Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Drosophila Merlin and Expanded are shown to be components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes (Hamaratoglu, 2006).
Developmental and environmental signals control a precise program of growth, proliferation, and cell death. This program ensures that animals reach, but do not exceed, their typical size. Understanding how cells sense the limits of tissue size and respond accordingly by exiting the cell cycle or undergoing apoptosis has important implications for both developmental and cancer biology. The Hippo (Hpo) pathway comprises the kinases Hpo and Warts/Lats (Wts), the adaptors Salvador (Sav) and Mob1 as a tumor suppressor (Mats), the cytoskeletal proteins Expanded and Merlin, and the transcriptional cofactor Yorkie (Yki). This pathway has been shown to restrict cell division and promote apoptosis. The caspase repressor DIAP1 appears to be a primary target of the Hpo pathway in cell-death control. Firstly, Hpo promotes DIAP1 phosphorylation, likely decreasing its stability. Secondly, Wts phosphorylates and inactivates Yki, decreasing DIAP1 transcription. Although some of the events downstream of the Hpo kinase are understood, its mode of activation remains mysterious. This study shows that Hpo can be activated by Ionizing Radiations (IR) in a p53-dependent manner and that Hpo is required (though not absolutely) for the cell death response elicited by IR or p53 ectopic expression (Colombani, 2006).
Hpo is the ortholog of the Mammalian Sterile Twenty-like (MST) kinases, which belong to the Ste20 family of kinases. MSTs are highly similar to Hippo (Hpo) in their N-terminal serine/threonine kinase domains as well as in the C-terminal Salvador (Sav) binding region (or SARAH domain). MST1 functions both downstream and upstream of caspases to promote chromatin condensation and nuclear fragmentation, as well as activation of the JNK (Jun N-terminal kinase) and p38 pathways. Like most Ste20 family kinases, MST1/2 auto- or trans-phosphorylates at a number of residues. One of these, T183 in the activation loop, has been shown to be required for full kinase activity and has been used as a useful marker of MST1 activation in cultured cells. In order to study events upstream of Hpo, antibodies that have previously been shown to recognize MST1/2 phosphorylated on T183 were tested for their ability to cross-react with Hpo on the equivalent residue (T195). Interestingly, it was found antibodies that specifically recognized the phosphorylated form of Hpo upon treatment with staurosporine (sts), a known activator of MST1/2. This signal is abolished by RNAi-mediated Hpo depletion and disappears upon phosphatase treatment. Moreover, the antibodies recognize overexpressed tagged Hpo before immunoprecipitation. By contrast, the antibodies did not recognize a nonphosphorylable (T195A) Hpo mutant protein. Myc-tagged wild-type and T195A Hpo were immunoprecipitated and their auto-kinase activity and their activity on an exogenous substrate (Histone H2B, not shown) were measured in both the presence and absence of sts. As has been observed for MST1/2, overexpression of Hpo leads to its activation, presumably via trans-phosphorylation. Sts treatment potently stimulates Hpo kinase activity (5-fold). By contrast, the T195A mutant is severely compromised both in its unstimulated and stimulated activities, suggesting that T195 phosphorylation is crucial to normal Hpo kinase activity. Thus, these phospho-specific antibodies can be used as readouts of Hpo pathway activity (Colombani, 2006).
In the course of testing stimuli that would activate Hpo in tissue culture, it was observed that γ-irradiation potently and rapidly induced Hpo activation. The fly p53 ortholog has been shown to mediate cell death upon ionizing radiation (IR)-induced DNA damage. Although the pro-apoptotic genes reaper (rpr), hid, and sickle are p53 transcriptional targets, removal of these three proteins via chromosomal deficiencies only partially suppresses the cell-death effects of IR in embryos, suggesting that additional death signals act downstream of p53. This prompted an examination of whether the Hpo pathway could function downstream of Drosophila p53 in the response to IR (Colombani, 2006).
Initially, wing imaginal discs (the larval precursors of the adult wing) containing clones of hpo, wts, and sav mutant cells were treated with γ-rays and cell death was examined by staining for activated caspases. Interestingly, although caspase activation was efficiently induced in wild-type tissue or control discs, cell death was severely reduced in hpo, wts, and sav mutant clones and in p53 mutant discs. Quantification of the caspase staining indicated that apoptosis was reduced by 2- to 3-fold in hpo, wts, and sav clones compared to wild-type tissue. This was also true in eye imaginal discs (Colombani, 2006).
Overexpression of p53 in the posterior portion of late larval eye imaginal dics was sufficient to induce apoptosis. Loss of function of hpo, wts, and sav decreased cell death in this context, although the effect was less pronounced in sav clones, perhaps as a reflection of the weaker phenotype of the sav mutants. This suggests that the Hpo complex may function as an effector in the p53-mediated response to IR. To test this hypothesis, Hpo activation was measured in cultured cells treated with γ-rays in the presence or absence of dsRNAs directed against p53. Excitingly, depletion of Dmp53 markedly reduced Hpo phosphorylation by IR. The residual level of Hpo activation observed in p53-depleted cells can probably be explained by the fact that the dsRNA-mediated p53 depletion was never complete, as measured by RT-PCR. To check that the increased Hpo phosphorylation observed corresponded to increased activity, IP kinase assays were performed on cells expressing ectopic Hpo. It was observed that IR treatment potently induced Hpo kinase activity. Furthermore, p53 expression alone, in the absence of IR, was sufficient to activate Hpo phosphorylation. Finally, it was determined whether p53-dependent Hpo activation could be observed in vivo by taking advantage of the fact that p53 is not required for viability. Dissected ovaries from p53 mutant and wild-type flies were treated with γ-rays and examin Hpo activity was examined by Western blotting. Interestingly, although γ-rays potently activated Hpo in wild-type flies, this response was abolished in p53 mutant animals. p53 expression in the ovaries was able to induce apoptosis, ovary degeneration, and total loss of fecundity. It is concluded that Hpo is activated as part of a p53-dependent DNA-damage response both in cultured cells and in vivo (Colombani, 2006).
MST1 and 2 are known to be activated by caspase 3 through proteolytic cleavage. Therefore, the possibility exists that the Hpo activation observed is merely a by-product of Rpr-dependent caspase activation. Several lines of evidence suggest that this is not the case. First, reaper overexpression in S2 cells did not increase Hpo activity. Second, depletion of DIAP1 from cultured cells, which potently induces caspase activation, fails to trigger detectable Hpo activation. Third, the phospho-Hpo signal detected corresponds to full-length Hpo rather than a caspase-cleaved fragment. In fact, the caspase cleavage site present in the MSTs is not thought to be conserved in Hpo, and no evidence was seen of Hpo cleavage upon apoptotic stimuli. Fourth, treatment of cultured cells with caspase inhibitors did not affect Hpo activation by IR. Thus, it is unlikely that Hpo is stimulated via p53-dependent caspase activation (Colombani, 2006).
The time course of Hpo activation by IR (2–3 hr for maximal activation) suggests that transcription may be required for this response. Indeed, treatment of cells with IR in the presence of the transcription inhibitor Actinomycin D (ActD) abolishes Hpo activation. Thus, Hpo activation in response to IR requires new gene transcription, which could be mediated, at least in part, by p53. Hpo activity is induced by p53 expression, but Hpo protein itself does not appear to be a target of p53 because Hpo levels are not detectably upregulated when p53 is expressed in the posterior portion of the eye imaginal disc or in Dmp53-expressing clones in the wing disc. Future studies will be aimed at determining the exact mechanism through which Dmp53 promotes Hpo activation (Colombani, 2006).
This study has demonstrate by genetic and biochemical approaches not only that the Hpo pathway is required for the full apoptotic response induced by γ-ray irradiation but also that DNA damage triggers Hpo kinase activity in a p53-dependent manner both in vivo and in vitro. The apoptosis induced by p53 overexpression is strongly affected in hpo, wts, and sav mutant clones and p53 does not modulate Hpo levels. This study constitutes the first description of an upstream activating signal of the Hpo complex in vivo and during organism development (Colombani, 2006).
It is noted that the blockage of p53-induced apoptosis is not complete in hpo clones; this incomplete blockage likely reflects the role of other pro-apoptotic proteins, such as Reaper, Hid, and Sickle, in this process. Thus, it is proposed that, after exposure to ionizing radiations, the ATM, Chk2, p53 signaling pathway is activated and induces apoptosis by targeting expression of pro-apoptotic effectors such as Reaper, as well as by activating the Hpo pathway. This cell-death response to irradiation requires the caspase DRONC and leads to upregulation of JNK activity in a p53-dependent manner. Because Hpo has been shown to induce JNK activation when overexpressed in vivo, it will be interesting to determine whether Hpo is necessary for IR-induced JNK activation (Colombani, 2006).
