RESUMO
Adherens junctions (AJs) and cell polarity complexes are key players in the establishment and maintenance of apical-basal cell polarity. Loss of AJs or basolateral polarity components promotes tumor formation and metastasis. Recent studies in vertebrate models show that loss of AJs or loss of the basolateral component Scribble (Scrib) cause deregulation of the Hippo tumor suppressor pathway and hyperactivation of its downstream effectors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ). However, whether AJs and Scrib act through the same or independent mechanisms to regulate Hippo pathway activity is not known. Here, we dissect how disruption of AJs or loss of basolateral components affect the activity of the Drosophila YAP homolog Yorkie (Yki) during imaginal disc development. Surprisingly, disruption of AJs and loss of basolateral proteins produced very different effects on Yki activity. Yki activity was cell-autonomously decreased but non-cell-autonomously elevated in tissues where the AJ components E-cadherin (E-cad) or α-catenin (α-cat) were knocked down. In contrast, scrib knockdown caused a predominantly cell-autonomous activation of Yki. Moreover, disruption of AJs or basolateral proteins had different effects on cell polarity and tissue size. Simultaneous knockdown of α-cat and scrib induced both cell-autonomous and non-cell-autonomous Yki activity. In mammalian cells, knockdown of E-cad or α-cat caused nuclear accumulation and activation of YAP without overt effects on Scrib localization and vice versa. Therefore, our results indicate the existence of multiple, genetically separable inputs from AJs and cell polarity complexes into Yki/YAP regulation.
Assuntos
Junções Aderentes/metabolismo , Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Morfogênese/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Células CACO-2 , Caderinas/genética , Moléculas de Adesão Celular/genética , Cruzamentos Genéticos , Primers do DNA/genética , Cães , Drosophila , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP , alfa Catenina/genéticaRESUMO
The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F-actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F-actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie-orthologue Yap is modulated by changes in F-actin. Thus, regulators of F-actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.
Assuntos
Actinas/genética , Proteínas de Drosophila/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Actinas/biossíntese , Actinas/química , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Citoesqueleto/química , Citoesqueleto/genética , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/química , Drosophila melanogaster/química , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Forminas , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Especificidade de Órgãos/genética , Fenótipo , Proteínas Serina-Treonina Quinases/química , Capuzes de RNA/antagonistas & inibidores , Capuzes de RNA/química , Capuzes de RNA/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Asas de Animais/químicaRESUMO
Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. Here we investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib(-) cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does not explain how scrib(-) cells are prevented from overproliferating. We report that cell competition between scrib(-) and wild-type cells prevents hyperproliferation by suppressing Yki activity in scrib(-) cells. Suppressing Yki activation is critical for scrib(-) clone elimination by cell competition, and experimental elevation of Yki activity in scrib(-) cells is sufficient to fuel their neoplastic growth. Thus, cell competition acts as a tumor-suppressing mechanism by regulating the Hippo pathway in scrib(-) cells.
Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Genótipo , Discos Imaginais/citologia , Discos Imaginais/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
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. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.
Assuntos
Apoptose , Proliferação de Células , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Genes da Neurofibromatose 2 , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Ciclo Celular , Ciclina E/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/fisiologia , Mutação , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Proteínas de Sinalização YAPRESUMO
Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants can cause neoplastic phenotypes, including loss of cell polarity and overproliferation. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crumbs (Crb) affects growth in Drosophila imaginal discs. Overexpression of Crb causes severe overproliferation, and we found that loss of Crb similarly results in overgrowth of imaginal discs. Crb gain and loss of function caused defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. A juxtamembrane FERM domain-binding motif is responsible for growth regulation and induction of Hippo target gene expression, whereas Crb uses a PDZ-binding motif to form a complex with other polarity factors. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components, Fat and Merlin, are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a previously undescribed upstream input into the Hippo pathway.
Assuntos
Polaridade Celular/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Proliferação de Células , DrosophilaRESUMO
Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumour growth. Here we show that Hippo (Hpo), the Drosophila homologue of the mammalian Ste20-like kinases, MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development. hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells. Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis. The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts. We find that Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Proteínas Inibidoras de Apoptose , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase Quinases , Masculino , Morfogênese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologia , Asas de Animais/anatomia & histologiaRESUMO
The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. Here, we report that Ds and Fj regulate Hippo signaling to control growth. Interestingly, we found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, we found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size.
