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. 2014 Sep 18;513(7518):440-3.
doi: 10.1038/nature13492. Epub 2014 Jul 13.

Coordinated regulation of protein synthesis and degradation by mTORC1

Yinan Zhang  1 , Justin Nicholatos  1 , John R Dreier  2 , Stéphane J H Ricoult  1 , Scott B Widenmaier  1 , Gökhan S Hotamisligil  1 , David J Kwiatkowski  2 , Brendan D Manning  1
Affiliations

Coordinated regulation of protein synthesis and degradation by mTORC1

Yinan Zhang et al. Nature. .

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 synthesis. 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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Figures

Extended Data Figure 1
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-hour 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
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. b, The same experiment as Fig. 1b was performed, except a pair of wild-type and Atg7−/− MEFs were used. Data are means ± 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 c, panel b or d, Fig. 1d. 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 panel h. The indicated cells were starved for 16 h in the presence of vehicle (DMSO) or 20 nM rapamycin, prior to 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 evaluated with a two-tailed Student’s t test.
Extended Data Figure 3
Extended Data Figure 3. mTORC1 signaling 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 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 analyzed in the same littermate-derived pair of Tsc2+/+ p53−/− and Tsc2−/− p53−/− MEFs use 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 analyzed 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 16 h then stimulated with 10% serum in the presence of vehicle (DMSO), 20 nM rapamycin, or 250 nM Torin1. Transcript levels are shown as mean ± s.e.m. relative to vehicle (n=3). b–d, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Extended Data Figure 4
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). 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 means ± 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 2e, 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 evaluated with a two-tailed Student’s t test.
Extended Data Figure 5
Extended Data Figure 5. Genetic and growth factor-stimulation of mTORC1 signaling 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. 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) 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 ELISA and are presented as the mean ± s.e.m (n=3). **p < 0.01, ##p < 0.01. f, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Extended Data Figure 6
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; thapsigargin, 1 μM; MG132, 0.5 μM; bortezomib, 0.5 μM). Whole-cell lysates were immunoblotted with the indicated antibodies. b, The same cells in a were serum starved for 24 h in the presence of vehicle or rapamycin. Cytoplasmic and nuclear 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
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 means ± 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 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 mature SREBP1c or empty vector. Known SREBP1 target sites on SCD served as a positive control, with GAPDH and NFE2L2/NRF2 promoters as negative controls. Data were normalized to the levels of bound DNA in control IgG IPs and are shown as mean ± s.e.m (n=3).
Extended Data Figure 8
Extended Data Figure 8. mTORC1 signaling 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). *p < 0.05 compared to fasted mice, #p < 0.05 compared to refed, vehicle-treated mice. Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Extended Data Figure 9
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 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 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 1x and represents the concentration in BME (“Low AAs” in Fig. 4h and panel d), with DMEM being 4x and twice that (8x) being the concentration denoted as “High AAs” in Fig. 4h and panel d.
Extended Data Figure 10
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 a, MEFs and b, MCF10As 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.
Figure 1
Figure 1. mTORC1 enhances protein degradation through an increase in proteasome levels
a, Tsc2−/− MEFs expressing TSC2 or empty vector (Vec) were serum starved 16 h with vehicle or 20 nM rapamycin, and protein synthesis was measured with 35S-Met incorporation (20 min). Data are mean ± s.e.m. (n=3). **P<0.01, #P<0.05. b, Cells treated as in a were pulse labeled 30 min and chased in medium containing vehicle or rapamycin. The rate of protein degradation is shown as the fraction of radiolabeled protein remaining over time. c–d, Cells were treated as in b, except that c, 10 μM chloroquine or d, 0.02 μM bortezomib was present in the chase media. b–d, Data are means ± s.e.m (n=3, note: small error bars are masked by line symbols). *P<0.05, **P<0.01, at 48h. e, The cells from a were serum starved 16 h with vehicle or 20 nM rapamycin, or treated for 2 h with MG132 (0.1 μM) or bortezomib (0.2 μM). Proteasome activity is presented as mean ± s.e.m. relative to vehicle-treated cells expressing TSC2 (n=3). *P<0.05, ##P<0.01. f, Cells were serum starved 24 h with vehicle, 20 nM rapamycin, or 2.5 μM PP242. Intact proteasome levels are presented as mean ± s.e.m. relative to vehicle-treated TSC2-expressing cells (n=3). Graphs labeled as in e; *P<0.05, #P<0.05, ##P<0.01. a–e, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Figure 2