Several reports have suggested that the mammalian homologs of members of the Hpo pathway might behave as tumor suppressors in humans. In addition, mice lacking the Wts homolog mLats1 are more sensitive to tumor-inducing agents. The current data suggest that one effect of mutations in Hpo-pathway members may be to protect these cells from DNA-damage-induced apoptosis and thus promote tumor progression and the accumulation of additional mutations. Further work on the Hpo pathway should further understanding of the DNA-damage response and its role in the transformation process (Colombani, 2006).
The 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).
Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).
Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).
Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).
In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).
In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).
In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).
To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).
The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).
The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).
Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).
Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).
To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).
Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of 32P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).
The conserved Hippo tumor suppressor pathway is a key kinase cascade that controls tissue growth by regulating the nuclear import and activity of the transcription co-activator Yorkie. This study reports that the actin-Capping Protein αβ heterodimer, which regulates actin polymerization, also functions to suppress inappropriate tissue growth by inhibiting Yorkie activity. Loss of Capping Protein activity results in abnormal accumulation of apical F-actin, reduced Hippo pathway activity and the ectopic expression of several Yorkie target genes that promote cell survival and proliferation. Reduction of two other actin-regulatory proteins, Cofilin and the cyclase-associated protein Capulet, cause abnormal F-actin accumulation, but only the loss of Capulet, like that of Capping Protein, induces ectopic Yorkie activity. Interestingly, F-actin also accumulates abnormally when Hippo pathway activity is reduced or abolished, independently of Yorkie activity, whereas overexpression of the Hippo pathway component expanded can partially reverse the abnormal accumulation of F-actin in cells depleted for Capping Protein. Taken together, these findings indicate a novel interplay between Hippo pathway activity and actin filament dynamics that is essential for normal growth control (Fernández, 2011).
The Hippo pathway has emerged as a crucial regulator of tissue size in both Drosophila and mammals. In Drosophila, the Hpo pathway comprises a kinase cascade in which the Hpo kinase binds the WW domain adaptor protein Salvador (Sav) to phosphorylate and activate the Warts (Wts) kinase. Wts, in turn, facilitated by Mats, phosphorylates and prevents nuclear translocation of the transcriptional co-activator Yorkie (Yki). This leads to transcriptional downregulation of target genes that positively regulate cell growth, survival and proliferation, including the Drosophila inhibitor of apoptosis protein 1 (Diap1; thread - FlyBase), expanded (ex), Merlin (Mer) and wingless (wg) in the inner distal ring, within the proximal wing imaginal disc. The upstream components Ex, Hpo and Wts are also thought to regulate Yki through a phosphorylation-independent mechanism, by directly binding to Yki, sequestering it in the cytosol and thereby abrogating its nuclear activity (Fernández, 2011).
Multiple upstream inputs are known to regulate the core Hpo kinase cassette at various levels. Thus, the atypical cadherin Fat was identified as an upstream component of the Hpo pathway and was proposed to transduce signals from the atypical cadherin Dachsous (Ds) and Four-jointed (Fj), a Golgi-resident kinase that phosphorylates Fat and Ds. Moreover, the two Ezrin-Radixin-Moesin (ERM) family members, Ex and Mer have been reported to lie upstream of the Hpo pathway. Mer and Ex can heterodimerize and are believed to exert their growth suppression activity by activating the Hpo kinase. However, how the different inputs that feed into the core kinase cassette are coordinated to regulate Yki activity is unknown (Fernández, 2011).
ERM proteins form a structural linkage between transmembrane components and actin filaments (F-actin). For instance, mammalian Mer binds numerous cytoskeletal factors, including actin, and appears to act as an inhibitor of actin polymerization. Interestingly, the Merlin-actin cytoskeleton association is required for growth suppression and inhibition of epidermal growth factor (EGFR) signaling. Moreover, F-actin depolymerization promotes activation of the Hpo orthologs MST1 and MST2 in mouse fibroblasts (Densham, 2009). These observations suggest a role for F-actin dynamics in modulating Hpo pathway activity (Fernández, 2011).
Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among these, the Capping Protein (CP) heterodimer, composed of α (Cpa) and β (Cpb) subunits, acts as a functional heterodimer to restrict the accessibility of the filament barbed end, inhibiting addition or loss of actin monomers (Cooper, 2008). In Drosophila, mutations in either cpa or cpb, lead to accumulation of F-actin within the cell and give rise to identical developmental phenotypes that are tissue specific. In the wing blade (BL), the most distal domain of the imaginal disc, cpa and cpb prevent cell extrusion and death, but they are not required for this function in the most proximal domain, the prospective body wall and the hinge wing imaginal disc (Janody, 2006). The Cofilin homolog Twinstar (Tsr) and the Cyclase-associated protein Capulet (Capt) also restrict actin polymerization: Tsr severs filaments and enhances dissociation of actin monomers from the pointed end, whereas Capt sequesters actin monomers, preventing their incorporation into filaments (Fernández, 2011).
This study investigated the relationship between the control of the actin cytoskeleton and Hpo pathway activity. Actin-binding proteins CP and Capt, but not Tsr, were shown to enhance Hpo signaling activity. Moreover, a new relationship was uncovered between the Hpo pathway and the machinery that regulates F-actin, and it was revealed that Hpo signaling activity, like CP, limits F-actin accumulation at apical sites independently of Yki. Finally, it is proposed that regulation of an apical F-actin network by Hpo signaling activity and CP sustains Hpo pathway activity, thereby limiting Yki nuclear import and the activation of proliferation and survival genes (Fernández, 2011).
This report shows an interdependency between Hpo signaling activity and F-actin dynamics in which CP and Hpo pathway activities inhibit F-actin accumulation, and the reduction in F-actin in turn sustains Hpo pathway activity, preventing Yki nuclear translocation and upregulation of proliferation and survival genes (Fernández, 2011).
It is suggested that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki. ERM proteins can form a structural linkage between transmembrane components and the actin cytoskeleton. Mammalian Mer appears to act as an inhibitor of actin polymerization. Moreover, the Mer-actin cytoskeleton association has a crucial role for growth suppression and inhibition of EGFR signaling. In Drosophila, Mer and Ex are structurally related and appear to have partially redundant functions but vary in their requirement depending on the tissue or developmental stage. In imaginal discs, loss of ex shows stronger phenotypes when compared with those of Mer. Ex might also have a stronger requirement on F-actin dynamics, as loss of ex, but not that of Mer, triggered F-actin accumulation. Surprisingly, loss of hpo, sav, mats or wts also triggered apical F-actin accumulation. Ex is likely to affect F-actin through activation of the Hpo kinase cassette because in most ex mutant clones, overexpressing hpo suppressed F-actin accumulation. Some clones seemed to contain increased F-actin. However, these clones also constricted apically, suggesting that the effect on F-actin levels results from a reduction of the apical cell surface and that in the absence of ex, differential activity of overexpressed hpo triggers cell shape changes. Together, these observations argue that Ex prevents apical F-actin accumulation through Hpo signaling activity but independently of Yki (Fernández, 2011).
Loss of Hpo pathway activity or CP triggerw apical F-actin accumulation. Ex localizes to the sub-apical region of epithelial cells, and colocalizes with an HA-tagged form of Cpa, Ex, Hpo, Sav and Wts all interact with each other through WW and PPXY motifs (Oh, 2009; Reddy, 2008). Therefore, a pool of Hpo, Sav and Wts, localized at apical sites, could directly regulate an actin-regulatory protein. Hpo pathway activity might act downstream of CP on F-actin. In agreement with this, ex overexpression significantly suppresses F-actin accumulation in cells with reduced CP levels. The role of Hpo signaling activity might be to inhibit an actin-nucleating factor, which adds new actin monomers to filament barbed ends free of the capping activity of CP. However, it cannot be excluded that ex overexpression enhances the activity of residual Cpa in cells knocked down using RNAi, nor that Hpo pathway activity acts in parallel to CP on F-actin. Interestingly, although endogenous Ex is upregulated in cells lacking CP, mutant cells still accumulated F-actin. wts mutant clones also upregulated Ex, which, when overexpressed, suppresses growth of wts mutant clones. Therefore, the increased levels of endogenous Ex in cells lacking either CP or wts appears to be insufficient to fully suppress the effects of loss of CP or wts on F-actin and growth, respectively (Fernández, 2011).
