Abstract
Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis1. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.
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References
Dibble, C. C. & Manning, B. D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nature Cell Biol. 15, 555–564 (2013)
Cornu, M., Albert, V. & Hall, M. N. mTOR in aging, metabolism, and cancer. Curr. Opin. Genet. Dev. 23, 53–62 (2013)
Suraweera, A., Munch, C., Hanssum, A. & Bertolotti, A. Failure of amino acid homeostasis causes cell death following proteasome inhibition. Mol. Cell 48, 242–253 (2012)
Fonseca, R., Vabulas, R. M., Hartl, F. U., Bonhoeffer, T. & Nagerl, U. V. A balance of protein synthesis and proteasome-dependent degradation determines the maintenance of LTP. Neuron 52, 239–245 (2006)
Singh, R. & Cuervo, A. M. Autophagy in the cellular energetic balance. Cell Metab. 13, 495–504 (2011)
Ebato, C. et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab. 8, 325–332 (2008)
Radhakrishnan, S. K. et al. Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells. Mol. Cell 38, 17–28 (2010)
Steffen, J., Seeger, M., Koch, A. & Kruger, E. Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop. Mol. Cell 40, 147–158 (2010)
Düvel, K. et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183 (2010)
Radhakrishnan, S. K., den Besten, W. & Deshaies, R. J. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition. eLife 3, e01856 (2014)
Ozcan, U. et al. Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis. Mol. Cell 29, 541–551 (2008)
Schröder, M. & Kaufman, R. J. The mammalian unfolded protein response. Annu. Rev. Biochem. 74, 739–789 (2005)
Rome, S. et al. Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle. Physiol. Genomics 34, 327–337 (2008)
Reed, B. D., Charos, A. E., Szekely, A. M., Weissman, S. M. & Snyder, M. Genome-wide occupancy of SREBP1 and its partners NFY and SP1 reveals novel functional roles and combinatorial regulation of distinct classes of genes. PLoS Genet. 4, e1000133 (2008)
Yuan, E. et al. Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features. Hum. Mol. Genet. 21, 4286–4300 (2012)
Lee, C. S., Ho, D. V. & Chan, J. Y. Nuclear factor-erythroid 2-related factor 1 regulates expression of proteasome genes in hepatocytes and protects against endoplasmic reticulum stress and steatosis in mice. FEBS J. 280, 3609–3620 (2013)
Yecies, J. L. et al. Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways. Cell Metab. 14, 21–32 (2011)
Toth, J. I., Datta, S., Athanikar, J. N., Freedman, L. P. & Osborne, T. F. Selective coactivator interactions in gene activation by SREBP-1a and -1c. Mol. Cell. Biol. 24, 8288–8300 (2004)
He, T. C. et al. A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95, 2509–2514 (1998)
Huang, J., Dibble, C. C., Matsuzaki, M. & Manning, B. D. The TSC1-TSC2 complex is required for proper activation of mTOR complex 2. Mol. Cell. Biol. 28, 4104–4115 (2008)
Kwiatkowski, D. J. et al. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum. Mol. Genet. 11, 525–534 (2002)
Zhang, H. et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233 (2003)
El-Hashemite, N., Zhang, H., Henske, E. P. & Kwiatkowski, D. J. Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma. Lancet 361, 1348–1349 (2003)
Cock, P. J. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009)
Zerenturk, E. J., Sharpe, L. J. & Brown, A. J. Sterols regulate 3β-hydroxysterol Δ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by sterol regulatory element binding proteins. Biochim. Biophys. Acta 1821, 1350–1360 (2012)
Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)
Acknowledgements
We thank I. Ben-Sahra and L. Yang for technical assistance. This work was supported in part by Department of Defense Tuberous Sclerosis Complex Research Program grant W81XWH-10-1-0861 (B.D.M.), National Institutes of Health grants CA122617 (B.D.M.) and CA120964 (B.D.M. and D.J.K.), the Ellison Medical Foundation (B.D.M.), National Science Foundation fellowship DGE-1144152 (S.J.H.R.), and a Canadian Institutes of Health Research fellowship (S.B.W.).
