RESUMO
TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In Drosophila, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of PTEN mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of Rheb is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, Tsc1 mutant cells show decreased PKB activity, and the extent of Tsc1 mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from Tsc1 mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of Tsc1 mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1-in contrast to PTEN-rarely become malignant.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Privação de Alimentos , Fatores de Transcrição Forkhead/genética , Genes de Insetos , Modelos Biológicos , Mutação , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The Target of Rapamycin complex 1 (TORC1) is an evolutionarily conserved kinase complex coordinating cellular growth with nutritional conditions and growth factor signaling, and its activity is elevated in many cancer types. The use of TORC1 inhibitors as anticancer drugs is, however, limited by unwanted side-effects and development of resistance. We therefore attempted to identify limiting modulators or downstream effectors of TORC1 that could serve as therapeutic targets. Drosophila epithelial tissues that lack the tumor suppressor Pten hyperproliferate upon nutrient restriction in a TORC1-dependent manner. We probed candidates of the TORC1 signaling network for factors limiting the overgrowth of Pten mutant tissues. The serine/arginine-rich splicing factor 2 (SF2) was identified as the most limiting factor: SF2 knockdown drives Pten mutant cells into apoptosis, while not affecting control tissue. SF2 acts downstream of or in parallel to TORC1 but is not required for the activation of the TORC1 target S6K. Transcriptomics analysis revealed transcripts with alternatively used exons regulated by SF2 in the tumor context, including p53. SF2 may therefore represent a highly specific therapeutic target for tumors with hyperactive TORC1 signaling.
Assuntos
Apoptose , Carcinogênese/patologia , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , PTEN Fosfo-Hidrolase/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Processamento de RNA/genética , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Resposta ao Choque Térmico/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Grânulos Citoplasmáticos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w + mW.hs ), or rosy (ry+ ) and neomycin (neoR ) transgenes are in common use. Parallel ovoD lines were developed using w + mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as ry+ , neoR FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry+ , neoR FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w + mW.hs FRT. Here we report the creation of a new ovoD line for the ry+ , neoR FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Infertilidade Feminina/genética , N-Acetilglucosaminiltransferases/genética , Alelos , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Mosaicismo/embriologia , Fenótipo , Mutações Sintéticas Letais/genética , TransgenesRESUMO
Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig), an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1) and Caprin (Capr) and directly interacts with and regulates the RNA-binding protein Rasputin (Rin) in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Drosophila/genética , Ecdisona/metabolismo , Ecdisterona/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Larva/crescimento & desenvolvimento , Mutação , Neurônios/metabolismo , Isoformas de Proteínas , Transdução de Sinais , Fatores de Transcrição/genética , TransgenesRESUMO
The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress.
Assuntos
Apoptose , Drosophila/enzimologia , Enterócitos/enzimologia , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Animais , Apoptose/genética , Drosophila/genética , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Mutação/genética , Fosforilação , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: Insulin/insulin-like growth factor signalling (IIS) has been described as one of the major pathways involved in growth control and homeostasis in multicellular organisms. Whereas its core components are well established, less is known about the molecular functions of IIS regulators. The adaptor molecule Lnk/SH2B has been implicated in IIS but the mechanism by which it promotes IIS activity has remained enigmatic. RESULTS: In this study, we analyse genetic and physical interactions among InR, Chico and Lnk in Drosophila tissues. FRET analysis reveals in vivo binding between all three molecules. Genetically, Lnk acts upstream of Chico. We demonstrate that Chico's plasma membrane localisation is ensured by both its PH domain and by the interaction with Lnk. Furthermore, Lnk is able to recruit an intracellular InR fragment to the membrane. CONCLUSIONS: Thus, by acting as a scaffolding molecule that ensures InR and Chico enrichment at the membrane, Lnk provides a fail-safe mechanism for IIS activation.
