RESUMEN
The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Aciltransferasas , Animales , Proteínas de Ciclo Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Humanos , Lisofosfolípidos/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Tamaño de los Órganos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Suero/química , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid-deficient conditions is not completely understood. Here, we identify ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature-sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Línea Celular , Femenino , Proteínas Activadoras de GTPasa/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidad , PronósticoRESUMEN
Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that is part of mTOR complex 1 (mTORC1), a master regulator that couples amino acid availability to cell growth and autophagy. Multiple cues modulate mTORC1 activity, such as growth factors, stress, energy status and amino acids. Although amino acids are key environmental stimuli, exactly how they are sensed and how they activate mTORC1 is not fully understood. Recently, a model has emerged whereby mTORC1 activation occurs at the lysosome and is mediated through an amino acid sensing cascade involving RAG GTPases, Ragulator and vacuolar H(+)-ATPase (v-ATPase).
Asunto(s)
Aminoácidos/metabolismo , Lisosomas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia , Proliferación Celular , Humanos , Leucina-ARNt Ligasa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Modelos Biológicos , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Neuropéptidos/metabolismo , Proteína Homóloga de Ras Enriquecida en el CerebroRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Some amino acids signal to mTORC1 through the Rag GTPase, whereas glutamine and asparagine activate mTORC1 through a Rag GTPase-independent pathway. Here, we show that the lysosomal glutamine and asparagine transporter SNAT7 activates mTORC1 after extracellular protein, such as albumin, is macropinocytosed. The N terminus of SNAT7 forms nutrient-sensitive interaction with mTORC1 and regulates mTORC1 activation independently of the Rag GTPases. Depletion of SNAT7 inhibits albumin-induced mTORC1 lysosomal localization and subsequent activation. Moreover, SNAT7 is essential to sustain KRAS-driven pancreatic cancer cell growth through mTORC1. Thus, SNAT7 links glutamine and asparagine signaling from extracellular protein to mTORC1 independently of the Rag GTPases and is required for macropinocytosis-mediated mTORC1 activation and pancreatic cancer cell growth.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Pinocitosis , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Asparagina/metabolismo , Glutamina/metabolismo , Humanos , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de SeñalRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HCT116 , Células HEK293 , Humanos , Ratones , Células PC-3 , Fosforilación/fisiologíaRESUMEN
The Rag GTPases are an evolutionarily conserved family that play a crucial role in amino acid sensing by the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is often referred to as the master regulator of cell growth. mTORC1 hyperactivation is observed in multiple diseases such as cancer, obesity, metabolic disorders, and neurodegeneration. The Rag GTPases sense amino acid levels and form heterodimers, where RagA or RagB binds to RagC or RagD, to recruit mTORC1 to the lysosome where it becomes activated. Here, we review amino acid signaling to mTORC1 through the Rag GTPases.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Complejos Multiproteicos , Complejos Multiproteicos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de Señal/fisiología , Aminoácidos/metabolismo , Lisosomas/metabolismoRESUMEN
In this issue of Molecular Cell, Ye et al. (2015) demonstrate that mTORC1 globally regulates miRNA biogenesis under nutrient-rich conditions via the E3 ubiquitin ligase Mdm2, which promotes Drosha degradation.
Asunto(s)
MicroARNs/biosíntesis , Complejos Multiproteicos/fisiología , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Ribonucleasa III/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Humanos , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejos Multiproteicos/metabolismo , Presión Osmótica/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Activación Enzimática , Eliminación de Gen , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Proteína Reguladora Asociada a mTORRESUMEN
The mammalian target of rapamycin (mTOR) senses upstream stimuli to regulate numerous cellular functions such as metabolism, growth, and autophagy. Increased activation of mTOR complex 1 (mTORC1) is typically observed in human disease and continues to be an important therapeutic target. Understanding the upstream regulators of mTORC1 will provide a crucial link in targeting hyperactivated mTORC1 in human disease. In this mini-review, we will discuss the regulation of mTORC1 by upstream stimuli, with a specific focus on G-protein coupled receptor signaling to mTORC1. SIGNIFICANCE STATEMENT: mTORC1 is a master regulator of many cellular processes and is often hyperactivated in human disease. Therefore, understanding the molecular underpinnings of G-protein coupled receptor signaling to mTORC1 will undoubtedly be beneficial for human disease.