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B.D.M. and Y.Z. designed and interpreted the experiments and wrote the manuscript. Y.Z., J.N., J.R.D., S.J.H.R. and S.B.W. performed the experiments. G.S.H. and D.J.K. provided key materials and technical guidance.
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Extended data figures and tables
Extended Data Figure 1 mTORC1 activation increases protein degradation.
a, Immunoblots of lysates from Fig. 1a are shown. b, The same experiment as in Fig. 1a except Tsc1+/+ and Tsc1−/− MEFs were used. Data are mean ± s.e.m. (n = 3). *P < 0.05, †P < 0.05. c, Schematic diagram of the experimental design for the pulse-chase measurements of protein turnover. d, An autoradiograph gel image representative of the three independent experiments quantified in Fig. 1b. e, The same experiment in Fig. 1b was performed, except with Tsc1+/+ and Tsc1−/− MEFs. Data are mean ± s.e.m. (n = 3). *P < 0.05 for the 48 h data point comparison. f, An autoradiograph gel image representative of the three independent experiments quantified in e. b, e, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t-test.
Extended Data Figure 2 mTORC1 activation enhances protein degradation in a proteasome-dependent manner.
a, An autoradiograph gel image representative of the three independent experiments quantified in Fig. 1c. Rap, rapamycin; WT, wild type. b, The same experiment as Fig. 1b was performed, except a pair of wild-type and Atg7−/− MEFs were used. Data are mean ± s.e.m. (n = 3; note: small error bars are masked by line symbols). ***P < 0.001 for 48 h time point. c, d, Autoradiograph gel images representative of the three independent experiments quantified and graphically represented in either panel b (c) or Fig. 1d (d). e, The same experiment in Fig. 1d was performed, except MG132 was used instead of bortezomib. f, The autoradiograph gel image quantified in e. g, Immunoblots demonstrating TSC2 loss and mTORC1 activation in the cells used in Fig. 1f and h. The indicated cells were starved for 16 h in the presence of vehicle (DMSO) or 20 nM rapamycin, before lysis. h, The same experiments in Fig. 1f were performed, except MCF10A and HeLa cells expressing non-targeting shRNAs (shCtl) or shRNAs targeting human TSC2 (shTSC2) were used. Data are presented as mean ± s.e.m. relative to vehicle-treated TSC2-expressing cells (n = 3). *P < 0.05, †P < 0.05, ††P < 0.01. b, h, Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test.
Extended Data Figure 3 mTORC1 signalling promotes PSM gene transcription.
a, PSM gene expression from a previous microarray experiment comparing expression in Tsc2−/− MEFs, over a time course of rapamycin treatment, to those in littermate Tsc2+/+ MEFs. Log2 expression levels provided are the average obtained from triplicate samples per time point of rapamycin treatment normalized to the expression levels in vehicle-treated wild-type (WT) cells. b, The expression levels of two additional PSM genes from the experiment in Fig. 2a are shown. Data are mean ± s.e.m. (n = 3). *P < 0.05 compared to vehicle-treated TSC2-expressing cells; †P < 0.05, ††P < 0.01 compared to vehicle-treated TSC2-deficient cells. c, The same experiment as Fig. 2a, except that PSM gene expression was analysed in the same littermate-derived pair of Tsc2+/+ p53−/− and Tsc2−/− p53−/− MEFs used in a. Data are mean ± s.e.m. (n = 3) relative to vehicle-treated Tsc2+/+ cells. *P < 0.05 or **P < 0.01 compared to vehicle-treated Tsc2+/+ cells; †P < 0.05, ††P < 0.01 or †††P < 0.001 compared to vehicle-treated Tsc2−/− cells. d, The same experiment shown in Fig. 2a, except that PSM gene expression was analysed in HeLa cells stably expressing shRNAs targeting firefly luciferase (shLUC) or those targeting human TSC2 (shTSC2). Data are mean ± s.e.m. (n = 3) relative to vehicle-treated shLUC-expressing cells. *P < 0.05 or **P < 0.01 compared to vehicle-treated shLUC-expressing cells; †P < 0.05, ††P < 0.01 or †††P < 0.001 compared to vehicle-treated shTSC2-expressing cells. e, Cells were serum starved for 16 h then stimulated with 10% serum in the presence of vehicle (DMSO), 20 nM rapamycin or 250 nM torin 1. Transcript levels are shown as mean ± s.e.m. relative to vehicle (n = 3). b–d, Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test.