RESUMO
Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Longevidade/fisiologia , Estresse Oxidativo , Animais , Tamanho Corporal , Drosophila melanogaster/genética , Feminino , Fertilidade , Regulação da Expressão Gênica , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mutação/genética , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Caracteres Sexuais , Inanição , Transcrição Gênica , Proteínas ras/metabolismoRESUMO
S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Drosophila/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Metamorfose Biológica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Quinases S6 Ribossômicas/genética , Transdução de SinaisRESUMO
Membrane potential is a property of all living cells1. However, its physiological role in non-excitable cells is poorly understood. Resting membrane potential is typically considered fixed for a given cell type and under tight homeostatic control2, akin to body temperature in mammals. Contrary to this widely accepted paradigm, we found that membrane potential is a dynamic property that directly reflects tissue density and mechanical forces acting on the cell. Serving as a quasi-instantaneous, global readout of density and mechanical pressure, membrane potential is integrated with signal transduction networks by affecting the conformation and clustering of proteins in the membrane3,4, as well as the transmembrane flux of key signaling ions5,6. Indeed, we show that important mechano-sensing pathways, YAP, Jnk and p387-121314, are directly controlled by membrane potential. We further show that mechano-transduction via membrane potential plays a critical role in the homeostasis of epithelial tissues, setting tissue density by controlling proliferation and cell extrusion of cells. Moreover, a wave of depolarization triggered by mechanical stretch enhances the speed of wound healing. Mechano-transduction via membrane potential likely constitutes an ancient homeostatic mechanism in multi-cellular organisms, potentially serving as a steppingstone for the evolution of excitable tissues and neuronal mechano-sensing. The breakdown of membrane potential mediated homeostatic regulation may contribute to tumor growth.
RESUMO
BACKGROUND: We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA). METHODS: The effect of NRBP1 expression on tumor cell growth was analyzed by using RNAi. NRBP1 protein expression was evaluated on two TMAs containing prostate samples from more than 1,000 patients. Associations with clinico-pathological features, the proliferation marker Ki67 and survival data were analyzed. RESULTS: RNAi mediated silencing of NRBP1 expression in prostate cancer cell lines resulted in reduced cell growth (P < 0.05). TMA analysis revealed NRBP1 protein expression in benign prostate hyperplasia in 6% as compared to 60% in both, high-grade intraepithelial neoplasia and prostate cancer samples. Strong NRBP1 protein expression was restricted to prostate cancer and correlated with higher expression of the proliferation marker Ki67 (P < 0.05). Further, patients with strong NRBP1 protein expression showed poor clinical outcomes (P < 0.05). Analysis of matched localized cancer tissues before and after castration revealed that post-therapy-related repression of NRBP1 expression was significantly associated with better overall survival. CONCLUSIONS: We demonstrate that expression of NRBP1 is up-regulated during the progression of prostate cancer and that high NRBP1 expression is linked with poor prognosis and enhanced tumor cell growth.
Assuntos
Adenocarcinoma/patologia , Expressão Gênica , Neoplasias da Próstata/patologia , Receptores Citoplasmáticos e Nucleares/genética , Proteínas de Transporte Vesicular/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Finlândia/epidemiologia , Humanos , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Prostatectomia , Hiperplasia Prostática/epidemiologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Taxa de Sobrevida , Suíça/epidemiologia , Análise Serial de Tecidos , Proteínas de Transporte Vesicular/metabolismoRESUMO
Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Receptor de Insulina/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Espectrometria de Massas , Mapas de Interação de Proteínas , Proteínas Quinases/genética , Proteoma/genética , Receptor de Insulina/genética , Transdução de Sinais , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismoRESUMO
Insulin/insulin-like growth factor signaling (IIS) plays a pivotal role in the regulation of growth at the cellular and the organismal level during animal development. Flies with impaired IIS are developmentally delayed and small due to fewer and smaller cells. In the search for new growth-promoting genes, we identified mutations in the gene encoding Lnk, the single fly member of the SH2B family of adaptor molecules. Flies lacking lnk function are viable but severely reduced in size. Furthermore, lnk mutants display phenotypes reminiscent of reduced IIS, such as developmental delay, female sterility, and accumulation of lipids. Genetic epistasis analysis places lnk downstream of the insulin receptor (InR) and upstream of phosphoinositide 3-kinase (PI3K) in the IIS cascade, at the same level as chico (encoding the single fly insulin receptor substrate [IRS] homolog). Both chico and lnk mutant larvae display a similar reduction in IIS activity as judged by the localization of a PIP(3) reporter and the phosphorylation of protein kinase B (PKB). Furthermore, chico; lnk double mutants are synthetically lethal, suggesting that Chico and Lnk fulfill independent but partially redundant functions in the activation of PI3K upon InR stimulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Família Multigênica , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Tamanho Corporal , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Insulina/genética , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Mutação , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Alinhamento de Sequência , Domínios de Homologia de srcRESUMO
BACKGROUND: Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. RESULTS: Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3ß ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3ß activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. CONCLUSIONS: Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3ß to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Genetic studies in Drosophila melanogaster underscore the importance of the insulin-signalling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homologue), dPI(3)K, dPKB, dPTEN, and dS6K. Mutations in any of these components have a striking effect on cell size and number, with the exception of dS6K. Mutants in dS6K affect cell size but not cell number, seemingly consistent with arguments that dS6K is a distal effector in the signalling pathway, directly controlled by dTOR, a downstream effector of dPI(3)K and dPKB. Unexpectedly, recent studies showed that dS6K activity is unimpaired in chico-deficient larvae, suggesting that dS6K activation may be mediated through the dPI(3)K docking sites of the Drosophila insulin receptor. Here, we show genetically, pharmacologically and biochemically that dS6K resides on an insulin signalling pathway distinct from that of dPKB, and surprisingly also from that of dPI(3)K. More striking, despite dPKB-dPI(3)K-independence, dS6K activity is dependent on the Drosophila homologue of the phosphoinositide-dependent protein kinase 1, dPDK1, demonstrating that both dPDK1, as well as dTOR, mediated dS6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Divisão Celular/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ativação Enzimática , Olho/crescimento & desenvolvimento , Genes de Insetos , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Receptor de Insulina/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimentoRESUMO
Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).
