ABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.
Subject(s)
Birt-Hogg-Dube Syndrome/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/deficiency , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Birt-Hogg-Dube Syndrome/genetics , Birt-Hogg-Dube Syndrome/pathology , Cell Line , Disease Models, Animal , Enzyme Activation , HeLa Cells , Humans , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Mice , Mice, Knockout , Monomeric GTP-Binding Proteins/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Ras Homolog Enriched in Brain Protein/metabolism , Substrate Specificity , Tuberous Sclerosis Complex 2 Protein/metabolism , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/geneticsABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.
Subject(s)
Feedback, Physiological/physiology , Gene Expression Regulation, Neoplastic , Mechanistic Target of Rapamycin Complex 1/metabolism , Neoplasms/physiopathology , Animals , Caloric Restriction , Cell Line, Tumor , Cell Proliferation/genetics , Cells, Cultured , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Liver/enzymology , Liver/physiopathology , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , Mice, Inbred C57BL , Neoplasms/enzymology , Signal TransductionABSTRACT
CONTEXT: Histone deacetylase inhibitors (HDACis) are anticancer agents that inhibit tumor cell growth and/or survival. However, their mechanism of action remains largely undefined. Recently, we have demonstrated that HDACis induce apoptosis in a model of rat thyroid cells transformed by the v-ras-Ki oncogene (FRTL-5 v-ras-Ki). The stabilization of TNF-related apoptosis-inducing ligand (TRAIL) protein, due to its reduced ubiquitination and proteasome degradation, accounts for the apoptotic effect induced specifically by suberoylanilide hydroxamic acid (SAHA, Vorinostat) in the v-ras-Ki thyroid transformed cells. OBJECTIVE: The aim of this work was to investigate whether SAHA may induce its cytotoxic effects also deregulating microRNA (miRNA) expression levels. DESIGN: We analyzed the miRNA expression profile of the thyroid transformed cells, FRTL-5 v-ras-Ki, upon SAHA treatment. RESULTS: Here we report that SAHA induces the down-regulation of 18 and the up-regulation of 11 miRNAs with a fold change higher than 2 in the transformed cells. Then, we focus on the miR-146b and miR-200b, respectively up-regulated and down-regulated by SAHA. We show that both these miRNAs target genes coding for proteins with a critical role in proteasome composition and ubiquitin degradation. CONCLUSION: These results suggest a role of miRNA deregulation in TRAIL protein stabilization responsible for SAHA-induced apoptotic effect in thyroid transformed cells.
