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).
Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. These results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).
Two protein kinases Hippo [Hpo and Warts (Wts)/large tumor suppressor (Lats)], and a scaffold protein Salvador (Sav)/Shar-pei, are key components of this pathway. Moreover, two FERM-domain proteins, Merlin (Mer) and Expanded (Ex), function upstream of Hpo, and Mob as tumor suppressor (Mats), associates with Wts to stimulate the catalytic activity of the Wts protein kinase. Recently, both putative receptor and ligand that function further upstream of, or in parallel with, Hpo signaling have been identified (Hariharan, 2006). A major signal output of this growth inhibitory pathway is to inactivate a transcription coactivator Yorkie (Yki) via phosphorylation by Wts kinase. In addition to Cyclin E and Drosophila inhibitor of apoptosis 1 (diap1), the bantam microRNA is also found to be a target of the Hpo pathway. Most components in this emerging signaling pathway are conserved from yeast to flies and humans, suggesting that this pathway plays a fundamental role in cellular regulation (Wei, 2007).
The function of Mob proteins has been better studied in yeast, Drosophila and mammalian cells, which revealed a conserved property of Mob proteins as a binding partner as well as a coactivator of protein kinases of the Ndr (nuclear Dbf2-related) family (Hergovich, 2006b). As stated above, Drosophila Mats/dMob1 is required for mediating Hpo signaling by regulating Wts kinase activity in growth inhibition and tumor suppression. All four Drosophila mob genes dMob1-4 genetically interact with trc (tricornered) (He, 2005a), the fly Ndr homolog important for maintaining integrity of epidermal outgrowths and regulating dentritic tiling and branching (Emoto, 2004; He, 2005b). In the budding yeast Saccharomyces cerevisiae, Mob1 binds to and activates Dbf2/Dbf20 protein kinases for controlling mitotic exit and cytokinesis (Komarnitsky, 1998; Lee, 2001; Mah, 2001). Similarly, Mob1 is required for the activation of Sid2, an Ndr family kinase in the fission yeast Schizosaccharomyces pombe essential for cytokinesis (Hou, 2000; Hou, 2004). In human, hLats1 preferentially interacts with hMob1/hMats, but not hMob2 protein, and appeared to be required for promoting mitotic exit (Bothos, 2005), as well as cytokinesis (Yang, 2004). Importantly, the function of Mob proteins has been highly conserved in evolution. For instance, the human Mob1A/Mats1 protein has been shown to act as a kinase activator and can rescue the lethality and tumor phenotypes ofDrosophila mats mutants (Lai, 2005; Wei, 2007 and references therein).
Structural analysis of a human Mob1 protein, Mob1A/Mats1, revealed several important features of Mob family proteins (Stavridi, 2003). One is that several highly conserved residues are responsible for generating an atypical Cys2-His2 zinc-binding site, which is predicted to contribute to the stability of the Mob protein. Another striking feature is that there is a flat surface rich in acidic residues on one side of the protein. This property provides the structural basis for a Mob protein to interact with its partner, such as Ndr family kinases through electrostatic forces. Indeed, a 65-amino-acid region rich in basic residues exists in the N-terminal side of the kinase domain of Ndr family kinases, and alterations in the basic residues can prevent the kinases from binding to Mob proteins (Bichsel, 2004; Bothos, 2005; Hergovich, 2006b). Finally, hMob1A adopts a globular structure involving residues throughout the polypeptide. Mob proteins are small and usually do not carry any other structural motifs other than the Mob domain (Wei, 2007).
Although previous studies suggest that Ndr family kinases can be activated by upstream regulators such as Cdc15, Hpo and Mst kinases via phosphorylation in yeast, flies or human cells, very little is known about how Mob is regulated. Studies carried out in yeast and mammalian cells suggested that Mob proteins may be regulated through phosphorylation. For instance, yeast Mob1 was shown to be essential for the phosphorylation of Dbf2 by an upstream protein kinase Cdc15 and Mob1 itself was also phosphorylated by Cdc15 (Mah, 2001). However, the functional significance of this modification has not been elucidated. Work on human Mob1A/Mats1 also suggested that phosphorylation might provide a mechanism for regulating hMob1A activity (Bichsel, 2004). This study has tested a hypothesis that Mats is directly activated by Hpo kinase to regulate Wts kinase activity for growth inhibition and tumor suppression. Using the Drosophila system, it was found that Mats can be complexed with Hpo and is a target of the Hpo protein kinase. Similarly, human Mats1 is also a target protein of mammalian Mst kinases. Mats phosphorylation by Hpo increases its affinity with Wts protein kinase and ability to increase Wts activity to target Yki. Moreover, epistatic analysis suggested that mats acts downstream of hpo. Genetic analysis indicated that Mats functions together with Hpo for mediating growth inhibition of developing organs. Therefore, the Mob as tumor suppressor protein, Mats, functions as a critical component of the Hpo signaling pathway. The results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).
Recent studies have defined an emerging growth inhibitory pathway mediated by Fat, Mer/Ex, Hpo/Sav and Wts/Mats proteins in tissue growth and organ size control in Drosophila. Previous work has shown that Mats functions as a coactivator of the Wts protein kinase (Lai, 2005). This study has focused on addressing how Mats is activated to regulate Wts kinase activity. Fenetic analysis suggests that Mats acts downstream of Hpo and is a critical component of the Hpo signaling pathway. Moreover, evidence is provided that Hpo-mediated phosphorylation increases Mats's activity as a coactivator of the Wts protein kinase, and this regulatory mechanism is conserved from flies to humans. Therefore, Hpo-mediated phosphorylation of Mats significantly contributes to Wts activation. In a simple model, Hpo needs to directly phosphorylate Wts as well as Mats in order for Wts kinase to be fully activated. Although both Wts and Mats are activated by Hpo-mediated phosphorylation, further investigations are needed to address how Hpo phosphorylation and Mats binding are coordinated for Wts activation (Wei, 2007).