The data indicate that CP inhibits Yki nuclear accumulation, activation of Yki target genes, and consequently overgrowth of the proximal wing epithelium. Interestingly, Yki was also found to accumulate in nuclei of wild-type cells adjacent to the clone border. Consistent with a non-autonomous effect of CP loss on Hpo pathway activity, ex-lacZ and diap1-lacZ were upregulated in wild-type cells adjacent to CP mutant clones. However, Ex levels were reduced in wild-type neighboring cells. Cells expressing different amounts of ds and fj also upregulate ex-lacZ, but show reduced levels of Ex. Therefore, loss of CP might affect Fj or Ds levels, creating a sharp boundary of their expression. However, in contrast to clones overexpressing ds or mutant for fj, cell lacking CP also upregulated Ex and Mer inside the mutant clones, indicating that CP also acts cell-autonomously to promote Hpo signaling activity. CP might facilitate Yki phosphorylation by the Hpo kinase cassette as cpa-depleted tissues contain decreased phospho-Yki levels. But, the possibility cannot be excluded that CP also favors the direct binding of non-phosphorylated Yki to Ex, Hpo or Wts (Oh, 2009). Further analysis will be required to elucidate the mechanisms by which CP restricts Yki activity cell autonomously and in wild-type neighboring cells (Fernández, 2011).
The results argue for a constitutive role of CP in Hpo pathway activity, since Yki target genes are upregulated in the whole wing and eye imaginal discs. However, loss of CP did not fully recapitulate the phenotype for core components of the hpo pathway. Despite that, on average, cpb mutant clones located in the proximal wing disc domain were 25% larger than wild-type twin spots; 60% of mutant clones failed to grow. Moreover, in the distal wing epithelium, reducing CP levels induces mislocalization of the adherens junction components Armadillo and DE-Cadherin, extrude and death. Furthermore, in Drosophila, CP also prevents retinal degeneration (Delalle, 2005; Johnson, 2008). This indicates that although loss of CP can, under certain conditions, result in tissue overgrowth due to inhibition of Hpo pathway activity, other factors such as the polarity status, also determine the survival and growth of the mutant tissue. Therefore, in addition to promoting Hpo pathway activity, CP has additional developmental functions in epithelia. However, the possibility cannot be excluded that, like most upstream inputs that feed into the Hpo pathway, CP has a tissue-specific requirement in Hpo pathway activity (Fernández, 2011).
CP, Capt and Tsr all restrict F-actin assembly directly. CP and Capt control F-actin formation near the apical surface and inhibit ectopic expression of Yki target genes, whereas Tsr acts around the entire cell cortex and has no effect on Yki target genes. This argues that Hpo signaling activity is not affected by the excess of F-actin per se but provides significant support to the view that stabilization of an apical F-actin network by CP, Capt and Hpo signaling activity feeds back on the Hpo pathway to sustain its activity (Fernández, 2011).
These findings do not lead to an understanding of where F-actin accumulation intersects Hpo signaling activity because both Hpo signaling activity and F-actin dynamics feedback to each other. For instance, hpo or ex overexpression suppressed growth of CP-depleted cells. But, overexpressed ex and possibly hpo also suppress F-actin accumulation of Cpa-depleted cells. The control of F-actin by Hpo signaling activity and CP might constitute a parallel input, which sustains Hpo pathway activity. Alternatively, F-actin could act upstream of the core kinase cascade, which in turn feeds back to F-actin, to maintain its activity. The identification of additional actin cytoskeletal components that either promote Hpo pathway activity or act downstream of Hpo pathway activity on F-actin would help to discriminate between these possibilities (Fernández, 2011).
How F-actin influences Hpo signaling activity remains to be determined. The apical F-actin network, which regulates the formation and movement of endocytic vesicles from the plasma membrane, might promote the recycling or degradation of Hpo pathway components. Increased F-actin at apical sites would, therefore, affect protein turnover. Alternatively, apical F-actin might act as a scaffold to tether Hpo pathway components apically. In support of this, Ex, Hpo, Sav, Wts and Yki could all interact between each other through WW and PPXY motifs at apical sites (Oh, 2009; Reddy, 2008). Moreover, expression of a membrane-targeted form of Mats enhances Hpo signaling (Ho, 2010). Although Ex and Mer are properly localized in CP mutant cells, other members of the pathway might be mislocalized in the presence of excess F-actin. Interestingly, in mouse fibroblasts, the Hpo orthologs MST1 and MST2 colocalize with F-actin structures and are activated upon F-actin depolymerization (Densham, 2009), suggesting that by tethering Hpo pathway components, F-actin dynamics modulates their activity. Finally, the F-actin network might act as a mechanical transducer. Most of the mechanosensitive responses require tethering to force-bearing actin filaments. Tissue surface tension has been proposed to be a stimulus for a feedback mechanism that could regulate tissue growth. The tension exerted by neighboring cells might be sensed at the cell membrane by the actin cytoskeleton and translated to the regulation of cell proliferation through the Hpo signaling pathway (Fernández, 2011).
In a genetic screen mutations were isolated in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha). PI4KIIIalpha was found to be is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. This analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade (Yan, 2011).
DV asymmetry of the Drosophila oocyte is established during mid-oogenesis through a repolarization process initiated in the posterior follicle cells PFCs. In response to an unknown signal from the PFCs the oocyte nucleus migrates from the posterior end to the dorsal-anterior corner of the oocyte. As a consequence, the Gurken (Grk) protein no longer accumulates at the posterior cortex of the oocyte, but is now found in the dorsal-anterior membrane overlying the oocyte nucleus where it activates EGFR to initiate DV patterning. In a genetic screen directed at FC components affecting this repolarization process, a complementation group with six lethal mutant alleles was isolated, and initially named after a representative allele, GS27. When the PFCs were mutant for the GS27 gene product, the oocyte nucleus frequently remained at the posterior end of the oocyte. This phenotype was confirmed by the abnormal posterior localization of Grk in late egg chambers (Yan, 2011).
The lethality of the GS27 complementation group was mapped through duplication and deficiency mapping to the X-chromosomal region 3A4-3A8, which contains 16 genes. Sequencing of candidate genes showed that four alleles of the GS27 complementation group contained mutations that lead to premature stop codons in the coding region of CG10260, a predicted phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the generation of PIP4. Phosphoinositides, including PIP4, are important phospholipids in the cell membrane that participate in numerous signaling events. Four classes of PI4Ks have been identified in mammalian cells that localize to different cellular compartments and are likely to perform non-redundant functions. Three PI4K genes have been annotated in the fly genome: four wheel drive (fwd; PI4KIIIbeta), CG2929 (PI4KIIalpha) and CG10260 (PI4KIIIalpha) (Yan, 2011).
To investigate the oocyte polarization defects caused by PI4KIIIalpha mutations, the localization of well-established oocyte polarity markers was examined. The microtubule cytoskeleton is polarized in the oocyte. The microtubule plus-end marker Kinesin (Kin, or Khc) fused to β-gal (Kin-β-gal), which normally forms a crescent at the posterior of the oocyte after stage 8, was examined. When the PFCs were mutant for PI4KIIIalpha, Kin-β-gal either localized to the center of the oocyte or was diffuse in the oocyte. Staufen localizes to the posterior pole of wild-type oocytes after stage 8 and is required for the localization of maternal RNAs. In PFC clones mutant for PI4KIIIalpha, Staufen also frequently mislocalized to the center of the oocyte or became dispersed in the oocyte. Therefore, in combination with the mislocalization of the oocyte nucleus, these results demonstrate that PI4KIIIalpha is required in the PFCs for all aspects of the establishment of correct oocyte polarity (Yan, 2011).
Oocyte polarization relies on the integrity of four signaling pathways in the PFCs: Notch, JAK/STAT, EGFR and Hippo. To examine whether the polarization defect observed in PI4KIIIalpha mutants was caused by disruption of one of these signaling pathways, well-established downstream targets of each pathway were examined in PI4KIIIalpha mutants (Yan, 2011).
The EGFR signaling reporter kekkon-lacZ (kek-lacZ) is highly expressed in the PFCs at stage 7 and 8 as a result of EFGR activation by Grk. In PFCs mutant for PI4KIIIalpha, the kek-lacZ expression level was comparable to that of wild-type PFCs, indicating that EFGR signaling was unaffected. The JAK/STAT signaling reporter 10×STAT92E-GFP is normally turned on in the PFCs during stage 7 and 8 in response to JAK/STAT activation. Apparently normal levels of GFP were detected in the nuclei of PI4KIIIalpha mutant PFCs, suggesting that JAK/STAT signaling was also intact (Yan, 2011).
