RESUMEN
Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis.
Asunto(s)
Fibroblastos/metabolismo , Hexosaminas/biosíntesis , Complejos Multiproteicos/metabolismo , Transferasas de Grupos Nitrogenados/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Fibroblastos/citología , Regulación de la Expresión Génica , Glucosa/metabolismo , Glutamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Células HeLa , Homeostasis , Humanos , Ácidos Cetoglutáricos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Metaboloma/genética , Metabolómica , Ratones , Complejos Multiproteicos/genética , Transferasas de Grupos Nitrogenados/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
The mammalian target of rapamycin (mTOR) integrates signals from nutrients and insulin via two distinct complexes, mTORC1 and mTORC2. Disruption of mTORC2 impairs the insulin-induced activation of Akt, an mTORC2 substrate. Here, we found that mTORC2 can also regulate insulin signaling at the level of insulin receptor substrate-1 (IRS-1). Despite phosphorylation at the mTORC1-mediated serine sites, which supposedly triggers IRS-1 downregulation, inactive IRS-1 accumulated in mTORC2-disrupted cells. Defective IRS-1 degradation was due to attenuated expression and phosphorylation of the ubiquitin ligase substrate-targeting subunit, Fbw8. mTORC2 stabilizes Fbw8 by phosphorylation at Ser86, allowing the insulin-induced translocation of Fbw8 to the cytosol where it mediates IRS-1 degradation. Thus, mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that in addition to persistent mTORC1 signaling, heightened mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1.
Asunto(s)
Proteínas F-Box/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Complejos Multiproteicos/metabolismo , Procesamiento Proteico-Postraduccional , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Activación Enzimática , Proteínas F-Box/genética , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Semivida , Insulina/fisiología , Proteínas Sustrato del Receptor de Insulina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Complejos Multiproteicos/antagonistas & inhibidores , Fosforilación , Proteína Quinasa C/metabolismo , Estabilidad Proteica , Proteínas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.