This report provides evidence that Mats is a target of Hpo/Mst protein kinases and Hpo/Mst-mediated phosphorylation positively regulates Mats protein's coactivator activity for Wts protein kinase. Importantly, it was found that Mats exists as a phosphoprotein in living cells, indicating that Mats phosphorylation occurs under physiological conditions. In addition to Hpo/Mst, Wts kinase has also been shown to target Mats for phosphorylation (Lai, 2005), although the physiological effect of this modification has not been elucidated. In S. cerevisiae, the founding member of the Mob superfamily Mob1 was found to be a phosphoprotein and a substrate for the Mps1 kinase. Mob1 is also phosphorylated by an upstream regulator Cdc15 kinase (Mah, 2001). However, the role of Cdc15 in Mob1 phosphorylation has not been revealed even though Mob1 is known to be required for Cdc15-mediated activation of its binding partner Dbf2 kinase. In mammalian cells, protein phosphatase 2A inhibition by OA treatment caused phosphorylation of a Mob family protein (Moreno, 2001). Moreover, OA-induced modification on hMob1 was shown to be critical for its binding to its partner Ndr kinase (Bichsel, 2004). Thus, phosphorylation appears to be a common mechanism for Mob regulation (Wei, 2007).
Consistent with the finding that Mats is activated by Hpo via phosphorylation for upregulating Wts kinase activity, epistatic analysis suggests that Mats is acting downstream of Hpo. This is the first case that Ste20 family protein kinase-mediated phosphorylation of Mob is critical for regulating the catalytic activity of Ndr family protein kinase such as Wts. At this point, it is not clear how Mob proteins function to activate Ndr family kinases. Based on the results from recent studies of human Mob1 and Ndr family kinases, a potential mechanism is that Ndr family kinase is rapidly recruited by hMob1 to the plasma membrane for activation (Hergovich, 2005; Hergovich, 2006a). It is speculate dthat Hpo phosphorylation might facilitate Mats to associate to the membrane through an unknown mechanism, which in turn recruits Wts to the membrane as evidenced by the observation that Hpo phosphorylated Mats has an increased affinity to Wts. Subsequently, Wts is activated by phosphorylations mediated by protein kinases such as Hpo. Mats as a target of Hpo kinase, is able to associate with Hpo in a protein complex. Since Hpo/Mst1 kinase was not present in the Mats/Wts protein complex (Lai, 2005), it appears that Mats simultaneously cannot associate with Hpo and Wts in the same protein complex (Wei, 2007).
In addition to the membrane recruitment model, the data also support an active and more direct role of Mats in upregulating Wts kinase. From in vitro kinase assays, it was found that Hpo-mediated phosphorylation increases the affinity between Mats and Wts, as well as the ability of Mats to activate Wts kinase activity in the absence of any membrane structures. The results support a model in which Mats binding likely causes a conformational change of Wts for Wts activation. In the case of human Ndr kinase, an autoinhibitory effect of hNdr can be released by hMob1 binding (Bichsel, 2004), which presumably induces a conformational change in hNdr for its activation. Finally, it was found that Mats increases the steady level of Wts protein, which contributes to the increase in Wts activity. Further investigation is needed to understand how Mats is able to stabilize and/or increase the production of Wts protein (Wei, 2007).
Previous work has shown that Mats negatively regulates tissue growth by binding to another tumor suppressor Wts and subsequently activating the catalytic activity of Wts kinase (Lai, 2005). Since loss of mats function leads to tissue overgrowth and tumor development, it suggests that Wts alone is not sufficient to inhibit tissue growth in the absence of Mats. Therefore, Mats is an indispensable component of the Hpo pathway, and Wts activation is dependent not only on Hpo-mediated phosphorylation, but also on Mats binding. Further studies are needed to understand how exactly Wts activation is coordinated by Hpo phosphorylation and Mats binding. This work has provide evidence that Mats activation can be mediated by Hpo phosphorylation (Wei, 2007).
The Hpo signaling pathway plays an important role in growth inhibition and tumor suppression in Drosophila, and this pathway appears to be also critical for tissue growth control and tumor suppression in mammals. For instance, mammalian NF2 tumor suppressor is a homolog of Drosophila Mer and Ex proteins, which are upstream regulators of the Hpo signaling pathway. Moreover, loss of Lats1 function in mouse causes soft tissue sarcomas and ovarian tumors. Recently, it was found that hMats1 can functionally replace fly Mats to suppress tumor development, and Mats1 is mutated in mammalian tumors (Lai, 2005). Thus, mechanisms for the control of Hpo signaling might be commonly used across species, and understanding such mechanisms should provide insights into tumor development in mammals. As shown in this report, one mechanism by which Hpo functions to control tissue growth is to target Mats for phosphorylation, and, consequently, Mats is activated to upregulate Wts kinase. Because mammalian Hpo orthologs, Mst kinases, regulates hMats1 in a similar manner, this mechanism is likely used in mammalian cells as well. Therefore, by understanding how Hpo/Mst kinases regulate Mats and Wts/Lats in normal as well as tumor cells, valuable insights will be gained into tissue growth inhibition and tumor suppression (Wei, 2007).