Notch signaling is required for FCs to exit the mitotic cell cycle at stage 6 and switch to an endocycle. PI4KIIIalpha mutant PFCs maintained a mitotic cell cycle after stage 6, as indicated by the sustained staining of the mitotic marker phosphorylated Histone H3 (PH3), which is only seen up to stage 6 in wild-type FCs. Consistent with a failure to exit the mitotic cycle, the PI4KIIIalpha mutant PFCs often lost their monolayered epithelial structure and had smaller nuclei than neighboring cells. The expression of two Notch signaling targets, Cut and Hindsight (Hnt; Pebbled) was examined. In wild-type FCs, Cut expression is downregulated whereas Hnt expression is upregulated upon Notch activation at stage 6. PI4KIIIalpha mutant PFCs frequently failed to downregulate Cut and upregulate Hnt expression. Interestingly, PI4KIIIalpha mutant cells on the lateral side of the egg chambers showed no defect in Notch signaling. These results suggest that PI4KIIIalpha mutations compromise Notch signaling in the PFCs only (Yan, 2011).
The phenotypes described above are similar to those caused by mutations in Hippo pathway components. In particular, the observation that only PFCs appear affected is characteristic of mutations in the Hippo pathway, which are reported to affect Notch signaling only in this group of FCs. When the expression of a Hippo pathway target, ex, was checked using the enhancer trap line ex-lacZ, a much higher level of β-galin PI4KIIIalpha mutant FCs was detected than in wild-type cells. This upregulation was observed in all FCs, regardless of their position. Another Hippo pathway target, Diap1, monitored with the enhancer trap line diap1-lacZ, was mildly upregulated in the PI4KIIIalpha mutant FCs. These results indicate that the polarization defect in the PI4KIIIalpha mutants is likely to be caused by defective Hippo signaling (Yan, 2011).
Multiple lines of evidence suggest that the apical localization of the Expanded-Merlin-Kibra complex is crucial for Hippo signaling activity as it is proposed to function as a platform to bring the core Hippo components into close proximity and facilitate the phosphorylation reactions. In addition, it has been reported that Expanded directly interacts with Yki and functions to sequester Yki in the cytoplasm (Yan, 2011).
To investigate how mutations in PI4KIIIalpha lead to defective Hippo signaling, the apical localization of the Merlin-Expanded-Kibra complex was exmined. The complex is confined to the apical domain in wild-type FCs. In the PI4KIIIalpha mutant cells, a loss of apical Merlin staining was observed, whereas Expanded and Kibra were upregulated at the apical membrane. In addition to being Hippo pathway regulators, Expanded and Kibra are also targets of the Hippo signaling pathway. Mutations in Hippo pathway components lead to upregulation of Expanded and Kibra. In addition, it has been reported that the apical sorting of Merlin, Expanded and Kibra occur independently of each other. Therefore, the absence of Merlin from the apical membrane in PI4KIIIalpha mutant cells is the likely cause of the signaling defect, and the upregulation of Expanded and Kibra would be an expected secondary consequence of the disrupted Hippo signaling (Yan, 2011).
When PI4KIIIalpha mutant clones were examined in the imaginal eye discs of early second instar larvae, an absence of Merlin from the apical and junctional region was observed. However, no overgrowth phenotype typical of Hippo pathway mutations was observed. In fact, adults with mutant eye clones had smaller eyes than wild-type adults. Eye discs from late L2 larvae exhibited pyknotic nuclei staining in PI4KIIIalpha mutant clones, indicating death of the mutant cells (data not shown) (Yan, 2011).
Multiple classes of PI4Ks exist in eukaryotic cells that participate in producing various phosphoinositide species in distinct cellular compartments. Three PI4K genes have been annotated in the fly genome. When the intracellular distribution and level of PIP2 was examined using a Ubi-PH-PLCδ-GFP reporter, a complete absence of PIP2 from PI4KIIIalpha mutant FCs was observed in rare cases. In most cases, the PIP2 reporter was specifically lost from the apical plasma membrane in the mutant cells. The yeast homolog of PI4KIIIalpha, Stt4p, localizes to patches on the plasma membrane where it is required for normal actin cytoskeleton organization. When the actin cytoskeleton of PI4KIIIalpha mutant FCs was examined by phalloidin staining, they exhibited abnormal actin-enriched spike structures on their apical domain that were positively marked by the microvillus marker Cad99C, suggesting that the spikes were malformed microvilli. As mutations in the Hippo pathway have been reported to lead to apical domain expansion, one possibility is that the malformed microvilli are caused by defective Hippo signaling. However, the morphology of the actin-enriched spikes in PI4KIIIalpha mutant cells is distinct from that caused by mutations in the Hippo pathway, suggesting that the loss of PI4KIIIalpha might also have a Hippo-independent effect on apical membrane structure (Yan, 2011).
How could PI4KIIIalpha mutations cause Merlin mislocalization? Expanded and Merlin are ERM (Ezrin, Radixin and Moesin)-related proteins, which are key linkers of the plasma membrane and cytoskeleton. Classical ERM proteins bind to PIP2 in the membrane to switch from a closed to an open conformation for their activation. Significantly, in PI4KIIIalpha mutant cells, phosphorylated ERM proteins were absent from the apical microvilli region as indicated by a phospho-ERM-specific antibody. The malformed microvillus structure might therefore indicate a general failure of ERM protein activation in the PI4KIIIalpha mutant cells. For Merlin, the closed conformation is the active form, opposite to other ERM proteins. Nevertheless, Merlin undergoes a similar conformational switch to the other ERM proteins and contains an ERM PIP2-binding site. Given these observations, it is possible that PIP2 binding activates and/or stabilizes Merlin in the apical membrane, and a depletion of this lipid species due to the absence of PI4KIIIalpha might directly lead to the loss of Merlin (Yan, 2011).
In summary, this study has shown that PI4KIIIalpha is required in the FCs for Merlin localization and Hippo signaling. PI4KIIIalpha mutations in the PFCs lead to a Notch signaling defect and the subsequent failure of oocyte repolarization, which are precisely the phenotypes reported for Hippo mutations in the FCs. This effect is likely to be caused by a change in lipid composition in the membrane. How the abnormal actin structures are generated in the mutant cells, and whether they have a direct role in Merlin localization, remain to be investigated (Yan, 2011).
The dMST mutant phenotypes closely resemble those caused by sav or wts mutations. It has been shown that Sav and Wts physically and genetically interact, suggesting that they may act in common pathways. To determine if dMST could act in the same pathways, coimmunoprecipitation assays with Sav and Wts were performed. S2 cells were transfected with DNA constructs expressing HA-tagged Sav and Flag-tagged full-length dMST (dMSTf), its N-terminal fragment containing the kinase domain (dMSTn), or its C-terminal fragment containing the regulatory domains (dMSTc). Both dMSTf and dMSTc, but not dMSTn, coimmunoprecipitate with Sav, suggesting that dMST binds Sav through its C-terminal regulatory region. The dimerization domain at the C terminus of dMST appears to be essential for interaction as deletion of this domain from dMSTc abolishes its ability to bind Sav (Jia, 2003).
To define the domain in Sav that binds dMST, a series of truncated forms of Sav were generated. Both C-terminal fragments, SavC1 and SavC2, bind dMST. In contrast, all the C-terminally truncated fragments, including SavDeltaC1, SavDeltaC2, and SavDeltaC3, fail to bind dMST, suggesting that dMST binds the C-terminal region of Sav and the coiled-coil domain of Sav is essential (Jia, 2003).
dMST also binds Wts. S2 cells were transfected with DNA constructs expressing Myc-tagged Wts and Flag-tagged dMSTf, dMSTn, or dMSTc. Wts binds dMSTf and dMSTn, but not dMSTc, suggesting that Wts interacts with the N-terminal region of dMST. The interaction between Wts and dMST is not affected by Sav, since coexpression of Sav does not increase the amount of dMSTf coimmunoprecipitated with Wts. However, it remains possible that Sav might regulate dMST/Wts interaction in vivo at physiological concentration. Taken together, these results suggest that dMST, Wts, and Sav form a complex in which Wts and Sav bind the kinase and regulatory domains of dMST, respectively (Jia, 2003).
Sav quantities increase markedly in the presence of Hpo. Furthermore, Sav mobility on acrylamide gels shifts toward higher molecular weights. Sav immunoprecipitated from lysates containing Hpo was treated with phosphatases and it was found that this band shift disappeared, confirming that Sav becomes phosphorylated in the presence of Hpo. Sav is also stabilized by treatment with the proteasome inhibitor LLnL, suggesting that Sav is normally targeted for degradation by the proteasome. Unexpectedly, kinase-dead Hpo also induces stabilization and a mobility shift of Sav, whereas Hpo lacking the Sav-binding domain has little effect (Pantalacci, 2003).