Assuntos
Caderinas/metabolismo , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de SinaisRESUMO
The Notch signaling pathway plays a central role in animal growth and patterning, and its deregulation leads to many human diseases, including cancer. Mutations in the tumor suppressor lethal giant discs (lgd) induce strong Notch activation and hyperplastic overgrowth of Drosophila imaginal discs. However, the gene that encodes Lgd and its function in the Notch pathway have not yet been identified. Here, we report that Lgd is a novel, conserved C2-domain protein that regulates Notch receptor trafficking. Notch accumulates on early endosomes in lgd mutant cells and signals in a ligand-independent manner. This phenotype is similar to that seen when cells lose endosomal-pathway components such as Erupted and Vps25. Interestingly, Notch activation in lgd mutant cells requires the early endosomal component Hrs, indicating that Hrs is epistatic to Lgd. These data suggest that Lgd affects Notch trafficking between the actions of Hrs and the late endosomal component Vps25. Taken together, our data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Endocitose/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Drosophila/fisiologia , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Imuno-Histoquímica , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: 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. RESULTS: Here, we report 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. We found that hippo mutant cells had elevated levels of bantam activity and that bantam was required for Yorkie-driven overgrowth. Additionally, overexpression of bantam was 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. CONCLUSIONS: Our data indicate that bantam is an essential target of the Hippo signaling pathway to regulate cell proliferation, cell death, and thus tissue size.
Assuntos
Ciclinas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose , Proliferação de Células , Ciclina E/genética , Ciclina E/metabolismo , Ciclinas/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Retina/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Merlin, the Drosophila homolog of the human Neurofibromatosis type-2 (NF2) tumor-suppressor gene, and the related protein Expanded are the most upstream components of the Hippo pathway identified so far. However, components acting upstream of Expanded and Merlin, such as transmembrane receptors, have not yet been identified. RESULTS: Here, we report that the protocadherin Fat acts as an upstream component in the Hippo pathway. Fat is a known tumor-suppressor gene in Drosophila, and fat mutants have severely overgrown imaginal discs. We found that the overgrowth phenotypes of fat mutants are similar to those of mutants in Hippo pathway components: fat mutant cells continued to proliferate after wild-type cells stopped proliferating, and fat mutant cells deregulated Hippo target genes such as cyclin E and diap1. Fat acts genetically and biochemically upstream of other Hippo pathway components such as Expanded, the Hippo and Warts kinases, and the transcriptional coactivator Yorkie. Fat is required for the stability of Expanded and its localization to the plasma membrane. In contrast, Fat is not required for Merlin localization, and Fat and Merlin act in parallel in growth regulation. CONCLUSIONS: Taken together, our data identify a cell-surface molecule that may act as a receptor of the Hippo signaling pathway.
Assuntos
Moléculas de Adesão Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Transdução de Sinais , Animais , Caderinas/genética , Caderinas/fisiologia , Moléculas de Adesão Celular/genética , Proliferação de Células , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olho/embriologia , Olho/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Neurofibromina 2/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/metabolismo , Asas de Animais/anatomia & histologia , Proteínas de Sinalização YAPRESUMO
A novel zinc gallophosphate zeolitic material |(C12H14N2)4F1.33|[Ga13.33Zn6.67(PO4)20] (denoted as JU101) has been prepared by using in situ generated methyl viologen (MV) as the template. The framework of JU101 features two building units including an unprecedented fused d6r and a novel [412·64·82·102] cavity. The connection of these two building units forms a 3D intersecting pore system containing 8-rings along the [010] direction, and 10-rings along the [001] and [100] directions. The MV-templated JU101 zeolitic material offers a new type of electron transfer system, which endows the material with interesting photochromism, thermochromism, and tuneable photovoltaic activity in response to light and heating. Importantly, JU101 shows an extended photochromism range from UV to visible light, high thermal stability, as well as a long-lived charge-separated state for potential application in solar energy conversion.
RESUMO
A new method for selenium speciation in fermented bean curd wastewater and juice was described. This method involved sample extraction with 5-sulfosalicylic acid (SSA)-functionalized silica-coated magnetic nanoparticles (SMNPs), capillary electrophoresis (CE) separation, and online detection with a modified electrothermal atomic absorption spectrometry (ETAAS) system. The modified interface for ETAAS allowed for the introduction of CE effluent directly through the end of the graphite tube. Elimination of the upper injection hole of the graphite tube reduced the loss of the anlayte and enhanced the detection sensitivity. The SSA-SMNPs were synthesized and used to extract trace amounts of selenite [Se(IV)], selenite [Se(VI)], selenomethionine (SeMet), and selenocystine (SeCys2) from dilute samples. The concentration enrichment factors for Se(VI), Se(IV), SeMet, and SeCys2 were 21, 29, 18, and 12, respectively, using the SSA-SMNPs extraction. The limits of detection for Se(VI), Se(IV), SeMet, and SeCys2 were 0.18, 0.17, 0.54, 0.49ngmL(-1), respectively. The RSD values (n=6) of method for intraday were observed between 0.7% and 2.9%. The RSD values of method for interday were less than 3.5%. The linear range of Se(VI) and Se(IV) were in the range of 0.5-200ngmL(-1), and the linear ranges of SeMet and SeCys2 were 2-500 and 2-1000ngmL(-1), respectively. The detection limits of this method were improved by 10 times due to the enrichment by the SSA-SMNP extraction. The contents of Se(VI) and Se(IV) in fermented bean curd wastewater were measured as 3.83 and 2.62ngmL(-1), respectively. The contents of Se(VI), Se(IV), SeMet, and SeCys2 in fermented bean curd juice were determined as 6.39, 4.08, 2.77, and 4.00ngmL(-1), respectively. The recoveries were in the range of 99.14-104.5% and the RSDs (n=6) of recoveries between 0.82% and 3.5%.