In Drosophila, Scalloped (Sd) belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain. Sd physically interacts with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation. The Vg-SD dimer activates the transcription of several specific wing genes, including sd and vg themselves. The dimer drives cell-cycle progression by inducing expression of the dE2F1 transcription factor, which regulates genes involved in DNA replication and cell-cycle progression. Yorkie (Yki) is a transcriptional coactivator that is the downstream effector of the Hippo signaling pathway, which controls cell proliferation and apoptosis in Drosophila. This study identified Sd as a partner for Yki. Interaction between Yki and Sd increases Sd transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes Yki nuclear localization. Yki overexpression induces vg and dE2F1 expression, and proliferation induced by Yki or by a dominant-negative form of Fat in wing disc is significantly reduced in a sd hypomorphic mutant context. Contrary to Yki, Sd is not required in all imaginal tissues. This indicates that Yki-Sd interaction acts in a tissue-specific fashion and that other Yki partners must exist (Goulev, 2008).
Yorkie (Yki) encodes a transcriptional coactivator that is the downstream effector of the Salvador-Warts-Hippo signaling pathway. This pathway consists of the two serine-threonine kinases, Hippo and Warts (Wts, also known as Large Tumor Suppressor), the adaptator molecules Salvador (Sav) and Mob as Tumor Suppressor (MATS), the FERM domain proteins Expanded (Ex) and Merlin (Mer), and the protocadherin Fat (Ft). In this pathway, the Hpo kinase phosphorylates Wts, which in turn phosphorylates and inactivates Yki by excluding it from the nucleus. Mer and Es colocalize at the cell cortex and act upstream of Hpo to regulate the activity of Yki. Fat acts upstream of the Hippo signaling cascade by recruiting Ex to the apical plasma membrane and modulating the abundance of Wts. Inactivation of ft, hpo, wts, and sav, inactivation of both ex and mer, and overexpression of yki all lead to increased proliferation and reduced apoptosis, This pathway acts through Yki to regulate the expression of cyclin E (cycE) and the Drosophila inhibitor of apoptosis 1 (diap-1), which are involved in cell-cycle progression and cell-death inhibition, respectively, and the microRNA bantam, which can promote growth and inhibit apoptosis. The Yki peptide shares significant homology with the mammalian Yes Associated Protein 65 (YAP) (31% identity with human YAP). A particular region near the amino terminus of Yki (amino acids 57-114) displays the highest homology with YAP (56.2% identity). This region in YAP corresponds to a domain that binds all mammalian transcription-enhancer factors (TEFs), which are the homologs of the Drosophila Sd protein (Goulev, 2008).
Conservation between mammalian YAP and Yki of the YAP domain that interacts with TEF suggests that Yki might interact with Sd. To investigate this possibility, glutathione S-transferase (GST)-pull-down experiments were conducted. 35S-labeled Yki specifically binds to GST-Sd beads (Goulev, 2008).
These results are in good accordance with a large two-hybrid screen in which Sd was identified as a binding partner for Yki. To refine the Sd domain that binds Yki and the region of Yki involved in the interaction with Sd, the binding of deleted Sd or Yki peptides was examined. Results that were obtained indicate a requirement of the N terminus of Yki and the C terminus domain of Sd. These results implicate Yki as an auxiliary protein that specifically interacts with Sd and demonstrate that this ability to interact is conserved between TEA factors (TEF-1 and Sd) and YAP factors (YAP and Yki) (Goulev, 2008).
To determine whether Sd, which possesses a putative nuclear localization signal (NLS) within the TEA domain, can modify Yki localization, transfection experiments were performed with plasmids expressing Sd fused to FLAG sequence and Yki fused to hemagglutinin (HA) sequence. In Drosophila S2 cells, Yki is mainly cytoplasmic. S2 cells were transfected with Yki and Sd and it was observed that Yki is exclusively nuclear. The nuclear translocation of Yki when Sd is expressed argues in favor of the idea that Sd and Yki interact ex vivo. Co-transfection experiments indicate that Yki interacts in a cellular context with Sd to stimulate the transcriptional activity conferred by Sd and additional experiments indicate that Vg and Yki do not compete for binding to Sd (Goulev, 2008).
It has been shown that the transcriptional activity of Yki is suppressed by coexpression of upstream components of the Hippo pathway. The transcriptional activity of Sd-Vg complex was analyzed when Hpo was overexpressed. Coexpression of GAL4db-Sd and Vg results in a powerful induction of luciferase activity. This induction was suppressed when Hpo was overexpressed. Although it cannot be excluded that Hpo used a mechanism other than Yki inhibition, the most likely expanation for these results is that the Hippo pathway, and thus Yki function, is required for Sd-Vg activity (Goulev, 2008).
It has been proposed that, in the Sd-Vg complex, Vg provides the activating component, and that the binding of Vg to Sd switches the target selectivity of Sd. If it is assumed that the Sd-Vg-Yki complex is able to form into a cell, one attractive explanation for the role of interaction between Yki and Sd is that Yki provides the main activating component and Vg undergoes a conformational change in Sd ensuring accurate DNA target selection (Goulev, 2008).
To explore the possibility that Yki function is evolutionarily conserved, the effects of Yki on TEF transcriptional activity was tested in a HeLa human cell line. Yki, similarly to YAP, stimulate TEF1 transcriptional activity. These results reveal an evolutionarily conserved regulatory mechanism. Interestingly, it has recently been shown that YAP is the effector of the mammalian Hippo pathway. It is amplified in mouse tumor models and has several oncogenic properties. Furthermore, YAP can functionally substitute for Yki in Drosophila. The relevance of the YAP-TEF interaction in this pathway, however, remains to be determined (Goulev, 2008).
To determine whether Yki-Sd interaction regulates Sd transcriptional activity in vivo in Drosophila, a sensor transgene, 'hsp70-GAL4db-sd', expressing a modified Sd protein was used, in which the TEA DNA-binding domain has been replaced by that of the yeast GAL4 DNA-binding domain, under the control of the hsp70 promoter. GAL4::Sd activity, monitored by the UAS-lacZ-responsive reporter transgene, is restricted to the wing pouch, where Sd dimerizes with Vg. A significant reduction in lacZ activity was observed in wing discs heterozygous for the amorphic ykiB5 allele compared to control wing discs. The fact that Sd activity is sensitive to yki dosage in wing imaginal discs supports the idea that Yki is required for Sd transcriptional activity in vivo (Goulev, 2008).
