ABSTRACT
The mTOR complex 1 (mTORC1) is an essential metabolic hub that coordinates cellular metabolism with the availability of nutrients, including amino acids. Sestrin2 has been identified as a cytosolic leucine sensor that transmits leucine status signals to mTORC1. In this study, we identify an E3 ubiquitin ligase RING finger protein 167 (RNF167) and a deubiquitinase STAMBPL1 that function in concert to control the polyubiquitination level of Sestrin2 in response to leucine availability. Ubiquitination of Sestrin2 promotes its interaction with GATOR2 and inhibits mTORC1 signaling. Bioinformatic analysis reveals decreased RNF167 expression and increased STAMBPL1 expression in gastric and colorectal tumors. Knockout of STAMBPL1 or correction of the heterozygous STAMBPL1 mutation in a human colon cancer cell line suppresses xenograft tumor growth. Lastly, a cell-permeable peptide that blocks the STAMBPL1-Sestrin2 interaction inhibits mTORC1 and provides a potential option for cancer therapy.
Subject(s)
Colorectal Neoplasms/enzymology , Peptide Hydrolases/metabolism , Stomach Neoplasms/enzymology , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Caco-2 Cells , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Disease Progression , Gene Expression Regulation, Neoplastic , HCT116 Cells , HEK293 Cells , Humans , Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice, Inbred BALB C , Mice, Nude , Mutation , Nuclear Proteins/metabolism , Peptide Hydrolases/genetics , Signal Transduction , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Tumor Burden , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.
Subject(s)
Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Signal Transduction , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Conserved Sequence , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HEK293 Cells , Humans , Leucine/deficiency , Longevity/genetics , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mechanistic Target of Rapamycin Complex 1/agonists , Mice , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/deficiency , Monomeric GTP-Binding Proteins/genetics , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Protein Binding , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that responds to a diverse set of environmental cues, including amino acids1,2. Deregulation of mTORC1 has been linked with metabolic diseases, cancer and ageing2-4. In response to amino acids, mTORC1 is recruited by the Rag GTPases to the lysosome, its site of activation5,6. The GATOR1 complex, consisting of DEPDC5, NPRL3 and NPRL2, displays GAP activity to inactivate Rag GTPases under amino-acid-deficient conditions 7 . However, it is unclear how the inhibitory function of GATOR1 is released upon amino acid stimulation. Here we find that in response to amino acids, the CUL3-KLHL22 E3 ubiquitin ligase promotes K48-linked polyubiquitination and degradation of DEPDC5, an essential subunit of GATOR1. KLHL22 plays a conserved role to mediate the activation of mTORC1 and downstream events in mammals and nematodes. Depletion of MEL-26, the Caenorhabditis elegans orthologue of KLHL22, extends worm lifespan. Moreover, KLHL22 levels are elevated in tumours of breast cancer patients, whereas DEPDC5 levels are correspondingly reduced. Depletion of KLHL22 in breast cancer cells suppresses tumour growth in nude mice. Therefore, pharmacological interventions targeting KLHL22 may have therapeutic potential for the treatment of breast cancer and age-related diseases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Aging/metabolism , Carcinogenesis/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/deficiency , Aging/genetics , Animals , Autophagy , Breast Neoplasms/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Carcinogenesis/genetics , Cell Line , Cell Proliferation , Cullin Proteins/metabolism , Female , GTPase-Activating Proteins/metabolism , Humans , Longevity/genetics , Mice , Mice, Nude , Multiprotein Complexes/metabolism , Oncogenes , Proteolysis , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Metformin, a widely used first-line drug for treatment of type 2 diabetes (T2D), has been shown to extend lifespan and delay the onset of age-related diseases. However, its primary locus of action remains unclear. Using a pure in vitro reconstitution system, we demonstrate that metformin acts through the v-ATPase-Ragulator lysosomal pathway to coordinate mTORC1 and AMPK, two hubs governing metabolic programs. We further show in Caenorhabditis elegans that both v-ATPase-mediated TORC1 inhibition and v-ATPase-AXIN/LKB1-mediated AMPK activation contribute to the lifespan extension effect of metformin. Elucidating the molecular mechanism of metformin regulated healthspan extension will boost its therapeutic application in the treatment of human aging and age-related diseases.