Extended Data Figure 4 NRF1 knockdown decreases the mTORC1-stimulated expression of PSM genes and protein degradation.
a, The expression levels of an additional PSM gene from the experiment in Fig. 2b is shown. *P < 0.05 compared to vehicle-treated TSC2-expressing cells; ††P < 0.01 compared to vehicle-treated vector-expressing cells. Data are mean ± s.e.m. (n = 3). Vec, vector. b, The same experiment shown in Fig. 2b, except the littermate-derived pair of Tsc2+/+ p53−/− and Tsc2−/− p53−/− MEFs were used. Data are shown as the mean ± s.e.m. (n = 3). *P < 0.05 or **P < 0.01; ††P < 0.01 or †††P < 0.001. c, MEF cell lysates obtained from the experiment in Fig. 2c were subjected to immunoblotting. d, The same experiment in Fig. 2c was performed, except HeLa cells expressing non-targeting shRNAs (shLUC) or shRNAs targeting human TSC2 (shTSC2) were used. Data are presented as mean ± s.e.m. relative to vehicle-treated shLUC-expressing cells (n = 3). *P < 0.05, †P < 0.05. e, f, Cell lysates obtained from the experiment in Fig. 2d and e, respectively, were subjected to immunoblotting. g, Autoradiograph of gel, representative of three independent experiments, corresponding to the data graphically represented in Fig. 2e. a, b, d, Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test.
Extended Data Figure 5 Genetic and growth-factor stimulation of mTORC1 signalling increases the protein levels of NRF1.
a, HEK293 cells were transfected with RHEB or empty vector and serum starved for 16 h in the presence of vehicle or rapamycin (Rap; 20 nM) before lysis and immunoblotting. Phosphorylated (p) S6K1 is shown as a marker of mTORC1 activity. b, The same experiment shown in Fig. 3a, except MCF10A cells expressing non-targeting shRNAs (−) or shRNAs targeting human TSC2 (+) were used and were stimulated with full serum (10% FBS) or EGF (10 ng ml−1) for the indicated durations after 16 h serum starvation. c, Same as b, except HeLa cells were used and were stimulated with insulin (100 nM). d, The normalized cell lysates from the experiment shown in Fig. 3a, with just the starved and 24-h-stimulated samples, along with the vector-expressing Tsc2-null cells, were run on a 4–12% continuous gradient NuPAGE gel, followed by immunoblotting. e, Lysates from the insulin-stimulated cells obtained in Fig. 3a were subjected to additional immunoblotting. f, Tsc2-null MEFs reconstituted with wild-type TSC2 were stimulated with insulin (100 nM for 24 h), and intact proteasome levels were measured by enzyme-linked immunosorbent assay (ELISA) and are presented as the mean ± s.e.m. (n = 3). **P < 0.01, ††P < 0.01. f, Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test.
Extended Data Figure 6 NRF1 activation downstream of mTORC1 is independent of ER stress, proteasome inhibition, and distribution between the cytosol and nucleus.
a, MCF10A cells stably expressing non-targeting shRNAs (−) or shRNAs targeting human TSC2 (+) were serum starved for 24 h in the presence of vehicle or the compounds indicated (tunicamycin, 0.5 µg ml−1; thapsigargin, 1 µM; MG132, 0.5 µM; bortezomib, 0.5 µM). Whole-cell lysates were immunoblotted with the indicated antibodies. Phosphorylated (p) PERK is shown as a marker of the unfolded protein response. b, The same cells in a were serum starved for 24 h in the presence of vehicle or rapamycin. Cytoplasmic (c) and nuclear (n) extracts were isolated and immunoblotted. c, HEK293 cells transiently expressing His–NRF1–Flag were serum starved for 24 h in the presence of vehicle or rapamycin, and subject to cytoplasmic/nuclear fractionation and immunoblotting.