To better understand the functional relationship between Yki and Sd during wing development, genetic interactions between these genes was assessed. The results indicate that reduction of both vg and yki dosage enhances the sd phenotype and support the hypothesis that Yki works together with Sd-Vg during wing development (Goulev, 2008).
It has been shown that binding sites for Sd and Sd-Vg are necessary for regulation of vg expression and thus may regulate by a feedback mechanism vg expression. Results obtained in S2 cells indicate that Yki increases Sd transcriptional activity. This prompted an investigation of whether yki overexpression in imaginal discs enhances Vg-Sd activity, thus regulating vg expression and vgQE activity. yki overexpression was drived by using a UAS-GAL4 system or by flip-out (FLP-FRT) recombination in clones. In both cases, overexpression of yki was shown to induce vg and vgQE expression in a cell-autonomous manner in wing discs and in haltere discs (Goulev, 2008).
Several sets of data argue strongly in favor of an involvement of Vg-Sd in cell proliferation and cell-cycle progression in wing imaginal discs. vgnull cell clones do not proliferate in the wing pouch, whereas they can be recovered in the notum region, where vg is not expressed, indicating that vg is required for the proliferation of wing blade cells or for their survival. In contrast, vg ectopic expression induces wing outgrowths and dE2F1 expression. dE2F1 is the Drosophila homolog of the E2F transcription-factor family that plays a pivotal role during cell-cycle progression in controlling expression of different genes involved in G1-S transition (including cycE, a target gene of the Hippo pathway) and DNA replication. It was found that Yki overexpression with the ptc-GAL4 driver clearly upregulates expression of the dE2F1-LacZ reporter strain in a cell-autonomous manner. This indicates that Yki and Vg-Sd both activate dE2F1 expression and might work together in this process. It can be hypothesized either that Sd-Vg is required for dE2F1 induction by Yki or that Yki induces dE2F1 independently from Vg-Sd and reinforces dE2F1 expression through Sd-Vg induction (Goulev, 2008).
To determine more precisely the relationship between Sd and Yki in cell proliferation, the effect of Yki overexpression was tested in a mutant context for sd. Strong hypomorph alleles of sd are characterized by an absence of wing pouch cells. In contrast, sdETX4 corresponds to a weak allele of sd in which a large number of wing pouch cells are still present. This allele was used to exclude the possibility that the observed effect resulted from an absence of cell proliferation in the wing pouch because of a lack of sd expression. Wing discs overexpressing yki with the en-GAL4 driver exhibit clear increases in size of the posterior region, resulting in massive overgrowth. In contrast, the size of the posterior compartment was almost normal in wing discs from flies hemizygous for sdETX4 and overexpressing yki with the en-GAL4 driver. This result shows that Yki needs Sd to fully induce cell proliferation. Next, it was asked whether Sd-Yki interaction is acting downstream of the Hippo pathway. To test this, use was made of a dominant-negative form of Ft that lacks the intracellular domain (FTΔICD) . Indeed, Ft is an upstream component of the Hippo pathway, and overexpression of FTΔICD induces overgrowth and the expression of Hippo target genes. A significant reduction was observed of induced overgrowth in sdETX4 wing discs. This result strongly suggests that Sd-Yki interaction acts downstream of the Hippo pathway in wing discs (Goulev, 2008).
This paper has addressed the role of Sd-Yki interaction in the wing disc. The role of Sd in other imaginal tissues is poorly understood. Strong alleles of sd are associated with lethality in early larval stage. In the eye disc, clones homozygous for a strong allele of sd die or poorly survived, whereas they are associated with truncated legs when generated in the leg disc, suggesting that Sd should play a role in cell survival in these tissues. In the wing disc, sd clones die in the wing pouch but can be easily recovered in regions that will give rise to the notum, whereas Yki is required in the entire wing disc. This implies that other partners must interact with Yki to promote tissue growth in other structures. However, the induction of vg that was observed in cells overexpressing yki and the results showing that proliferation induced by Yki in the wing disc is significantly reduced in sd mutant context suggest that Sd-Vg is required for Yki function in the wing disc (Goulev, 2008).
The Hippo (Hpo) kinase cascade restricts tissue growth by inactivating the transcriptional coactivator Yorkie (Yki), which regulates the expression of target genes such as the cell death inhibitor diap1 by unknown mechanisms. The TEAD/TEF family protein Scalloped (Sd) is a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th) locus harbors a minimal Sd-binding Hpo Responsive Element (HRE) that mediates transcriptional regulation by the Hpo pathway. Sd binds directly to Yki, and a Yki missense mutation that abrogates Sd-Yki binding also inactivates Yki function in vivo. sd is required for yki-induced tissue overgrowth and target gene expression, and that sd activity is conserved in its mammalian homolog. These results uncover a heretofore missing link in the Hpo signaling pathway and provide a glimpse of the molecular events on a Hpo-responsive enhancer element (Wu, 2008).
The Hpo signaling pathway has emerged as a central and highly conserved mechanism that regulates organ size in animals. At the core of this pathway is a kinase cascade that impinges on the transcriptional coactivator Yki to regulate the transcription of target genes involved in cell growth, proliferation, and survival. Given Yki's pivotal position in the Hpo pathway, understanding the mechanisms by which Yki regulates target gene expression should provide important mechanistic insights that can facilitate therapeutic manipulation of this crucial size-control pathway (Wu, 2008).
