The Protein Complex Ragulator is the Substrate-specific Scaffold for mTORC1 to Regulate Transcription Factor EB
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ABSTRACT:
PROVIDER: PRJNA719171 | ENA |
REPOSITORIES: ENA
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Project description:Mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is activated by nutrition sufficiency signals and extracellular growth signals. mTORC1 acts the hub that integrates these inputs to orchestrate number of cellular responses such as translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, the scaffold protein which specifically regulates any single downstream signaling molecule has not been identified to date. Here we show the heteropentamer protein complex Ragulator is critically required to regulate nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacks Ragulator but maintained total mTORC1 activity. The clone showed a markedly enhanced nuclear translocation of TFEB even in nutrition-sufficient state, despite the full mTORC1 activity. As a cellular phenotype, the number of lysosomes were increased by 10 times in the Ragulator-deficient clone. These findings suggest that mTORC1 essentially requires the scaffold Ragulator to regulate the subcellular location of TFEB. Our finding implicates that mTORC1 has other scaffold proteins that regulate downstream molecules specifically.
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Project description:The RNA biding protein, LARP1, has been proposed to function downstream of mTORC1 to positively regulate the translation of 5M-bM-^@M-^YTOP mRNAs such as ribosome protein (RP) mRNAs. However, its regulatory roles in mTORC1-mediated translation remain unclear. PAR-CLIP of LARP1 revealed its direct and dynamic interactions with RP mRNAs through pyrimidine-enriched sequences in the 5M-bM-^@M-^YUTR of RP mRNAs when mTOR activity is inhibited. Importantly, this LARP1 is a direct substrate of mTORC1 and S6K1/Akt, and phosphorylated LARP1 scaffolds mTORC1 on translation-competent mRNAs to facilitate 4EBP1 and S6K1 phosphorylation. Ablation of LARP1 causes multiple defects in the processes of translation including abnormal eIF4G1 interaction with RP mRNAs and inefficient RP mRNA elongation thereby reducing ribosome biogenesis and cell proliferation. These observations illustrate that LARP1 functions both an effector and a regulator for local mTORC1 activity, and acts as a molecular switch for ribosome biogenesis by sensing growth factor/nutrient signaling. LARP1-bound RNA regions were sequenced from HEK293T cells under growing or mTOR-inactive conditions. In parallel, mRNA abundance was quantified, in biological duplicate, from HEK293T cells under the same conditions.
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Project description:The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.
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Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-bM-^@M-^\low/low/high/lowM-bM-^@M-^] or M-bM-^@M-^\LLHLM-bM-^@M-^]. We found that mTORC1 regulated the expression of, among other lysosomal genes, V-ATPases through the transcription factor EB (TFEB, Tcfeb in the mouse). The knockdown of Tfeb resulted in the 'flattening' of the LLHL pattern and allowed the identification of genes regulated by mTORC1 through Tfeb Mouse embryo fibroblasts (MEFs) wild type or deficient in Tsc2 expressing a Tfeb shRNA or scrambled shRNA vector were treated with 25 nM rapamycin or vehicle (methanol) for 24 h under low serum conditions (0.1% FBS)
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Project description:The RNA biding protein, LARP1, has been proposed to function downstream of mTORC1 to positively regulate the translation of 5’TOP mRNAs such as ribosome protein (RP) mRNAs. However, its regulatory roles in mTORC1-mediated translation remain unclear. PAR-CLIP of LARP1 revealed its direct and dynamic interactions with RP mRNAs through pyrimidine-enriched sequences in the 5’UTR of RP mRNAs when mTOR activity is inhibited. Importantly, this LARP1 is a direct substrate of mTORC1 and S6K1/Akt, and phosphorylated LARP1 scaffolds mTORC1 on translation-competent mRNAs to facilitate 4EBP1 and S6K1 phosphorylation. Ablation of LARP1 causes multiple defects in the processes of translation including abnormal eIF4G1 interaction with RP mRNAs and inefficient RP mRNA elongation thereby reducing ribosome biogenesis and cell proliferation. These observations illustrate that LARP1 functions both an effector and a regulator for local mTORC1 activity, and acts as a molecular switch for ribosome biogenesis by sensing growth factor/nutrient signaling.
2014-07-31 | GSE59599 | GEO
Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a “low/low/high/low” or “LLHL”. We found that mTORC1 regulated the expression of, among other lysosomal genes, V-ATPases through the transcription factor EB (TFEB, Tcfeb in the mouse). The knockdown of Tfeb resulted in the 'flattening' of the LLHL pattern and allowed the identification of genes regulated by mTORC1 through Tfeb
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