mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress

Abstract

Highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are central to cellular metabolism and cell proliferation1,2, and their dysregulation is implicated in the pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex 1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly downregulates AMPK signalling by phosphorylating the evolutionarily conserved residue S367 in the fission yeast AMPK catalytic subunit Ssp2 and AMPK α1 S347 and α2 S345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop T172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP/ATP ratios, which under nutrient stress conditions was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental bidirectional regulation between two major metabolic signalling networks and uncover new opportunities for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumour microenvironment.

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Fig. 1: Ssp2 S367A is associated with increased activating phosphorylation and reduced cell growth under nutrient stress.
Fig. 2: TORC1-dependent regulation of Ssp2 S367 phosphorylation.
Fig. 3: mTORC1 phosphorylation of mammalian AMPK α subunit at S345 negatively regulates cellular phosphorylation at T172 and AMPK signalling.
Fig. 4: α2 S345 phosphorylation promotes cell proliferation under conditions of nutrient stress.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 14 and Extended Data Figs. 110 are presented with the paper.

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Acknowledgements

We thank K. Gull (Oxford University, UK) and S. Lim (St. Vincent’s Institute, Australia) for antibodies, M. Balasubramanian (Warwick University, UK), K. Shiozaki (Nara University, Japan) and S. Moreno (IBFG Salamanca, Spain) for yeast strains, M. Hall (University of Basel, Switzerland) for iRapKO MEFs, I. Hagan for stimulating discussions and C. Proud and J. Murphy for critical evaluation of the manuscript. C.G.L. was supported by an Early Career Fellowship from the National Health and Medical Research Council (NHMRC; 1143080). J.S.O. was supported by a Future Fellowship from the Australian Research Council (ARC; FT130100988), the NHMRC (1098459), St Vincent’s Institute of Medical Research (Australia) and in part by the Victorian Government’s Operational Infrastructure Support Program. J.P. was supported by a Cancer Research UK senior fellowship (C10888/A11178), Cancer Council Australia (1125662), Worldwide Cancer Research (16-0052), the NHMRC (1161262), the ARC (DP180101682), a Flinders Foundation seeding grant, Manchester (UK), and Flinders University (Australia).

Author information

N.X.Y.L., A.K., A.H., E.D., K.R.W.N., K.R.M., W.J.S., G.M.F., T.W., S.L., T.A.D., J.S.O. and J.P. performed the experiments. C.G.L. and J.W.S. provided reagents and intellectual input. J.S.O. and J.P. designed and coordinated the study and wrote the manuscript. All authors read and approved the manuscript.

Correspondence to Jonathan S. Oakhill or Janni Petersen.

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The authors declare no competing interests.

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Peer review information Primary Handling Editors: Ana Mateus; Christoph Schmitt.

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Extended data

Extended Data Fig. 1 Analysis of S. pombe Ssp2-S367A/D KI mutants and validation of S. pombe temperature-sensitive TORC1 expression mutant tor2.ts.

Ssp2 dependent phosphorylation of the Scr1 transcription factor is elevated in S. pombe expressing Ssp2-S367A mutant. a) Lysates were prepared from WT S. pombe and indicated ssp2 mutants in which Scr1 was tagged with GPF and immunoblotted for anti-GFP, Ponceau S staining shows total protein. Error bars, mean Ssp2 specific phosphorylation (signal absent in the kinase inactive Ssp2-T189A mutant) vs. WT ± s.e.m., n = 4. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. b) Lysates from WT S. pombe and and S367A/D mutants, treated as indicated, were prepared and immunoblotted for Maf1-PK. Increased hypo-phosphorylation is indicated by lower arrow. Error bars, mean fold change in phosphorylation ± s.e.m., n = 3. Statistical significance was calculated using by two-way ANOVA with Sidak’s multiple comparisons test. n represent independent experiments. c) The yeast S6 ribosomal protein Rps6 is not phosphorylated in the temperature-sensitive TORC1 mutant tor2.ts at restricted temperature or when torin1 is added to WT cells. Lysates were prepared from WT S. pombe and indicated mutants, under conditions indicated, and immunoblotted for pRps6 and total Rps6. Error bars, mean fold change in pRps6 ± s.e.m., n = 3 independent experiments. Statistical significance was calculated using by one-way ANOVA with Sidak’s multiple comparisons test. d) Heat stress of wild type cells at 37 ˚C for 3 h does not affect phosphorylation of Ssp2-S367. Lysates were prepared from WT S. pombe and immunoblotted for pS367 and total Ssp2. e) Lysates were prepared from WT S. pombe and indicated mutants and immunoblotted for Maf1 with anti-PK antibodies. MAF1 hypo-phosphorylation is indicated by lower arrow. For d, e) similar results were obtained from 3 independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 2 AMPK α2-S345 is basally phosphorylated in a variety of mammalian cell lines and tissues and is an mTORC1 substrate using purified enzymes.