A major gap in understanding of the Hpo signaling pathway concerns how Yki regulates target gene transcription. This study shows that Sd represents a crucial missing link between Yki and the regulatory DNA of Hpo pathway target genes. First, an unbiased dissection of diap1 regulatory region revealed a minimal Sd-binding enhancer element (HRE) that confers Hpo-responsive regulation. The HRE not only responded to Yki activity in vivo, but also conferred Sd-dependent and Yki-dependent transcriptional activity in cultured Drosophila cells. In a parallel line of experiments, Sd was identified in an unbiased screen for proteins that bind to a critical N-terminal Yki domain defined by a missense allele, ykiP88L. The fact that this missense mutation disrupted binding of Yki to Sd supports the physiological relevance of a Sd-Yki transcription complex in vivo. The identification of Sd as a cognate Yki partner using two unbiased approaches, combined with the genetic interactions between sd and yki, provide strong evidence that Sd is a critical DNA-binding factor that mediates the transcriptional output of the Hpo signaling pathway. It is worth noting that the requirement for sd in yki-driven overgrowth is highly specific, since the same sd mutation had no effect on overgrowth driven by the activated Ras oncogene. The observation that Yki, but not Sd, can be overexpressed or mutated to elicit tissue overgrowth further suggests that Sd is normally present in excess, and the activity of the Sd-Yki complex is regulated through modulation of Yki activity effected by the Hpo kinase cascade (Wu, 2008).
The founding member of the TEAD family transcription factors, TEAD-1/TEF-1, was initially identified based on its binding to the GTIIC motif of the simian virus 40 (SV40) enhancer. The TEAD family transcription factors have been mostly studied in the context of muscle-specific gene transcription, and their roles in cell proliferation and cell survival are poorly understood. The observation that TEAD-2 and YAP have similar activity to Sd and Yki, respectively, suggests that the growth-regulatory activity of the Sd-Yki complex is likely conserved in the mammalian Hpo pathway. It is also worth noting that besides growth regulation, the Hpo pathway has also been implicated in controlling other biological processes such as rhodopsin gene expression in mature photoreceptors and dendrite morphogenesis in postmitotic neurons. It remains to be determined whether Yki partners with Sd or other (unknown) DNA-binding factors in such nongrowth contexts (Wu, 2008).
Despite their elevated transcription upon inactivation of Hpo pathway tumor suppressors or activation of the Yki oncoprotein, it was previously unknown whether Yki regulates the known Hpo pathway target genes directly or indirectly through intermediary transcriptional regulators. This study has taken an unbiased approach to this question by isolating a HRE for diap1. This DNA element provided several important insights into how Hpo signaling activity is converted into transcriptional output. First, the Sd protein directly binds to the HRE and activates an HRE-luciferase reporter in cell culture in conjunction with Yki, supporting the notion that Yki directly regulates the transcription of the diap1 gene. Second, the minimal diap1 HRE contains non-Sd-binding sequence that is indispensable for HRE activity, suggesting that the HRE likely binds to additional transcription factors besides Sd in vivo. This latter characteristic is not unique to the HRE, but is a general feature that has been observed for many signaling pathways in Drosophila. For example, Notch-regulated enhancers contain not only binding sites for the signal-regulated transcription factor Suppressor of Hairless, but also binding sites for additional cofactors whose activity is Notch independent. It will be important to identify the factors that bind to the non-Sd sequence in the diap1 HRE, and to investigate whether such factors play a general role in mediating the Hpo responsiveness of other target genes (Wu, 2008).
The identification of a minimal HRE makes possible several new avenues of investigation to better understand the Hpo signaling pathway. The minimal HRE revealed in this study should facilitate a comprehensive cataloguing of Hpo pathway target genes, many of which remain to be identified. It also provides a useful tool for constructing reporters that can be used to monitor the specific activity of the Hpo pathway in vivo. Furthermore, this work will facilitate cell-based RNAi screens for components or modulators of the Hpo pathway, as illustrated by the successful use of pathway-specific luciferase reporters for interrogating other signaling pathway (Wu, 2008).
An interesting and somewhat unexpected finding from this study concerns the differential requirement of yki for the basal expression of diap1 and expanded (ex), with the former being yki dependent and the latter being yki independent, respectively. Thus, different Hpo target genes, and by inference different enhancer elements or their combinations, can respond to different threshold levels of Yki activity. It is suggest the basal expression of ex is mediated by non-HRE sequence in the ex locus and is therefore independent of yki. Excessive yki activity, either directly via an HRE in the ex locus or indirectly by turning on another factor, promotes ex transcription above the basal level. In contrast, Yki (through Sd, non-Sd DNA-binding factors, or both), regulates the basal level transcription of diap1. It is noted that the basal transcription of diap1 is not necessarily regulated through the HRE, which was identified by virtue of reporter expression under hyperactive Yki activities. Indeed, it was found that although the HRE is responsive to yki overexpression, it is largely unresponsive to loss of yki. Thus, the diap1 HRE revealed in this study is uniquely sensitive to unrestrained Yki activity (Wu, 2008).
The exquisite sensitivity of yki-induced overgrowth to sd dosage suggests that Sd/TEAD could be specifically targeted to ablate certain unwanted tissue growth, such as that caused by aberrant Hpo signaling, with minimal effect on normal growth. Thus, Sd/TEAD belongs to a growing list of genes that cause 'non-oncogene addition' -- genes that cannot be mutated or overexpressed to an extent that directly promotes tumorigenesis, but are still rate limiting to their specific signaling pathways. The requirement of such non-oncogenes in tumor cells makes them excellent targets for the development of new cancer therapeutics (Wu, 2008).