Because Hpo is a kinase, the possibility that it might phosphorylate DIAP1 was investigated. Epitope-tagged Hpo was immunoprecipitatedfrom S2 cell lysates, and the ability of these immunoprecipitates to phosphorylate bacterially expressed DIAP1 was tested. Whereas Hpo induces DIAP1 phosphorylation in this assay, a kinase-dead (K71R) mutant of Hpo (HpoKD) does not. This activity is unimpaired by removal of the Sav-binding domain (amino acids 602-669) of Hpo (HpoC). DIAP1 phosphorylation by Hpo might be direct because bacterially expressed Hpo is also able to phosphorylate DIAP1 tagged with glutathione S-transferase (GST) in vitro. In addition, Hpo can autophosphorylate, as has been reported for its mammalian ortholog (Dan, 2001). Constructs expressing epitope-tagged Hpo and DIAP1 were transfected into S2 cells, and the cells were subjected to metabolic labelling. When normalized for the reduction in DIAP1 expression on cotransfection of Hpo, DIAP1 labelling increased 2.5-fold in the presence of Hpo. Thus, Hpo-dependent phosphorylation of DIAP1 can occur in live cells (Pantalacci, 2003).
The effect of Hpo on DIAP1 quantities was examined. Constructs expressing epitope-tagged DIAP1 plus Hpo and/or Sav, as well as a LacZ control plasmid, were transfected into S2 cells. On co-transfection of DIAP1 and Hpo, quantities of DIAP1 were reduced, and this effect was increased to more than twofold in the presence of Sav. By contrast, Sav alone or plus HpoC had only a minor effect on DIAP1, whereas Wts and HpoKD had no effect. To characterize further the effect of Hpo and Sav on DIAP1, S2 cells transfected with constructs expressing DIAP1 plus/minus Hpo and Sav were treated with the translation inhibitor cycloheximide (CHX). This experiment was done in the presence of caspase inhibitors to block caspase-induced DIAP1 degradation. The efficiency of caspase blockage was verified by Western blot analysis of DIAP1. Under these conditions, overexpressed DIAP1 is stable (95% remaining after 6 h). In the presence of Hpo and Sav, however, DIAP1 is less abundant and considerably destabilized (35% remaining after 6 h). Thus, Hpo decreases the stability of DIAP1 in S2 cells (Pantalacci, 2003).
Two independent clones of hpo were isolated in a yeast two-hybrid screen by using Sav as bait. Both clones contained the autoregulatory and dimerization domains. To characterize this interaction further, 35S-labelled Hpo and Sav proteins were produced by coupled in vitro transcription and translation, and they were assayed for complex formation by co-immunoprecipitation. Full-length Sav co-purifies Hpo, and systematic deletion analyses show that the dimerization domain of Hpo and the conserved Sav-specific domain (SD) of Sav are both necessary and sufficient for this interaction. The dimerization domain of Hpo is also necessary and sufficient for dimerization of Hpo itself, similar to its vertebrate homolog MST1. However, Sav and Hpo do not form tetrameric complexes in this assay. The interaction between Hpo and Sav is probably direct because it is also observed in yeast, which lacks this signalling pathway. All four hpo alleles produce C-terminally truncated proteins in which the region that interacts with Sav is deleted, indicating that the interaction between Hpo and Sav is important in vivo. The previously identified savshrp allele specifically deletes the SD domain and causes phenotypes that are as strong as those of sav null alleles, further supporting this conclusion. The other conserved domains of Sav, the two WW domains, bind to Wts. Thus, Sav may act as a scaffold to assemble signalling complexes comprising Hpo and Wts kinases (Udan, 2003).
The similarity of the wts, sav, and hpo mutant phenotypes suggests that the three genes function in the same pathway and that all three proteins may exist in the same protein complex. The Sav protein has domains capable of interacting with other proteins, including a WW domain and a C-terminal portion that is predicted to form a coiled-coil. Sav may therefore function as a scaffold in a multiprotein complex. The WW domains of Sav have been shown to bind to the PPXY motifs in the N-terminal portion of Wts. Whether Hpo can also bind to Sav was tested. Sav was expressed in E. coli as a fusion with maltose binding protein (MBP). After stringent washing, MBP-tagged Sav (MBP-Sav) coupled to amylose resin showed significant binding to in vitro translated Hpo but MBP coupled to amylose resin did not. Hpo also bound to in vitro translated Sav expressed as a Myc-tagged fusion (MTSav). Parallel binding experiments with proteins corresponding to the truncated versions of Hpo generated by the hpoMGH1, hpoMGH2, and hpoMGH3 alleles showed that all three mutant proteins had markedly reduced in vitro binding to both MT-Sav and MBP-Sav. These experiments indicate that the C-terminal portion of Hpo is necessary for binding to Sav. Since the protein encoded by the hpoMGH3 allele lacks only the C-terminal 49 amino acids, it is likely that Sav interacts with the conserved C-terminal domain of Hpo. Alternatively, conformational changes induced by the deletion of the C-terminal portion may preclude interaction with Sav (Harvey, 2003).
To determine if Sav and Hpo interact directly or via an accessory protein found in reticulocyte lysates, MBP-Sav coupled to amylose resin was incubated with either partially purified bacterial His-tagged Hpo or a control bacterial lysate. Immunoblotting with an anti-His antibody showed that Hpo interacted with MBP-Sav but not to MBP alone, suggesting that Hpo and Sav can interact with each other directly (Harvey, 2003).
To define the portion of Sav that is capable of interaction with Hpo, different domains of the Sav protein were expressed as fusions with MBP. These fusion proteins were tested for their ability to bind in vitro translated Hpo. Binding of the portion of Sav N-terminal to the WW domain (Sav-N) was comparable to that of full-length Sav (Sav), however no binding was detected with the fusion proteins containing the WW domains of Sav (Sav-WW) or the C-terminal coiled-coil domain (Sav-CC). Thus, Hpo appears to bind predominantly to the N-terminal portion of Sav. In further experiments using in vitro translated MTSav domains, some binding of full-length Hpo to the coiled-coil region of Sav was observed. This might imply a second site of interaction between the two proteins or be the result of the 'stickiness' of the coiled-coil domain (Harvey, 2003).
To examine whether Hpo and Wts bind competitively to Sav or at distinct sites, increasing amounts of in vitro translated Wts were added to the binding reaction. Would this addition of Wts reduce the binding of Hpo to Sav? Even the addition of four times the amount of Wts as Hpo had no effect on the binding of Hpo to Sav. This is consistent with the notion that Hpo and Wts bind to different portions of Sav, i.e., the N-terminal portion and the WW domains respectively and is consistent with the possibility that the three proteins can form a ternary complex (Harvey, 2003).
The interaction of hpo with sav and wts was examined in vivo. Combined overexpression of sav and wts in the eye generates a small rough eye phenotype, which is mostly due to increased cell death. When hpo clones were generated in eyes overexpressing both sav and wts, eye size was significantly restored. This suggests that hpo is required for the ability of sav and wts to induce cell death. If, as the binding data suggest, Hpo functions in a complex with Sav and Wts, then Hpo function appears necessary for the proapoptotic activity of the complex (Harvey, 2003).
At a stage of development (38 hr APF) when excess interommatidial cells are eliminated by apoptosis, most of the cell death in discs containing hpo clones was restricted to wild-type portions of the disc. The DIAP1 protein, an antagonist of caspases, is elevated in sav clones. hpo clones posterior to the MF also have elevated DIAP1 levels. Regulation of DIAP1 by hpo is likely to occur largely at the posttranscriptional level since DIAP1 RNA expression, as assessed by either in situ hybridization or by RT-PCR, was not obviously elevated in eyes composed almost entirely of hpo mutant tissue. DIAP1 RNA expression was also examined in S2 cells treated with RNAi designed to reduce expression of Hpo. While DIAP1 protein levels were increased 1.5-fold in cells treated with Hpo RNAi, no obvious difference in DIAP1 RNA expression was seen. DIAP1 levels are also elevated in wts cells. Therefore the defect in apoptosis in sav, wts, and hpo tissue is likely due to the result of elevated levels of DIAP1 in mutant clones that renders caspases inactive in these cells (Harvey, 2003).
Proteins such as Hid, Rpr, and Grim are thought to downregulate DIAP1 levels by stimulating its autoubiquitination or by repressing general protein translation, which has the greatest effect on short-lived proteins such as DIAP1. To investigate whether hpo can modify the function of such proteins, hpo clones were generated in flies overexpressing the grim gene under the control of the GMR promoter. When overexpressed in the Drosophila eye, grim induces extensive cell death as visualized by TUNEL, which results in a small, rough eye. When hpo clones were generated in eyes overexpressing Grim, eye size was significantly restored and Grim-induced cell death was greatly reduced in hpo mutant clones. sav and wts clones are relatively resistant to cell death induced by Rpr or Hid. The increased basal level of DIAP1 found in sav, wts, or hpo clones may make it more difficult for proteins such as Rpr, Hid, or Grim to reduce DIAP1 levels sufficiently to activate caspases in these cells (Harvey, 2003).