Asunto(s)
Transducción de Señal , Serina-Treonina Quinasas TOR , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
mTOR complex 1 (mTORC1) senses nutrients to mediate anabolic processes within the cell. Exactly how mTORC1 promotes cell growth remains unclear. Here, we identified a novel mTORC1-interacting protein called protein kinase A anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth, and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit Iα. Reintroducing full-length AKAP8L into cells restored mTORC1-regulated processes, whereas reintroduction of AKAP8L missing the N-terminal region that confers the interaction with mTORC1 did not. Our results suggest a multifaceted role for AKAPs in the cell. We conclude that mTORC1 appears to regulate cell growth, perhaps in part through AKAP8L.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Nucleares/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Proteínas Nucleares/deficienciaRESUMEN
Nutrient sensing by cells is crucial, and when this sensing mechanism is disturbed, human disease can occur. mTOR complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Leucine, arginine, and methionine signal to mTORC1 through the well-characterized Rag GTPase signaling pathway. In contrast, glutamine activates mTORC1 through a Rag GTPase-independent mechanism that requires ADP-ribosylation factor 1 (Arf1). Here, using several biochemical and genetic approaches, we show that eight amino acids filter through the Rag GTPase pathway. Like glutamine, asparagine signals to mTORC1 through Arf1 in the absence of the Rag GTPases. Both the Rag-dependent and Rag-independent pathways required the lysosome and lysosomal function for mTORC1 activation. Our results show that mTORC1 is differentially regulated by amino acids through two distinct pathways.
Asunto(s)
Asparagina/metabolismo , Glutamina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminoácidos/química , Aminoácidos/farmacología , Animales , Asparagina/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Medios de Cultivo/química , Medios de Cultivo/farmacología , Glutamina/química , Células HEK293 , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
The mammalian target of rapamycin (mTOR) senses nutrients and growth factors to coordinate cell growth, metabolism and autophagy. Extensive research has mapped the signaling pathways regulated by mTOR that are involved in human diseases, such as cancer, and in diabetes and ageing. Recently, however, new studies have demonstrated important roles for mTOR in promoting the differentiation of adult stem cells, driving the growth and proliferation of stem and progenitor cells, and dictating the differentiation program of multipotent stem cell populations. Here, we review these advances, providing an overview of mTOR signaling and its role in murine and human stem and progenitor cells.
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Células Madre Adultas/metabolismo , Células Madre Multipotentes/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Células Madre Adultas/patología , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Humanos , Células Madre Multipotentes/patología , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, the ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino) ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.
Asunto(s)
Isoquinolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Línea Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo-YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.
Asunto(s)
Diferenciación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Aciltransferasas , Adipogénesis , Animales , Línea Celular , Proliferación Celular , AMP Cíclico/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Drosophila melanogaster/enzimología , Drosophila melanogaster/metabolismo , Activación Enzimática , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosforilación , Sistemas de Mensajero Secundario/fisiología , Serina-Treonina Quinasa 3 , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The mammalian target of rapamycin (mTOR) is a conserved protein kinase involved in a multitude of cellular processes including cell growth. Increased mTOR activation is observed in multiple human cancers and inhibition of mTOR has proven efficacious in numerous clinical trials. mTOR comprises two complexes, termed mTORC1 and mTORC2. Both complexes respond to growth factors, whereas only mTORC1 is controlled by nutrients, such as glucose and amino acids. Since the discovery of mTOR, extensive studies have intricately detailed the molecular mechanisms by which mTORC1 is regulated. Somewhat paradoxically, amino acid (AA)-induced mTORC1 activation -arguably the most essential stimulus leading to mTORC1 activation - is the least understood. Here we review the current knowledge of nutrient-dependent regulation of mTORC1.
Asunto(s)
Aminoácidos/metabolismo , Glucosa/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proliferación Celular , Metabolismo Energético , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
The intestine is a proliferative tissue subjected to a variety of stresses that disturb its homeostasis. A recent study by Tucker et al. demonstrated that loss of the tumor suppressor SIRT4 leads to increased cell proliferation via the de novo nucleotide biosynthesis pathway over the salvage pathway after ionizing irradiation (IR).
RESUMEN
Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.