The Hippo (Hpo) signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes; however, the transcription factor of the pathway has remained elusive. This study provides evidence that the TEAD/TEF family transcription factor Scalloped (Sd) acts together with the coactivator Yorkie (Yki) to regulate Hpo pathway-responsive genes. Sd and Yki form a transcriptional complex whose activity is inhibited by Hpo signaling. Sd overexpression enhances, whereas its inactivation suppresses, tissue overgrowth caused by Yki overexpression or tumor suppressor mutations in the Hpo pathway. Inactivation of Sd diminishes Hpo target gene expression and reduces organ size, whereas a constitutively active Sd promotes tissue overgrowth. Sd promotes Yki nuclear localization, whereas Hpo signaling retains Yki in the cytoplasm by phosphorylating Yki at S168. Finally, Sd recruits Yki to the enhancer of the pathway-responsive gene diap1, suggesting that diap1 is a direct transcriptional target of the Hpo pathway (Zhang, 2008).
The Hpo pathway has emerged as a conserved signaling pathway that plays a critical role in controlling tissue growth and organ size. Despite the growing recognition of the importance of this pathway in development and cancer, the transcription factor that links the cytoplasmic components to the nuclear events has remained elusive and thus represents a major gap in the pathway. This study demonstrates Sd is the missing transcription factor of the Hpo pathway based on several lines of genetic and biochemical evidence. (1) Sd and Yki form a transcriptional complex to activate a reporter gene in S2 cells and this transcriptional activity is inhibited by Hpo signaling. Furthermore, Sd and Yki synergize in vivo to promote Hpo target gene expression and tissue overgrowth. (2) More importantly, loss of Sd function suppresses tissue overgrowth induced by Yki overexpression or loss-of-function mutations in hpo, sav, and wts. In addition, Sd inactivation either by RNAi or a genetic mutation blocks the ectopic expression of Hpo responsive genes induced by excessive Yki activity. (3) RNAi knockdown of Sd phenocopies knockdown of Yki, which is manifested by reduced organ size and diminished expression of Hpo pathway-responsive genes. (4) A constitutively active form of Sd activates multiple Hpo pathway-responsive genes and promotes tissue overgrowth. (5) Sd promotes Yki nuclear translocation and recruited Yki to the diap1 enhancer (Zhang, 2008).
Several sd null alleles were generated to further explore the consequence of loss of Sd. sd null clones located in the wing pouch region were found to exhibit growth deficit such that early-induced clones (48-72 hrs AEL) were eliminated by the end of late third instar. However, late-induced clones (72-96 hrs AEL) survived and exhibited diminished expression of diap1. In contrast, early-induced clones were recovered in the notal region of wing discs and in eye discs without showing discernible change in Diap1 levels. However, a previous study showed that yki mutant clones exhibited reduced diap1 expression in eye discs. It is possible that low levels of residual Sd activity persist in sd mutant clones, which are sufficient to support the basal expression of the Hpo target genes. Alternatively, Yki may act through another transcription factor to maintain the basal expression of Hpo target genes. Nevertheless, sd null mutation suppresses the overgrowth phenotype and ectopic cycE expression induced by excessive Yki activity, suggesting the residual Sd in sd mutant clones is insufficient to support the elevated Yki activity (Zhang, 2008).
The identification of Hpo pathway transcription factor provided an opportunity to assess direct transcriptional targets of the pathway. To this end, the diap1 enhancer was characterized, and a 1.8 kb enhancer element critical for diap1 expression was identified. This region contains a total of seventeen predicted Sd binding sites. Using the ChIP assay, it was demonstrated that both Sd and Yki physically interact with the 1.8 kb diap1 enhancer and the association of Yki with the diap1 enhancer is mediated by Sd. These results suggest that Sd recruits Yki to the diap1 enhancer to activate its transcription (Zhang, 2008).
It has been shown that Sd acts in conjunction with Vg to promote wing development by directly regulating the expression of wing patterning genes. This study has demonstrated that Sd acts in conjunction with Yki to control organ size by regulating the expression of genes involved in cell proliferation, cell growth, and apoptosis. These observations raise an important question of how Yki-Sd and Vg-Sd transcriptional complexes specifically select their targets. One possibility is that Vg-Sd and Yki-Sd prefer to interact with distinct Sd binding sites. Indeed, a previous study showed that binding of Vg to Sd modulates the DNA binding selectivity of Sd. Another possibility is that target selectivity could be influenced by cofactors that bind in the vicinity of Sd binding sites. In support of this notion, previous studies have shown that wing specific enhancers contain both Sd binding sites and binding sites for transcription factors that mediate specific signaling pathways. It is also possible that Vg-Sd and Yki-Sd may share common targets. For example, diap1 could be activated by Vg-Sd in the wing pouch, which might explain why sd mutant clones in this region exhibits diminished diap1 expression (Zhang, 2008).
In principle, the Hpo pathway could regulate the activity of Yki-Sd transcriptional complex at several levels. For example, Hpo signaling could regulate the formation Yki-Sd complex or the recruitment of other factor(s) to the Yki-Sd transcriptional complex. Alternatively, Hpo signaling could regulate the nuclear-cytoplasmic transport of Yki. In support of the latter possibility, Yki exhibits elevated nuclear localization in wts or hpo mutant clones. In addition, coexpression of Hpo with Yki depletes nuclear Yki in S2 cells, suggesting that Hpo signaling impedes nuclear localization of Yki and thereby limits the amount of active Yki-Sd transcriptional complex (Zhang, 2008).
Mutating Yki S168 to Ala increases nuclear localization and growth promoting activity of Yki. In addition, it has been demonstrated that phosphorylation of Yki S168 was stimulated by Hpo. Phosphorylation of Yki by Hpo signaling increases their association with 14-3-3, which is abolished by mutating Yki S168 to Ala. Since 14-3-3 often regulates nuclear-cytoplasmic shuttling of its interacting proteins, these observations suggest that Hpo signaling inhibits Yki at least in part by phosphorylating Yki S168, which promotes 14-3-3 binding and cytoplasmic sequestration of Yki (Zhang, 2008).