Assuntos
Benzenossulfonatos/química , Técnicas de Química Analítica/métodos , Eletroforese Capilar , Nanopartículas de Magnetita/química , Salicilatos/química , Selênio/química , Espectrofotometria Atômica , Limite de Detecção , Selênio/isolamento & purificação , Dióxido de Silício/química , Águas Residuárias/químicaRESUMO
The paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL) for studying the interaction between human serum albumin (HSA) and trimetazidine dihydrochloride (TMZ). The time required for equilibrium by ultrasonic microdialysis was 45min, which was far less than that by traditional dialysis (240min). It took 80min to achieve the required combination equilibrium by normal incubation and only 20min by ultrasonic. Compared with traditional dialysis, the use of ultrasonic microdialysis simplified experimental procedures, shortened experimental time and saved consumption of sample. A simple, sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized. Under the optimized conditions, the linear range of TMZ was from 0.075 to 80µmol/L (r(2)=0.9974). The detection limit was 26nmol/L with RSD of 2.8%. The number of binding sites and binding constant were 1.54 and 15.17L/mol, respectively.
Assuntos
Eletroforese Capilar/métodos , Medições Luminescentes/métodos , Microdiálise/métodos , Albumina Sérica/metabolismo , Trimetazidina/metabolismo , Ultrassom , Sítios de Ligação , Soluções Tampão , Eletroquímica , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Lineares , Ligação Proteica , Reprodutibilidade dos Testes , Albumina Sérica/química , Integração de Sistemas , Fatores de Tempo , Trimetazidina/químicaRESUMO
The Drosophila Yorkie (Yki) protein and its mammalian homolog Yes-associated protein (YAP) are potent growth promoters, and YAP overexpression is associated with multiple types of cancer. Yki and YAP are transcriptional coactivators and function as downstream effectors of the Hippo tumor suppressor pathway. The regulation of Yki and YAP by the Hippo signaling pathway has been extensively investigated; however, how they regulate gene expression is poorly understood. To identify additional regulators of Yki activity, we performed a genome-wide RNAi screen in Drosophila S2 cells. In this screen, we identified the conserved protein Mask (Multiple ankyrin repeats single KH domain) as a novel promoter of Yki activity in vitro and validated this function in vivo in Drosophila. We found that Mask is required downstream of the Hippo pathway for Yki to induce target-gene expression and that Mask forms complexes with Yki. The human Mask homolog MASK1 complexes with YAP and is required for the full activity of YAP. Additionally, elevated MASK1 expression is associated with worsened outcomes for breast cancer patients. We conclude that Mask is a novel cofactor for Yki/YAP required for optimal Yki/YAP activity during development and oncogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
The Hippo tumor-suppressor pathway controls tissue growth in Drosophila and mammals by regulating cell proliferation and apoptosis. The Hippo pathway includes the Fat cadherin, a transmembrane protein, which acts upstream of several other components that form a kinase cascade that culminates in the regulation of gene expression through the transcriptional coactivator Yorkie (Yki). Our previous work in Drosophila indicated that Merlin (Mer) and Expanded (Ex) are members of the Hippo pathway and act upstream of the Hippo kinase. In contrast to this model, it was suggested that Mer and Ex primarily regulate membrane dynamics and receptor trafficking, thereby affecting Hippo pathway activity only indirectly. Here, we examined the effects of Mer, Ex and the Hippo pathway on the size of the apical membrane and on apical-basal polarity complexes. We found that mer;ex double mutant imaginal disc cells have significantly increased levels of apical membrane determinants, such as Crb, aPKC and Patj. These phenotypes were shared with mutations in other Hippo pathway components and required Yki, indicating that Mer and Ex signal through the Hippo pathway. Interestingly, however, whereas Crb was required for the accumulation of other apical proteins and for the expansion of the apical domain observed in Hippo pathway mutants, its elimination did not significantly reverse the overgrowth phenotype of warts mutant cells. Therefore, Hippo signaling regulates cell polarity complexes in addition to and independently of its growth control function in imaginal disc cells.
Assuntos
Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurofibromina 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Caderinas/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Olho/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mutação , Neurofibromina 2/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Asas de Animais/metabolismo , Proteínas de Sinalização YAPRESUMO
During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.