a) Validation of the AMPK α2-S345 phospho-specific antibody. Lysates were prepared from FLAG-α2- (WT and S345A/E mutants) expressing α1-/-/α2-/- MEFs and immunoblotted as indicated. α2-S345 is phosphorylated under nutrient-replete/basal conditions in b) mouse liver and a range of mammalian cell lines (negative control: α2β2γ1 expressed and purified from E. coli, which is not phosphorylated on α2-S345), c) human vastus lateralis skeletal muscle, and d) all GST-fusion α2 AMPK complexes expressed in COS7 mammalian cells. e) mTORC1 phosphorylates α-S345 on purified, bacterial expressed recombinant AMPK (α1β2γ1 and α2β1γ1). Kinase inactive (KI) AMPK was used as the substrate to exclude autophosphorylation. f) LC-MS/MS analysis of mTORC1 treated KIα2β1γ1. The masses of the b and y ion series provide direct evidence for phosphate incorporation onto α2-S345. M(O): oxidised methionine; pS: pS345. For a-e), similar results were obtained from 3 independent experiments; for f), results were obtained from a single experiment. Representative immunoblots are shown. Source Data

Extended Data Fig. 3 Elevated AMPKα2 signalling in MEFs in response to pharmacological mTOR inhibition is mediated by α2-S345.

a) Lysates were prepared from FLAG-α2-(WT and S345A/E mutants) expressing α1-/-/α2-/- MEFs and immunoblotted for AMPK substrates TSC2-pT1387 (n = 6), raptor-pS792 (n = 6) and ULK1-pS555 (n = 3). Error bars, mean fold change in phosphorylation ± s.e.m. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. n represent independent experiments. b) Lysates were prepared from FLAG-α2- (WT or S345A/E mutants) expressing α1-/-/α2-/- MEFs, following incubation with mTOR inhibitors AZD8055 or INK128, and immunoblotted as indicated. Error bars, mean fold change in phosphorylation ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Black P values vs. vehicle; red P values vs. 1 μM AZD8055 treated; blue P values vs. 1 μM INK128. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 4 Elevated AMPKα2 signalling in MEFs in response to pharmacological mTOR inhibition is independent of AMP/ATP and ADP/ATP ratios but is synergistic with energy stress.

a) Adenine nucleotides were perchlorate extracted from lysates of FLAG-α2-expressing α1-/-/α2-/- MEFs, incubated for 1 h with rapamycin, AZD8055 or INK128, and measured by LC-MS. Error bars, mean adenylate energy charge ± s.e.m., n = 3. b) torin1 (2 h) treatment of FLAG-α2-expressing α1-/-/α2-/- MEFs. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. c) Lysates were prepared from FLAG-α2-expressing α1-/-/α2-/- MEFs, following 2 h incubation with torin1 ± phenformin, and immunoblotted as indicated. Error bars, mean fold change in phosphorylation ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Black P values vs. vehicle; red P values vs. 1 μM torin1 treated. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 5 Serum starvation suppresses mTORC1-mediated α2-S345 phosphorylation in MEFs.

FLAG-α2-expressing α1-/-/α2-/- MEFs were serum starved for 4 h, and α2-pS345 and α-pT172 tracked for 1 h following a) serum re-addition, or b) 100 nM insulin incubation. Error bars, mean fold change in phosphorylation ± s.e.m., n = 3. For a), statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Black P values vs. basal α2-pS345; red P values vs. basal α-pT172. For b), statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Black P values vs. serum-starved α2-pS345; red P values vs. serum-starved α-pT172. c) WT and inducible Raptor KO (iRapKO) MEFs were treated with 4-OHT and transduced with AMPK FLAG-α2 lentivirus. Cells at full confluence were serum starved overnight (DMEM only), followed by 20 min complete nutrient starvation (1xPBS only) ± subsequent serum/nutrient re-addition (DMEM, 10% FBS) for 60 min. Prepared lysates were immunoblotted for α2-pS345. Error bars, mean fold change in phosphorylation vs. serum/nutrient starved ± s.e.m., n = 4. Statistical significance was calculated using unpaired, two-tailed Student’s t test. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 6 mTORC1 inhibition in MEFs induces more robust dephosphorylation of α1-pS347 compared to α2-pS345.