The Hpo pathway appears to restrict cell growth and control organ size in mammals. The finding that Sd is critical for Yki-induced tissue growth has raised the interesting possibility that the effect of YAP in promoting tissue growth may rely on the TEAD/TEF family of transcription factors. Corroborating this hypothesis, TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with YAP (Vassilev, 2001). Furthermore, YAP can bind to and stimulate the trans-activating activity of all four TEAD/TEF family members (Vassilev, 2001). The TEAD/TEF family members exhibit overlapping but distinct spatiotemporal expression patterns and thus may have redundant but unique roles during development . It will be important to determine which TEAD/TEF family members are involved in the mammalian Hpo pathway and whether YAP employs distinct sets of TEAD/TEF transcription factors in different tissues. Since abnormal activation of YAP is associated with multiple types of cancer, disrupting YAP-TEAD/TEF interaction may provide a new strategy for cancer therapeutics (Zhang, 2008).
The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. This study demonstrates that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). This study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP (Zhao, 2008).
To investigate the function of TEAD in YAP-induced growth control, transgenic flies were generated that express human YAP-S127A (an active form) or YAP-S94A/S127A in developing eyes. YAP-S127A overexpression significantly increased eye size and the number of interommatidial cells. Mutation of S94A dramatically decreased the activity of YAP-S127A in promoting tissue growth. Scalloped (Sd) is the only TEAD homolog in Drosophila. Yki was found to directly interacted with Sd in an in vitro binding assay. Furthermore, Yki S97A mutation (equivalent to YAP-S94A) diminished its interaction with Sd. Moreover, this Sd-binding-defective Yki-S97A mutant was less potent in stimulating growth in vivo compared with wild-type Yki. The functional defect of the TEAD-binding-deficient YAP/Yki was further confirmed by generating overexpression flip-out clones in the Drosophila larval wing discs as labeled by positive GFP expression. Both YAP-S127A and Yki are potent in stimulating tissue growth as individual clones, and the whole discs were generally larger than wild-type clones or discs. However, neither YAP-S94A/S127A nor Yki-S97A showed a similar level of growth-promoting effect. These data indicate that TEAD/Sd binding is important for the physiological function of YAP/Yki (Zhao, 2008).
The genetic interaction between Yki and Sd was tested. A strong loss-of-function allele of sd dominantly suppressed the enlarged and rough eye phenotypes caused by Yki overexpression. Thus, the level of Sd is critical for Yki to promote tissue growth. Overexpression of Sd caused small eyes, presumably due to a dominant-negative effect, but it did not result in lethality. This phenotype was strongly enhanced by reduction of yki levels, such that all of these flies died at the late pupal stage and had no eyes. Furthermore, coexpression of Yki with Sd suppressed the reduced eye phenotype caused by Sd overexpression. In fact, the eyes of animals overexpressing both Yki and Sd were enlarged more than those of animals that only expressed Yki. Therefore, Sd overexpression enhanced the Yki overexpression phenotypes. Together, these results indicate that Sd is a critical functional partner of Yki, a conclusion consistent with TEAD as a critical downstream target transcription factor of YAP (Zhao, 2008).
The Hippo kinase pathway plays a central role in growth regulation and tumor suppression from flies to man. The Hippo/Mst kinase phosphorylates and activates the NDR family kinase Warts/Lats, which phosphorylates and inhibits the transcriptional activator Yorkie/YAP. Current models place the FERM-domain protein Expanded upstream of Hippo kinase in growth control. To understand how Expanded regulates Hippo pathway activity, affinity chromatography and mass spectrometry were used to identify Expanded-binding proteins. Surprisingly it was found that Yorkie is the major Expanded-binding protein in Drosophila S2 cells. Expanded binds Yorkie at endogenous levels via WW-domain-PPxY interactions, independently of Yorkie phosphorylation at S168, which is critical for 14-3-3 binding. Expanded relocalizes Yorkie from the nucleus, abrogating its nuclear activity, and it can regulate growth downstream of warts in vivo. These data lead to a new model whereby Expanded functions downstream of Warts, in concert with 14-3-3 proteins to sequester Yorkie in the cytoplasm, inhibiting growth activity of the Hippo pathway (Badouel, 2009).
Current models propose that ex and mer function together to restrict tissue growth upstream of hpo. Mer and Ex colocalize with cortical actin in the apical region of the cell. Both genetic and physical interactions have been observed between Mer and Ex: loss of one copy of mer dominantly enhanced wing overgrowth in ex mutants. In addition, fragments of Mer and Ex protein can interact physically with each other in far-western experiments or when overexpressed in cultured cells. Clones doubly mutant for mer and ex have more dramatic overgrowth than either single mutant, and mer,ex double mutants phenocopy hpo mutants (Badouel, 2009).
However, despite the widespread acceptance that ex and mer function upstream of Hpo, some data are difficult to reconcile with ex acting strictly upstream of hpo in activation of the pathway. Genetic analysis indicates that ex is downstream of dachs, which has been shown to be directly upstream of wts in growth control. Biochemical analysis of the effects of overexpressing Mer and Ex also suggested that ex may not act simply upstream of hpo. For example, overexpression of Mer in S2 cells leads to a shift in Wts mobility, whereas overexpression of Ex does not alter Wts mobility. In vivo analysis has also suggested that mer and ex may have different roles in controlling growth and apoptosis (Badouel, 2009).