Since DIAP1 protein levels are elevated in hpo clones and in S2 cells treated with hpo RNAi, the possibility that hpo might regulate DIAP1 stability was tested. When hpo is overexpressed in Drosophila S2 cells, endogenous DIAP1 protein is consistently reduced to approximately 60%-70% of normal levels, when normalized to loading controls. Since Wts and Hpo are both predicted to have kinase activity, it is possible that a complex consisting of Sav, Hpo, and Wts regulates the phosphorylation state of DIAP1 and hence regulates its turnover. Indeed, both Sav-associated and Hpo-associated complexes are capable of phosphorylating DIAP1 in vitro, and DIAP1 is destabilized in the presence of Hpo, presumably as a result of Hpo-dependent phosphorylation of DIAP1 (Harvey, 2003).
The coordination between cell proliferation and cell death is essential to maintain homeostasis within multicellular organisms. The mechanisms underlying this regulation are yet to be completely understood. hippo has been identified as a gene that regulates both cell proliferation and cell death in Drosophila. hpo encodes a Ste-20 family protein kinase that binds to and phosphorylates the tumor suppressor protein Salvador, which is known to interact with the Warts protein kinase. Loss of hpo results in elevated transcription of the cell cycle regulator cyclin E and the cell-death inhibitor diap1, leading to increased proliferation and reduced apoptosis. Further, hpo, sav, and wts define a pathway that regulates diap1 at the transcriptional level. A human homolog of hpo completely rescues the overgrowth phenotype of Drosophila hpo mutants, suggesting that hpo might play a conserved role for growth control in mammals (Wu, 2003).
A yeast two-hybrid screen was carried out in the hope of identifying Hpo binding proteins. Approximately 1 million cDNA clones were screened using as bait the noncatalytic C-terminal portion of Hpo. Interestingly, 6 out of 12 positive clones isolated from the screen corresponded to Sav, representing 3 different classes of clones. These Hpo-interacting Sav clones define the C-terminal half of Sav (residues 362-607) as an Hpo binding region. This region contains predicted Sav WW and coiled-coil domains. Another yeast two-hybrid screen was carried out using the C-terminal half of Sav as the bait. In this screen, 5 out of 45 positive clones isolated from the screen corresponded to Hpo, representing 4 different classes of clone. These Sav-interacting Hpo clones define the C-terminal portion of Hpo (residues 474-669) as a Sav binding region. The identification of Hpo and Sav as interacting proteins in unbiased yeast two-hybrid screens provides strong evidence that these proteins interact with each other in vivo. Consistent with this hypothesis, Hpo and Sav associate with each other in vitro. GST fusion protein containing full-length Sav, but not a control GST fusion protein, is able to specifically pull-down endogenous Hpo protein from S2 cell extracts. Hpo and Sav also interact with each other in coimmunoprecipitation assays (Wu, 2003).
Next, whether Hpo can function as a Sav kinase was tested. For this purpose, a cotransfection assay was established in S2 cells. Coexpression of Hpo and Sav results in retarded mobility of Sav, leading to the formation of multiple slower migrating bands. Phosphatase treatment abrogates this shift, suggesting that the mobility shift is due to protein phosphorylation. In contrast, coexpression of Sav and Wts, also a Ser/Thr kinase, does not result in Sav mobility shift, nor does expression of Wts affect the phosphorylation of Sav by Hpo. In vitro, myc-tagged Hpo protein specifically phosphorylates a GST fusion protein containing the Hpo binding region of Sav. Thus, Hpo phosphorylates Sav. These results presented above suggest a model wherein the C-terminal domain of Hpo associates with Sav and presents Sav to the Hpo kinase. If so, a kinase-dead mutant of Hpo, or the C-terminal noncatalytic domain of Hpo expressed alone, should behave as dominant-negative forms, since these variants should associate nonproductively with endogenous Sav and interfere with signal propagation. Indeed this is the case (Wu, 2003).
Having established a functional link between Hpo and Sav and given the results from a genetic analyses implicating hpo, sav, and wts in a common pathway, whether Wts might be regulated by Hpo and/or Sav was tested. In S2 cells, expression of Hpo results in retarded mobility of Wts, while coexpression of Hpo and Sav results in a further mobility shift of Wts. For simplicity, this further shift of Wts upon coexpression of Hpo and Sav is referred to as 'supershift' to be distinguished from the mobility shift caused by expression of Hpo alone. Both shifts are largely abolished by phosphatase treatment, confirming that the shifts are due to phosphorylation. Taken together, these data suggest that Sav increases the ability of Hpo to phosphorylate Wts (Wu, 2003).
The mobility shift assay described the narrowing down of the domain of Wts that is the target of Hpo-mediated phosphorylation to a region at the N-terminal noncatalytic portion (residues 68-414) of the Wts protein. In vitro, a GST fusion protein containing this region of Wts is phosphorylated by Hpo. Consistent with Wts as a kinase substrate of Hpo, the mobility of endogenous Wts protein on SDS-PAGE is increased in Hpo mutant animals (Wu, 2003).
These results suggest a model wherein Hpo associates with and phosphorylates Sav and interactions between Hpo and Sav facilitate Wts phosphorylation by Hpo. This model is consistent with a direct physical interaction between Sav and Wts. Thus, Sav could be viewed as an adaptor protein that brings Hpo in proximity to Wts to facilitate Wts phosphorylation. Since the Sav WW domains have been implicated in Sav/Wts interaction, it is speculated that the coiled-coil domain of Sav, located C-terminal to the WW domains, might be involved in Sav/Hpo interaction. Interestingly, the shrp6 allele of sav causes a frameshift mutation that truncates just the coiled-coil domain but leaves the WW domains intact. To pinpoint the functional defect of the savshrp6 allele, a mutant Sav protein, Savshrp6, was engineered that lacks the C-terminal 79 residues as seen in savshrp6, and the ability of this mutant protein to associate with Hpo and to facilitate Wts phosphorylation by Hpo was examined. Unlike wild-type Sav, Savshrp6 can not associate with Hpo, suggesting that the coiled-coil domain of Sav is required for Hpo/Sav interaction. Importantly, coexpression of Savshrp6 and Hpo can no longer cause the supershift of Wts as seen when wild-type Sav and Hpo are coexpressed. Thus, Hpo/Sav interaction is required for Sav to facilitate the phosphorylation of Wts by Hpo (Wu, 2003).
Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation. Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation. This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity. In this study it is shown that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor function (Polesello, 2006).
The mammalian RASSF family comprises six different loci encoding a variety of splice variants. Most transcripts encode proteins that contain a Ras association domain (RA), an N-terminal C1-type zinc finger, and a C-terminal SARAH (Sav RASSF Hippo) domain. RASSF family members, most notably RASSF1A, are frequently silenced in a variety of solid tumors. Thus, it has been proposed that RASSF genes act as tumor suppressors (Polesello, 2006).
The biological function of these genes is not well understood. RASSF1A and Nore1A have both been shown to interact with MST1 via its SARAH domain. Overexpression of RASSF1A or Nore1A inhibits MST1 activation, but coexpression of these RASSF proteins with Ras enhanced MST1 activity. RASSF1A knockout mice have mildly increased tumor susceptibility, confirming that RASSF genes can act as tumor suppressors. The weakness of the mouse phenotype, which is at odds with the frequency of RASSF1A inactivation in human tumors, can be ascribed to redundancy with other family members (Polesello, 2006).
By contrast, Drosophila melanogaster has a single RASSF family member, which is encoded by the CG4656 gene and which will be referred to as dRASSF. Like its vertebrate counterparts, dRASSF encodes a protein bearing an RA and SARAH domain at its C terminus. It also possesses a LIM domain that shares some similarities with C1 zinc fingers at its N terminus (Polesello, 2006).
Mutant alleles of dRASSF were generated by imprecise excision of two nearby transposons, GE23517 and EY2800. Multiple alleles, which delete up to the fourth intron, including the initiating ATG, were obtained. Some transcript was still detected in dRASSFX16, dRASSFX36, but a strong reduction was found in dRASSF44.2, which lacks the transcription start. However, antibodies raised against the C terminus (amino acids 792–806) and a nonconserved region (amino acids 294–308) of dRASSF showed that full-length dRASSF is absent in lysates from all mutant lines, suggesting the dRASSF mutants are indeed loss-of-function mutations for the locus. All of these alleles were viable and behaved identically in subsequent assays. In addition, dRASSF staining was severely reduced in FLP/FRT-generated dRASSF mutant clones in the eye-imaginal disc, the larval precursor to the adult eye (Polesello, 2006).