Assuntos
Divisão Celular/genética , Proteínas de Drosophila/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Neuropeptídeos/fisiologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Divisão Celular/fisiologia , Tamanho Celular/genética , Tamanho Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Olho/ultraestrutura , Feminino , Deleção de Genes , Genes de Insetos , Genes Supressores de Tumor , Substâncias de Crescimento/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Neuropeptídeos/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Transdução de Sinais , Ativação Transcricional , TransgenesRESUMO
TSC22D1, which encodes transforming growth factor beta-stimulated clone 22 (TSC-22), is thought to be a tumor suppressor because its expression is lost in many glioblastoma, salivary gland, and prostate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun family of leucine zipper transcription factors; its functions have not been investigated in a multicellular environment. Genetic studies in the model organism Drosophila melanogaster often provide fundamental insights into mechanisms disrupted in carcinogenesis, because of the strong evolutionary conservation of molecular mechanisms between flies and humans. Whereas humans and mice have four TSC-22 domain genes with numerous isoforms, Drosophila has only one TSC-22 domain gene, bunched (bun), which encodes both large and small protein isoforms. Surprisingly, Drosophila Bun proteins promote cellular growth and proliferation in ovarian follicle cells. Loss of both large isoforms has the strongest phenotypes, including increased apoptosis. Cultured S2 cells depleted for large Bun isoforms show increased apoptosis and less frequent cell division, with decreased cell size. Altogether, these data indicate that Drosophila TSC-22/DIP/Bun proteins are necessary for cellular growth, proliferation, and survival both in culture and in an epithelial context. Previous work demonstrated that bun prevents recruitment of epithelial cells to a migratory fate and, thus, maintains epithelial organization. We speculate that reduced TSC22D1 expression generally reduces cellular fitness and only contributes to carcinogenesis in specific tissue environments.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/química , Proteínas Supressoras de Tumor/fisiologia , Animais , Crescimento Celular , Sobrevivência Celular , Células Epiteliais/citologiaRESUMO
During the development of multicellular organisms the fate of individual cells is specified with great precision and reproducibility. Although classical genetic approaches led to the identification of many of the signaling pathways contributing to cell fate specification, they have provided little insight into the mechanisms that ensure robustness and reproducibility. We have used the specification of the R7 photoreceptor cells controlled by the Sevenless receptor tyrosine kinase (Sev) pathway to screen for modulators of pathway activity and to uncover the mechanisms underlying the robustness of cell fate decisions. Here we provide genetic evidence that the Drosophila SOCS36E adaptor protein containing an SH2 domain and a SOCS box acts as an attenuator of Sev signaling. Overexpression of Socs36E strongly suppresses the specification of extra R7 photoreceptor cells in response to constitutive activation of Sev, and loss of Socs36E function suppresses the loss of R7 cells when Sev activity is impaired. In a wild-type background, however, loss and gain of Socs36E function exhibits little effect on R7 specification. We also show that SH2 domain of SOCS36E is essential for this function in inhibiting Sev action and that Socs36E expression is suppressed by high Sev pathway activity. In our model, only the cell able to activate high levels of receptor tyrosine kinase signaling will repress SOCS36E expression, reduce the negative effect on Sev signaling and allow this cell to differentiate into R7. In contrast, the remaining cells fail to receive high signaling, and thus maintain high levels of SOCS36E. This represses residual Sev activity and blocks R7 development. Therefore, Socs36E constitutes a novel partially redundant feedback mechanism that contributes to the robustness of R7 specification. The SOCS family of adaptor proteins may have evolved as modulators of specific signaling pathways that contribute to the robustness and precision of cell fate specification.
Assuntos
Olho Composto de Artrópodes/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas do Olho/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sequência de Bases , Comunicação Celular/fisiologia , Olho Composto de Artrópodes/citologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Mutação , Células Fotorreceptoras/fisiologia , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.