RESUMO
Protein kinase B (PKB, also termed Akt) is a phosphatidylinositol 3' kinase (PI3'K)-dependent enzyme implicated in survival signaling and human tumorigenesis. To identify potential targets of this protein kinase, we employed a genetic screen in Drosophila. Among several genes that genetically interacted with PKB was trachealess (trh), which encodes a bHLH-PAS domain transcription factor required for development of the trachea and other tubular organs. Trh activates expression of the fibroblast growth factor receptor Breathless, which, in turn, is required for directed migration of all tracheal branches. Using a combination of biochemical and transgenic approaches, we show that direct phosphorylation of Trh by PKB at serine 665 is essential for nuclear localization and functional activation of this regulator of branching morphogenesis.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Traqueia/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Genes Reporter , Humanos , Masculino , Morfogênese/fisiologia , Mapeamento de Peptídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão/metabolismo , Traqueia/anatomia & histologia , Fatores de Transcrição/genéticaRESUMO
Akt (or PKB) is an oncogene involved in the regulation of cell survival. Akt is regulated by phosphatidylinositol 3-OH kinase (PI3'K) signaling and has shown to be hyperactivated through the loss of the PTEN tumor suppressor. In Drosophila, insulin signaling as studied using the Drosophila IRS-4 homolog (Chico) has been shown to be a crucial regulator of cell size. We have studied Drosophila Akt (Dakt1) and have shown that it is also involved in the regulation of cell size. Furthermore we have performed genetic epistasis tests to demonstrate that in Drosophila, PI3'K, PTEN and Akt comprise a signaling cassette that is utilized during multiple stages of development. In addition, we show that this signaling cassette is also involved in the regulation of cell survival during embryogenesis. This study therefore establishes the evolutionary conservation of this signaling pathway in Drosophila. Oncogene (2000) 19, 3971 - 3977.
Assuntos
Drosophila melanogaster/fisiologia , Proteínas de Insetos/fisiologia , Insulina/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor , Animais , Tamanho Celular , Sobrevivência Celular , Proteínas de Drosophila , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Epistasia Genética , Olho/embriologia , Feminino , Genes de Insetos , Masculino , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt , TransfecçãoRESUMO
We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.
Assuntos
Proteínas de Drosophila , Heparina/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Uridina Difosfato Glucose Desidrogenase/metabolismo , Proteínas de Peixe-Zebra , Sequência de Aminoácidos , Animais , Células Clonais , DNA Complementar/genética , Drosophila/embriologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Genes de Insetos , Biblioteca Genômica , Hibridização In Situ , Proteínas de Insetos/genética , Dados de Sequência Molecular , Fenótipo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Uridina Difosfato Glucose Desidrogenase/genética , Proteínas Wnt , Proteína Wnt1RESUMO
Feline leukaemia virus (FeLV) is epidemiologically associated with induction of the majority of lymphoid tumours of the domestic cat. However, about one-third of these tumours are devoid of exogenous virus or show evidence of virus integration only after tumour outgrowth. To help define the genetic mechanisms of feline lymphomagenesis we have explored here the possibility that cellular oncogenes (c-onc genes) are rearranged in tumour cell DNA. Of 16 FeLV-positive T-cell tumours among 31 naturally occurring lymphomas, 2 showed evidence of recombinant FeLV proviruses containing myc oncogene sequences. One of the two produced a transmissible myc-containing FeLV. In both cases c-myc and its surrounding DNA appeared unaltered. We believe that the association of myc with FeLV may result in its activation and play a part in the development of a significant fraction of cat T-cell lymphomas. Our findings contrast with studies of experimental induction of chicken lymphoma, in which myc activation occurs by retrovirus promoter insertion near c-myc (refs 3-5), rather than by incorporation into virus.
Assuntos
Doenças do Gato/microbiologia , Genes Virais , Vírus da Leucemia Felina/genética , Leucemia/veterinária , Oncogenes , Transdução Genética , Animais , Sequência de Bases , Gatos , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , DNA Viral/genética , Leucemia/microbiologiaRESUMO
During serial passages of an avian leukosis virus (the transformation-defective, src deletion mutant of Bratislava 77 avian sarcoma virus, designated tdB77) in chicken embryo fibroblasts, viruses which transformed chicken embryo fibroblasts in vitro emerged. Chicken embryo fibroblasts infected with these viruses (SK770 and Sk780) had a distinctive morphology, formed foci in monolayer cultures, and grew independent of anchorage in semisolid agar. Bone marrow cells were not transformed by these viruses. Another virus (SK790) with similar properties emerged during serial subcultures of chicken embryo fibroblasts after a single infection with tdB77. The 50S to RNAs isolated from these viruses contained a tdB77-sized genome (7.6 kilobases), 8.7- and 5.7-kilobase RNAs, and either a 4.1-kilobase RNA or a 4.6-kilobase RNA. These RNAs did not hybridize with cDNA's representing the src, erb, mac, and myb genes of avian acute transforming viruses. Cells transformed by any one of the Sk viruses (SK770, SK780, or SK790) synthesized two novel gag-related polyproteins having molecular weights of 110,000 (p110) and 125,000 (p125). We investigated the compositions of these proteins with monospecific antiviral protein sera. We found that p110 was a gag-pol fusion protein which contained antigenic determinants, leaving 49,000 daltons which was antigenically unrelated to the structural and replicative proteins of avian leukosis viruses. An analysis of the SK viral RNAs with specific DNA probes indicated that the 5.7-kilobase RNA contained gag sequences but lacked pol sequences and, therefore, probably encoded p125. The transforming ability, the deleted genome, and the induced polyproteins of the SK viruses were reminiscent of the properties of several replication-defective acute transforming viruses.