Although the dRASSF mutant flies are viable, they present a clear growth defect in comparison to wild-type animals when reared in carefully controlled conditions. dRASSF mutant flies were 15% lighter than their wild-type counterparts, a phenotype which was significantly rescued by introduction of a single copy of a dRASSF rescue construct, although wild-type levels of dRASSF were not fully restored. dRASSF mutant flies were fully fertile and normally proportioned but sensitive to γ-irradiation. Wing surface area was reduced by 8% in dRASSF mutant flies, whereas wing hair density was unaffected. This suggests that the growth defect of dRASSF mutant flies is due to a reduction in cell number and not a defect in cell size (Polesello, 2006).
In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity. Therefore whether dRASSF can interact with Hpo was tested. Coimmunoprecipitation (Co-IP) experiments were performed in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo. The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (HpoΔC) lacking this domain fails to bring down dRASSF. Thus, the Hpo SARAH domain can associate with both Sav and dRASSF (Polesello, 2006).
Sav is stabilized by the presence of Hpo. Therefore whether dRASSF levels are modulated by Hpo was tested. dRASSF immunostaining was reduced in clones mutant for a hpo allele that lacks the SARAH domain. In addition, RNAi-mediated depletion of Hpo from Drosophila Kc cells resulted in a reduction of endogenous dRASSF expression, whereas dRASSF transcripts were unaffected. By contrast, dRASSF levels were unaffected in clones mutant for other Hpo-pathway members, such as ex, sav, and wts. These results suggest that direct binding to Hpo through its SARAH domain, rather than signaling through the Hpo pathway, is necessary for dRASSF stability. This is analogous to the situation for Sav, which is also stabilized by a kinase-dead form of Hpo (Polesello, 2006).
Because Hpo, Sav, and dRASSF all contain a SARAH domain, it was speculated that dRASSF might also bind Sav. To test this, whether dRASSF interacts with Sav was investigated by co-IP but no such an interaction was detected. Because the possibility of a ternary complex had been raised by another study, whether the three proteins could be found in the same complex was tested. Hpo, Sav, and dRASSF were co-expressed in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF. However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav. Identical results were obtained with endogenous IPs by using dRASSF and Sav antibodies. These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes (Polesello, 2006).
Sav has been shown to be a positive regulator of the Hpo pathway, whereas genetic results suggest that dRASSF might antagonize Hpo function. It was therefore of interest to determining whether complexing with Sav or dRASSF might influence Hpo activity. Immunoprecipitates were probed with an phospho-MST1 antibody that recognizes phosphorylated (active) Hpo. Interestingly, although Hpo that was coimmunoprecipitated with dRASSF showed barely detectable levels of phosphorylation, the Sav-associated fraction was highly phosphorylated. Thus, Hpo can exist as two pools, a highly active Sav-associated pool and an inactive dRASSF-associated pool. This correlates with data showing that Nore1 can repress MST1 activity in mammalian cells. This also suggests that Sav can promote Hpo activation and provides the first direct evidence of a function for the Hpo/Sav interaction (Polesello, 2006).
Next, the prediction that dRASSF depletion would promote Hpo activation was tested. Like that of Hpo's mammalian counterparts, phosphorylation of endogenous Hpo can be potently stimulated by the drug Staurosporine (STS) in Kc cells. Although RNAi depletion of dRASSF alone was not able to induce Hpo phosphorylation, dRASSF depletion markedly potentiated STS-induced Hpo activation. Thus, dRASSF restricts Hpo activation in cultured cells (Polesello, 2006).
Given their opposing effects on Hpo activation, the relationship between Sav and dRASSF was investigated. Depletion of dRASSF in Kc cells gives rise to an increase in Sav protein levels. Although dRASSF levels were unaltered in sav mutant clones, overexpression of Sav in the wing disc results in a robust decrease of dRASSF staining. Whether dRASSF and Sav compete to bind Hpo was tested. To address this question, because Sav and dRASSF repress each other's expression and dRASSF has reduced affinity for phosphorylated Hpo, separate Kc cell lysates expressing a kinase-dead form of Hpo (HpoKD-Flag), Sav-HA, and HA-dRASSF were mixed and IPs were performed after the proteins were allowed to bind overnight. Both Sav and dRASSF were able to interact with Hpo. In these conditions, increasing the amount of Sav was able to displace the dRASSF fraction bound to Hpo, showing that Sav and dRASSF are competing to bind Hpo. The outcome of the competition probably determines the stability of Sav and dRASSF; both proteins are downregulated when Hpo is depleted by RNAi. Thus, it is suggested that interplay between the inhibitor dRASSF and the activator Sav determines the level of Hpo activation and therefore affects body size (Polesello, 2006).
This model was tested by performing genetic-interaction experiments. A mutant allele of hpo was crossed into the dRASSF mutant background and the adult body mass was measured. The body-mass reduction of dRASSF mutant flies (15% reduction) was substantially rescued by removal of just one copy of Hpo (8% reduction). Flies overexpressing Sav showed a reduction of 10% in weight and 5% in wing area, mimicking dRASSF loss of function. This wing defect was significantly increased in a dRASSF mutant background. In addition, misexpression of dRASSF was able to robustly rescue the rough-eye phenotype elicited by coexpression of Sav and Wts. These data support the notion that dRASSF can antagonize Sav-mediated Hpo activation in vivo (Polesello, 2006).
Though the results are consistent with biochemical data on mammalian RASSF family members, they are at odds with the fact that RASSF genes are commonly silenced in tumor cells. It has been proposed that one RASSF protein, Nore1, possesses a tumor-suppressor function that is independent of MST1 and MST2. Two lines of evidence to support this notion were found. First, in vivo clones were made in the head (by using the eyeless FLP system) that were mutant for two hpo hypomorphic alleles, hpo42–48 and hpoKC203, that remove the SARAH domain in a dRASSF mutant background. Interestingly, the overgrowth phenotype elicited by these hpo alleles was strongly enhanced by loss of dRASSF. By contrast, a hpo allele (hpo42–47) bearing an inactivating deletion in the kinase domain but an intact SARAH domain was barely if at all enhanced by dRASSF loss of function. This suggests that dRASSF may possess a tumor-suppressor function, which may be uncovered when the Hpo function is compromised (Polesello, 2006).
In addition, the relationship between Ras1 and dRASSF was examined because the mammalian RASSF proteins have all been shown to bind Ras proteins. In Drosophila imaginal tissues, Ras1 mutant clones grow poorly and are eliminated by apoptosis. When double-mutant clones for Ras1 and dRASSF were made in the developing eye, a substantial rescue was observed of the growth defect observed in clones mutant for Ras1 alone. This rescue of Ras loss of function was the result of both increased proliferation quantified with phosphorylated Histone 3 staining and a reduction of apoptosis visualized with a cleaved-Caspase 3 antibody. Thus, dRASSF appears to antagonize Ras1 signaling in growth control, which is again suggestive of a “tumour-suppressing” effect distinct from its “oncogenic” role in opposing the Hpo pathway. However, it has been suggest that NORE1 may also have both Ras- and MST-independent functions. Future experiments will therefore be aimed at gaining a better understanding of the RASSFs' growth-restricting functions. The fact that the dRASSF mutations are viable might therefore reflect the facts that its ability to regulate the Hpo pathway may be redundant with other modes of regulation and that loss of dRASSF's tumor-suppressive activity is balanced by loss of its growth-promoting activity. It has been proposed that MST2 may be inactivated by binding to Raf-1. It will be interesting to determine whether this mode of regulation is redundant with RASSF (Polesello, 2006).
In summary, mutant alleles of the sole Drosophila ortholog of the RASSF family of tumor suppressors were generated. Surprisingly, dRASSF mutant flies are smaller than control flies. This growth defect can probably be ascribed in part to dRASSF's ability to antagonize Hpo signaling by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor activity, which is uncovered when hpo or Ras1 function is compromised. It will be interesting to investigate whether some mammalian RASSF proteins share these properties (Polesello, 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; S. E. 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).
The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. This study identified Kibra as another upstream component of the Hippo signaling pathway. This study shows that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis (Yu, 2010).