Figure 2. mTORC1 induces proteasome gene expression and protein degradation through NRF1
a, Tsc2−/− MEFs expressing TSC2 or empty vector (Vec) were serum starved 16 h with vehicle or 20 nM rapamycin. Transcript levels are shown as mean ± s.e.m. relative to vehicle-treated TSC2-expressing cells (n=3). *P<0.05 or **P<0.01 compared to vehicle-treated TSC2-expressing cells; #P<0.05, ##P<0.01, or ###P<0.001 compared to vehicle-treated vector-expressing cells. b, siRNA-transfected cells were serum starved 16 h. Transcript levels are presented as mean ± s.e.m. relative to TSC2-expressing cells with control siRNAs (n=3). *P<0.05, ##P<0.01. c, Proteasome levels for cells treated as in b are presented as in b (n=3). **P<0.01, #P<0.05. d, Proteasome levels in HEK293 cells transfected with indicated plasmids and serum starved 16 h with vehicle or 20 nM rapamycin are shown as mean ± s.e.m relative to vehicle-treated vector-expressing cells. **P<0.01. e, Rates of protein degradation in serum-starved siRNA-transfected cells (control or Nrf1) treated with vehicle or rapamycin are shown as the fraction of radiolabeled protein remaining over time, presented as mean ± s.e.m (n=3, note: small error bars are masked by line symbols). **P<0.01 at 48h. a–e, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Figure 3
Figure 3. Growth factors stimulate an increase in NRF1 through mTORC1, which induces NRF1 transcription in an SREBP1-dependent manner
a, Tsc2−/− MEFs expressing TSC2 were serum starved 16 h and stimulated with 10% serum, 10 ng/ml EGF, or 100 nM insulin for the indicated durations with vehicle or 20 nM rapamycin. b, Nrf1 and Nrf2 transcript levels from cells serum starved 16 h with vehicle, 20 nM rapamycin, or 250 nM Torin1 are shown as mean ± s.e.m. relative to vehicle-treated TSC2-expressing cells (n=3). ***P<0.001 compared to vehicle-treated TSC2-expressing cells; ##P<0.01 or ###P<0.001 compared to vehicle-treated vector-expressing cells. c, siRNA transfected cells were treated as in b (Rap = 20 nM rapamycin). Nrf1 transcript levels are presented as mean ± s.e.m. relative to Tsc2-expressing cells with control siRNAs (n=3). ***P<0.001 compared to TSC2-expressing cells, ###P<0.001 compared to vector-expressing cells with control siRNAs. d, NRF1 protein levels from cells treated as in c or e, in HEK293 cells transfected with mature Flag-SREBP1c or empty vector and serum starved 16 h with vehicle or 20 nM rapamycin. f, ChIP from HEK293 cells transfected as in e with anti-Flag (SREBP1c) or Pol II. Bound DNA was measured by qRT-PCR for indicated promoter regions (left: 1, 2, 3) and normalized to control IgG immunoprecipitations. Data are mean ± s.e.m (n=3). b,c, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
Figure 4
Figure 4. NRF1 is induced upon mTORC1 activation in tissues and influences cellular amino acid levels and protein synthesis
a, Protein from indicated brain lysates are shown, with NRF1 levels normalized to actin below. b, Nrf1 and Nrf2 and c, PSM gene transcript levels from brain tissues in a are shown as mean ± s.e.m. of triplicate samples relative to Tsc2+/+ sample 1. d, Mice fasted overnight were refed (6 h) following 30-min pretreatment with vehicle or rapamycin (10 mg/kg). Protein from liver lysates are shown, with NRF1 levels normalized to actin graphed as mean ± s.e.m. relative to fasted mice (n=4 per condition). *P<0.05, ##P<0.01. e, Transcript levels from liver tissues in d are shown as mean ± s.e.m. relative to fasted mice. *P<0.05, #P<0.05. f, Tsc2−/− MEFs expressing TSC2 or empty vector transfected with Nrf1 (a and b) or control siRNAs were serum starved 16 h with vehicle or 20 nM rapamycin or treated 1 h with 100 nM bortezomib. Amino acid levels are shown as mean ± s.e.m. of triplicate samples relative to TSC2-expressing cells. *P<0.05 compared to TSC2-expressing cells, ##P<0.01 or ###P<0.001 compared to vehicle-treated vector-expressing cells. g, Rates of protein synthesis in cells treated as in f are shown as mean ± s.e.m relative to TSC2-expressing cells (n=3). **P<0.01 compared to TSC2-expressing cells; #P<0.05 or ##P<0.01 compared to vehicle-treated vector-expressing cells. h, Cells treated as in f were switched to low or high amino acid media overnight, and rates of protein synthesis are shown as the mean ± s.e.m relative to vehicle-treated cells (n=3). ***P<0.001 compared to vehicle-treated low AA cells; #P<0.05 or ##P<0.01 compared to vehicle-treated high AA cells. d–h, Statistical significance for pairwise comparisons evaluated with a two-tailed Student’s t test.
See this image and copyright information in PMC

Comment in

  • Control of proteasomal proteolysis by mTOR.
    Zhao J, Garcia GA, Goldberg AL. Zhao J, et al. Nature. 2016 Jan 21;529(7586):E1-2. doi: 10.1038/nature16472. Nature. 2016. PMID: 26791731 Free PMC article. No abstract available.
  • Zhang & Manning reply.
    Zhang Y, Manning BD. Zhang Y, et al. Nature. 2016 Jan 21;529(7586):E2-3. doi: 10.1038/nature16473. Nature. 2016. PMID: 26791732 Free PMC article. No abstract available.

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