Using biochemical purification and mass-spectrometic analysis, Yki as a major Ex-binding protein in Drosophila S2 cells was identified. Binding of Yki to Ex is direct and is mediated by a WW domain-PPxY interaction. This interaction is independent of Wts-dependent phosphorylation at S168, a site previously shown to be essential for strong interactions of Yki with 14-3-3 proteins. Consistent with the biochemical analysis, it was shown that ex can act downstream of wts in the regulation of growth in eye imaginal discs and can repress the pupal lethality caused by excessive growth of wts clones. In addition, it was found that loss of hpo does not alter the ability of ex to regulate yki activity, as indicated by transcriptional assays in S2 cells, and that expression of Ex is sufficient to relocalize Yki to the cytoplasm. These data lead to a model in which Ex functions to repress Yki activity at least in part by keeping Yki out of the nucleus. Intriguingly, the Yki homolog, YAP, was first identified as a protein that binds Src family kinases at the cell membrane. Subsequent studies have focused on the role of Yki in the nucleus. Interestingly, immunohistochemical analysis reveals that a portion of Yki colocalizes with Ex at the cell membrane in Drosophila imaginal discs (Badouel, 2009).
Once Yki is phosphorylated by Wts, it can bind 14-3-3 proteins and can be transported out of the nucleus. However, since 14-3-3-bound Yki can also shuttle back into the nucleus, Ex binding Yki provides an anchor that can effectively dampen Yki activity. 14-3-3 shuttling activity results in an equilibrium of distribution of Yki between the nucleus and the cytoplasm. This equilibrium is biased in favor of Yki in the cytoplasm in the presence of Ex acting as an anchor. The presence of a tether of Yki in the cytoplasm was already suggested based on the distribution of YkiS168A between the cytoplasm and the nucleus, instead of predominantly in the nucleus (Badouel, 2009).
The strong nuclear localization of Yki is seen in Drosophila tissues only in cases of pathological stimulation of growth, such as in wts loss-of-function clones, which lead to massive overgrowth. The lack of detectable Yki nuclear localization during normal growth regulation suggests that Yki is an exceedingly potent growth regulator, and points to why there are many layers of regulation of Yki localization. The need for Ex to dampen Yki signaling in the nucleus is reflected by the increase of Cyclin E and Diap1 transcription in ex mutant clones (Badouel, 2009).
It is speculated that the regulation of the Hpo pathway by combined loss of Ex and Mer is so potent because one acts as the brake and the other controls the accelerator. Ex restricts Yki to the cytoplasm, thus blocking activity downstream, whereas Mer activates Hpo activity, thereby restricting Yki via inhibitory phosphorylation. Thus, loss of Ex on its own does not have a dramatic effect on cell proliferation and apoptosis, since the activity of the kinase cascade is regulated via Mer. Conversely, as long as Ex is present, excessive pathway activity induced by loss of Mer can be effectively modulated by the dampening activity of Ex (Badouel, 2009).
The data strongly suggest that Ex regulates Yki activity downstream of wts, by directly binding Yki and inhibiting Yki nuclear localization and transcriptional activity. The possibility cannot, however, be excluded that Ex also has additional upstream roles in regulating Hpo activity. Interestingly, overexpressed Ex does not induce apoptosis in a wts mutant background, although it can block growth, suggesting that ex is upstream of wts in apoptosis control, yet downstream of wts in growth control. Genetic dissection of this pathway is complicated by the well-documented feedback loops in the Hpo pathway: for example, Yki regulates the expression of both mer and ex. In addition, genetic evidence suggests that ex and mer function together to regulate endocytosis and growth factor signaling. Further biochemical dissection of Hpo pathway activity will be required to fully elucidate the diverse ways in which growth and apoptosis are controlled in response to various developmental and environmental signals (Badouel, 2009).
The binding of Ex to Yki is likely to be 14-3-3 independent, as mutation of S168, the predominant Wts phosphorylation site, impairs 14-3-3 binding, yet does not affect the ability of Ex to bind Yki. Thus, Ex binding to Yki could provide a pool of Yki that is nonphosphorylated and poised for release by upstream growth regulators. The binding of 14-3-3 to Yki can protect Yki from dephosphorylation. This provides a problem for the cell, since 14-3-3 must dissociate from Yki to allow it to become dephosphorylated, thus releasing a potent activator of proliferation and inhibitor of cell death, allowing it to re-enter the nucleus. The ability of Ex to bind both phosphophorylated and dephosphorylated Yki provides a mechanism by which to anchor dephosphorylated Yki in the cytoplasm (Badouel, 2009).
An appealing model is that the binding of Ex to Yki may be modulated, once in the cytoplasm, as an additional control point for the Hpo pathway. FERM-domain proteins frequently form inhibitory intramolecular associations, blocking the activity of the protein until the repression is relieved. Modifications of the Ex FERM domain or linker region could thus alter the ability of Ex to bind Yki in vivo. Ex localization at apical junctions is at least partially dependent upon the atypical cadherin Fat, which can regulate Hpo pathway activity. The recruitment of Ex complexes (directly or indirectly) to Fat may modify the ability of Ex to interact with Yki (Badouel, 2009).
The in vivo analysis indicates that the N-terminal FERM domain of Ex contains apical localization elements, whereas the C-terminal region contains junctional localization elements. Thus, each of these localization elements might be regulated independently and might impact on the ability of Ex to sequester Yki. Identification of which protein(s) Ex binds at junctions may illuminate the mechanisms by which Ex responds to external inputs to regulate Yki activity (Badouel, 2009).
All of the components of the Hpo pathway are well conserved in mammals and have been shown to have conserved functions in regulating growth. Loss of Hpo and Wts orthologs and overexpression of the Yki ortholog, YAP, have been implicated in a variety of human cancers. FERM6, the human ortholog of Ex, also regulates Hpo pathway activity in mammals. Future studies will determine if FERM6 directly binds YAP, and if disrupting YAP-FERM6 interactions is a tumor-promoting event (Badouel, 2009).
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