RESUMO
The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Humanos , Metabolismo dos Lipídeos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Fosfatidato Fosfatase , Serina-Treonina Quinases TORRESUMO
The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1-S6K-RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling.
Assuntos
Proteínas Recombinantes/metabolismo , Proteína S6 Ribossômica/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Imunofluorescência , Redes Reguladoras de Genes , Genoma , Genômica , Humanos , Análise em Microsséries , Terapia de Alvo Molecular , Fosforilação , Interferência de RNA , Proteínas Recombinantes/genética , Proteína S6 Ribossômica/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
UNLABELLED: There is a strong and growing need in the biology research community for accurate, automated image analysis. Here, we describe CellProfiler 2.0, which has been engineered to meet the needs of its growing user base. It is more robust and user friendly, with new algorithms and features to facilitate high-throughput work. ImageJ plugins can now be run within a CellProfiler pipeline. AVAILABILITY AND IMPLEMENTATION: CellProfiler 2.0 is free and open source, available at http://www.cellprofiler.org under the GPL v. 2 license. It is available as a packaged application for Macintosh OS X and Microsoft Windows and can be compiled for Linux. CONTACT: anne@broadinstitute.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.