RESUMEN
Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.
Asunto(s)
Proteínas Portadoras/metabolismo , Distrofina/metabolismo , Músculo Esquelético/enzimología , Distrofia Muscular Animal/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Factores de Edad , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Células Cultivadas , Distrofina/genética , Electroporación , Metabolismo Energético , Activación Enzimática , Femenino , Glucosa/metabolismo , Glucógeno/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Musculares/enzimología , Contracción Muscular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatología , Mutación , Oxidación-Reducción , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Ratas , Proteína Reguladora Asociada a mTOR , Índice de Severidad de la Enfermedad , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Transducción Genética , Utrofina/metabolismoRESUMEN
The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.