In multicellular organisms, cell growth, proliferation, and death must be coordinated in order to attain proper organ size during development and to maintain tissue homeostasis in adult life. Recent studies in Drosophila have led to the discovery of the Hippo signaling pathway as a key mechanism that controls organ size by impinging on cell growth, proliferation, and apoptosis. Central to the Hippo pathway is a kinase cascade comprised of four tumor suppressors, including the Ste20-like kinase Hippo (Hpo) and its regulatory protein Salvador (Sav), the NDR family kinase Warts (Wts) and its regulatory protein Mats. The Hpo-Sav complex phosphorylates and activates the Wts-Mats complex, which in turn phosphorylates and inactivates the oncoprotein Yki, which normally functions as a coactivator for the TEAD/TEF family transcription factor Scalloped (Sd). Recent studies have also implicated the atypical cadherin Fat (Ft) as well as the membrane-associated FERM-domain proteins Expanded (Ex) and Merlin (Mer) as upstream components of the Hippo pathway. How these proteins are biochemically linked to the Hippo kinase cascade remains largely unknown, although Ex can at least partially regulate the Hippo pathway by directly binding and sequestering Yki in the cytoplasm. Ft differs from Ex, Mer, and core components of the Hippo kinase cascade in that, besides tissue growth, Ft also regulates planar cell polarity (PCP), for which it interacts with another cadherin Dachsous (Ds). Most recently, it was shown that a gradient of Ds activity in imaginal discs can modulate Hippo-mediated growth regulation, thus potentially linking PCP to the Hippo kinase cascade, although the biochemical mechanism of this linkage remains to be determined (Yu, 2010).
The physiological function of the Hippo pathway is best understood in Drosophila imaginal discs, where inactivation of the Hippo pathway tumor suppressors, or overexpression of the Yki oncoprotein, results in tissue overgrowth characterized by excessive cell proliferation, diminished apoptosis, and increased transcription of Hippo pathway target genes such as the cell death inhibitor diap1 and the microRNA bantam, as well as ex and mer as part of a negative feedback regulatory loop. Recent studies further implicated the Hippo pathway as a conserved mechanism of organ size control and tissue homeostasis in mammals. Thus, the mammalian homologs of Hpo (Mst1/2), Sav (WW45), Wts (Lats1/2), and Yki (YAP) constitute an analogous kinase cascade, and transgenic overexpression of YAP or inactivation of Mst1/2 led to massive organomegaly and rapid progression to tumorigenesis. Furthermore, NF2, the mammalian homolog of mer, is a well-established tumor suppressor gene whose mutations lead to neurofibromatosis (Yu, 2010 and references therein).
Besides its prominent role in controlling imaginal disc growth, the Hippo pathway is required during Drosophila oogenesis for the proper maturation of posterior follicle cells (PFCs). In the absence of Hippo signaling, the PFCs fail to undergo a Notch-mediated mitotic cycle-endocycle switch and accumulate in extra layers of follicular epithelium. The PFC maturation defects, in turn, lead to a disruption of the anterior-posterior (AP) polarity of the underlying oocyte, which manifests itself as mislocalization of the oocyte nucleus and AP axis determinants such as the RNA-binding protein Staufen (Stau). Interestingly, the oocyte polarity defect is observed in mutants for components of the Hippo kinase cascade as well as ex and mer, but not ft, suggesting that the canonical Hippo pathway may integrate different signals in different developmental contexts (Yu, 2010).
This study identifies Kibra as an upstream component of the Hippo pathway. Loss of kibra leads to oogenesis defects, imaginal disc overgrowth, and aberrant gene expression characteristic of defective Hippo signaling. Kibra functions together with Mer and Ex in an apical protein complex, which, through direct binding to the Hpo-Sav complex, regulates the Hippo kinase cascade and thus Yki phosphorylation. These findings uncover an important missing link in the Hippo signaling pathway and shed light on the molecular mechanism of the Ex and Mer tumor suppressor proteins (Yu, 2010).
In a genetic screen for oocyte polarity mutants based on FRT/FLP-induced mitotic clones in follicle cells, four lethal P element insertion lines on chromosome 3R were identified that caused mislocalization of Stau-GFP and Stau to the center of the oocyte when the PFCs were made homozygous mutant for the P element insertions. This polarity defect was observed with variable penetrance depending on the specific P element line analyzed, likely due to their hypomorphic nature. These lethal lines (264/09, 1156/7, f06952, and EP3494) fail to complement each other and all carry a P element insertion near the 5' UTR or within the first intron of CG33967. CG33967 encodes a 1288 amino acid protein that shares 39% identity with KIBRA, a cytoplasmic protein named after its predominant expression in kidney and brain in humans. Both CG33967 and KIBRA contain two N-terminal WW domains and one C-terminal C2 domain. CG33967 is referred to as kibra to distinguish it from its human ortholog KIBRA (Yu, 2010).
This study identifies Kibra as a tumor suppressor and an essential component of the Hippo pathway. A model is proposed in which Kibra functions together with Mer and Ex in an apical protein complex to transduce growth-regulatory signals to the Hpo-Sav complex, which, through the canonical Hippo kinase cascade, controls Yki phosphorylation and target gene transcription. Of note, the findings do not exclude the possibility that Kibra, Ex, or Mer may interact with additional Hippo pathway components besides Hpo-Sav, especially given the recent report that Ex can directly bind Yki. How Kibra, Ex, and Mer function together to integrate upstream signals remains to be determined. One possibility is that these proteins function redundantly in receiving signals from the same upstream regulator(s). Alternatively, each protein may be regulated by distinct upstream regulator(s) (Yu, 2010).
A commonly used assay for Hippo signaling in Drosophila S2 cells involves examining mobility shifts of the Wts protein on SDS-PAGE. Given its large size and that not all protein phosphorylation causes discernable mobility shift on SDS-PAGE, this assay is less sensitive in detecting Wts phosphorylation than the phospho-specific antibody used in the present study. Indeed, overexpression of Ex in S2 cells has no effect on Wts mobility, yet this study demonstrates that Ex induces robust Wts phosphorylation at its hydrophobic motif. The fact that this hydrophobic motif is a well-established direct phosphorylation site by Hpo homologs in mammalian cells further suggests that Ex, as well as Mer and Kibra, regulates Wts through the canonical Hippo kinase cascade. Indeed, it was found that Ex-induced Wts phosphorylation is Hpo dependent. These results are not incompatible with recent report that Ex can also regulate the Hippo pathway in a kinase-independent manner. Using a well-established assay for Yki transcriptional activity, this study found that while Ex, Mer plus Kibra, or Hpo could all suppress the activity of a Yki-Gal4 fusion protein, only Ex was able to suppress the activity of a Yki-Gal4 fusion protein in which all the possible Wts-phosphorylation sites are mutated. These observations are consistent with the view that Ex can regulate the Hippo pathway through both Wts-dependent and -independent mechanisms (Yu, 2010).
A comparison of the loss-of-function phenotypes of mer, ex, and kibra in egg chambers and imaginal discs reveals tissue-specific differences in the relative contribution of each gene to Hippo pathway regulation. For example, loss of ex alone, but not mer or kibra, is sufficient to cause robust diap1 upregulation in imaginal discs, suggesting that ex has a more essential role in diap1 transcriptional regulation. However, the converse is true in the ovary, where loss of mer or kibra results in stronger oocyte polarity and Notch signaling defects than loss of ex. In fact, the severity of mer or kibra mutant phenotypes in oogenesis are comparable to those of core components of the Hippo pathway such as hpo and sav, even though the former display much milder overgrowth than the latter in imaginal discs. Perhaps the most extreme case of tissue-specific requirement is provided by the ft tumor suppressor gene, which is required for Hippo pathway regulation in the imaginal discs but dispensable in developing egg chambers. While the underlying molecular basis remains to be determined, such tissue-specific requirements suggest that the core Hippo kinase cascade may function as a signal integrator of multiple inputs in a dynamic and versatile manner, and that additional cell surface receptors besides Ft may signal to the Hippo pathway (Yu, 2010).
Considerable efforts have been directed at identifying the key signaling pathways regulated by the NF2/Merlin tumor suppressor protein. These investigations have led to the identification of a number of effector mechanisms downstream of NF2/Merlin, such as growth control pathways mediated by Ras, Rac, STAT, or PI3K, contact inhibition mediated by cell surface receptors or adherens junctions, and endocytosis/degradation of various membrane proteins. The recent identification of Mer as an upstream regulator of Hpo in Drosophila provides yet another plausible mechanism through which Mer functions as a tumor suppressor protein. The identification of Kibra as a regulator of the Hippo pathway further strengthens the case for a functional link between NF2/Mer and the Hippo pathway. The observation that NF2/Mer and KIBRA can synergistically stimulate Lats1/2 phosphorylation in mammalian cells not only supports an NF2/Mer-Hippo connection, but further implicates KIBRA as a potential tumor suppressor in humans with relevance to neurofibromatosis (Yu, 2010).
The identification of Kibra as an upstream regulator of the Hippo pathway has implications for understanding memory-related functions of the human KIBRA gene. Besides its well-established roles in growth control, the Hippo pathway is also required for differentiation and morphogenesis of certain postmitotic neurons in Drosophila. It is speculated that modulation of the Hippo pathway may influence the growth or differentiation of memory-related neuronal structures, a hypothesis that can be directly tested by genetic manipulation of Hippo signaling activity in animal models (Yu, 2010).
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