a) Rapamycin or torin1 (2 h) treatment of FLAG-α-expressing α1-/-/α2-/- MEFs. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (α1-pS347 vehicle vs. rapamycin/torin1 treatment) or unpaired, two-tailed Student’s t test (α1-pS347 vs. α2-pS345). b) Rapamycin and/or phenformin (1 h) treatment of FLAG-α1-expressing α1-/-/α2-/- MEFs. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. c) Phenformin (1 h) treatment of FLAG-α2(S345E)-expressing α1-/-/α2-/- MEFs. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 3. Statistical significance was calculated using unpaired, two-tailed Student’s t test. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 7 Regulation of endogenous AMPK signalling in HEK293T cells in response to mTOR inhibitors and AMPK activating conditions.

a) Lysates were prepared from HEK293T cells, following incubation for 1-24 h with 0.25 or 1 μM INK128, and immunoblotted as indicated. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Black P values vs. vehicle α2-pS345; red P values vs. vehicle α-pT172. b) Lysates were prepared from HEK293T cells, treated with direct AMPK activators (A-769662, SC4) or indirect AMPK activating agents (phenformin, 2-deoxyglucose (2-DG), H2O2) as detailed, and immunoblotted as indicated. Error bars, mean fold change in phosphorylation vs. vehicle ± s.e.m., n = 4. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 8 S. pombe Ssp2-S367 is not regulated by a range of CDKs.

Lysates were prepared from WT S. pombe and indicated cdk mutants and immunoblotted for Ssp2-pS367 and total Ssp2. Error bars, mean fold change in phosphorylation vs. WT 25 °C ± s.e.m., n = 3 independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 9 Phosphorylation of AMPK α2-S345 does not influence AMP sensitivity and is not dependent on β-subunit myristoylation in HEK293T cells.

a) α2-pS345 does not affect AMP allosteric activation of AMPK. Lysates were prepared from FLAG-α2- (WT and S345A mutant) expressing α1-/-/α2-/- MEFs and AMPK immunoprecipitated using FLAG-agarose. AMPK activity was assayed ± AMP (0-50 μM). Error bars, fold change in AMPK activity vs. basal ± s.e.m., n = 3. α2-pS345 does not affect T172 phosphorylation by b) LKB1, or c) CaMKK2. Bacterial expressed α1β1γ1, incubated ± mTORC1 during purification (see Extended Data Fig. 2e) was treated with upstream kinase and immunoblotted for α-pT172. For b), Error bars, mean fold change in pT172 ± s.e.m., n = 6. For c), Error bars, mean pT172 (arbitrary units) ± s.e.m., n = 3. Statistical analyses performed by unpaired, two-tailed, Student’s t test. d) α2-pS345 does not affect rate of pT172 dephosphorylation by phosphatase PP2c. Bacterial expressed α1β1γ1, incubated with CaMKK2 and ± mTORC1 during purification, was incubated with PP2c and residual pT172 measured by immunoblot. Error bars, mean % residual pT172 ± s.e.m., n = 4. Analyses performed by unpaired, two-tailed, Student’s t test. e) Loss of AMPK β-subunit myristoylation (G2A mutant) does not affect basal or rapamycin-induced reductions in α2-S345 phosphorylation in HEK293T cells. Lysates were prepared from GST-α2β1γ1 AMPK- (WT or β1-G2A mutant) expressing HEK293T cells, following 1 h incubation with rapamycin, and immunoblotted for AMPK α2-pS345. Error bars, mean phosphorylation (arbitrary units) ± s.e.m., n = 3. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. n represent independent experiments. Representative immunoblots are shown. Source Data

Extended Data Fig. 10 Arg/Lys starvation suppresses AMPK α2-S345 phosphorylation in HEK293 cells and affects the ability of 2-DG to enhance cell proliferation.

See main Fig. 4. a–d, i, j) Real time proliferation analysis of HEK293 cells (untransfected or transiently expressing GFP-α2β1γ1 (WT or α2-S345A mutant)), treated as indicated. Error bars, relative confluence ± s.e.m. For a), n = 3; for b-d, i, j), n = 8. For c), ns P = 0.1819; for d), ns P > 0.9999. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. e–h) Lysates were prepared from GFP-α2β1γ1 AMPK- (WT or α2-S345A mutant) expressing HEK293 cells (treated as indicated) and immunoblotted for AMPKα2-pS345, S6K-pT389 and ACC-pS79/212. Error bars, mean fold change in phosphorylation vs. -Arg/Lys ± s.e.m., n = 3 (WT) and 4 (S345A). Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. n represent independent experiments. Representative immunoblots are shown. Source Data

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Ling, N.X.Y., Kaczmarek, A., Hoque, A. et al. mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress. Nat Metab 2, 41–49 (2020). https://doi.org/10.1038/s42255-019-0157-1

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