Extended Data Figure 7 mTORC1 activates NRF1 gene expression through SREBP1.
a, The same experiment shown in Fig. 3c, except with human TSC2−/− angiomyolipoma cells reconstituted with human TSC2 or empty vector (EV). Data are shown as the mean ± s.e.m. (n = 3). *P < 0.05 compared to TSC2-expressing cells transfected with control siRNAs; †P < 0.05 compared to vector-expressing cells transfected with control siRNAs. Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test. b, Consensus sterol regulatory elements (SREs) are conserved in the promoters of the human and rodent NRF1 genes. Forward (top left) and reverse (top right) position weight matrices based on SREs of twenty established SREBP targets are shown and were used to find putative SREs in the NRF1 promoter. The human, mouse and rat NRF1 promoters are aligned and numbered with their distance from the conserved translation start site. The two possible transcription start sites are depicted with a numbered arrow above the aligned sequences. Four SREs were found to be conserved in all three promoters in the region of these start sites. c, In the same samples described in Fig. 3f, ChIP analysis for SREBP1c and Pol II promoter occupancy of the given genes was performed using HEK293 cells expressing Flag-tagged (FL) mature SREBP1c or empty vector. Known SREBP1 target sites on SCD served as a positive control, with GAPDH and NRF2 promoters as negative controls. Ab, antibody. Data were normalized to the levels of bound DNA in control IgG immunoprecipitations and are shown as mean ± s.e.m. (n = 3).
Extended Data Figure 8 mTORC1 signalling influences proteasome subunit expression in vivo.
a, Some individual proteasome subunits are shown in the same brain lysates obtained in Fig. 4a. b, Expression of transcripts from representative PSM genes in the livers of the mice described in Fig. 4d, e were measured by qRT–PCR and are presented as mean ± s.e.m. relative to fasted controls (n = 4). Rap, rapamycin. *P < 0.05 compared to fasted mice, †P < 0.05 compared to refed, vehicle-treated mice. Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test.
Extended Data Figure 9 NRF1 and the proteasome influence intracellular amino acid levels and rates of protein synthesis.
a, Tsc2−/− MEFs reconstituted with human TSC2 or empty vector were serum starved for 16 h and treated for 1 h with the indicated compound. The total pool of intracellular amino acids was measured and is shown as mean ± s.e.m. of triplicate samples relative to untreated samples (veh). *P < 0.05 or **P < 0.01 compared to vehicle-treated TSC2-expressing cells; †P < 0.05 or ††P < 0.01 compared to vehicle-treated vector-expressing cells. Statistical significance for pairwise comparisons was evaluated with a two-tailed Student’s t-test. b, Immunoblot control for the experiment shown in Fig. 4g. c, d, Autoradiographs of gels, representative of three independent experiments each, corresponding to the protein synthesis data graphically represented in Fig. 4g and h, respectively. e, Tsc2−/− MEFs were grown in media containing increasing concentrations of amino acids for 16 h in the presence or absence of rapamycin. Immunoblots of lysates are shown. The physiological concentration of amino acids is indicated as 1× and represents the concentration in BME (‘Low AAs’ in Fig. 4h and d), with DMEM being 4× and twice that (8×) being the concentration denoted as ‘High AAs’ in Fig. 4h and d.
Extended Data Figure 10 TSC2-deficient MEFs and MCF10As exhibited increased sensitivity to NRF1 knockdown relative to their isogenic wild-type counterparts.
a, b, Viable counts of TSC2-expressing and -deficient MEFs (a) and MCF10As (b) transfected with siRNAs targeting Nrf1 are shown as mean ± s.e.m. relative to the same cells expressing control siRNAs (n = 3 technical replicates, representative of two independent experiments each). a, *P < 0.02; b, **P < 0.005. a, b, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t-test. c, Model of the parallel regulation of protein synthesis and degradation by mTORC1 described in this study. AAs, amino acids.
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Zhang, Y., Nicholatos, J., Dreier, J. et al. Coordinated regulation of protein synthesis and degradation by mTORC1. Nature 513, 440–443 (2014). https://doi.org/10.1038/nature13492
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DOI: https://doi.org/10.1038/nature13492
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