors. The finding that YAP overexpression leads to immediate organomegaly followed by tumor formation provides direct support for this hypothesis. The widespread upregulation of YAP in diverse tumor types further suggests that the Hippo pathway represents a common mutational target that allows cancer cells to evade the intrinsic size-control mechanisms that normally maintain tissue homeostasis in animals (Dong, 2007).
The observation of two distinct patterns of YAP distribution in tumor cells -- with or without nuclear accumulation -- implicates two possible mechanisms by which Hippo signaling may be dysregulated in cancer cells. Based on the mechanism of Yki/YAP inactivation by Hippo signaling as revealed by the current study, it is suggested that the former pattern could result from inactivation of tumor suppressors upstream of YAP, mutation of the S127 phosphorylation site, or perturbation of the nuclear-cytoplasmic shuttling machinery, whereas the latter pattern could be caused by YAP overabundance, either via gene amplification, increased transcription, or protein stabilization. It is further speculated that these mechanisms may also be employed in normal physiological contexts to regulate the activity of the Hippo pathway in flies and mammals. Thus, besides phosphorylation, mechanisms that regulate Yki/YAP transcription or stability are likely relevant to the modulation of Hippo signaling activity in vivo (Dong, 2007).
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