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AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth in response to amino acid and glucose levels. However, how mTORC1 senses glucose availability to regulate various downstream signalling pathways remains largely elusive. Here we report that AMP-activated protein kinase (AMPK)-mediated phosphorylation of WDR24, a core component of the GATOR2 complex, has a role in the glucose-sensing capability of mTORC1. Mechanistically, glucose deprivation activates AMPK, which directly phosphorylates WDR24 on S155, subsequently disrupting the integrity of the GATOR2 complex to suppress mTORC1 activation. Phosphomimetic Wdr24S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity. On the other hand, compared to wild-type littermates, phospho-deficient Wdr24S155A knock-in mice are more resistant to fasting and display elevated mTORC1 activity. Our findings reveal that AMPK-mediated phosphorylation of WDR24 modulates glucose-induced mTORC1 activation, thereby providing a rationale for targeting AMPK–WDR24 signalling to fine-tune mTORC1 activation as a potential therapeutic means to combat human diseases with aberrant activation of mTORC1 signalling including cancer.

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Fig. 1: The GATOR1/2 complexes regulate mTORC1 glucose sensing.
Fig. 2: The AMPK–GATOR1/2 axis regulates mTORC1 glucose sensing.
Fig. 3: AMPK phosphorylation of WDR24 on S155 is regulated by glucose availability.
Fig. 4: AMPK-mediated phosphorylation of WDR24 on S155 regulates mTORC1 kinase activity.
Fig. 5: Glucose regulates GATOR2 complex integrity through phosphorylation of WDR24 on S155.
Fig. 6: WDR24 phosphorylation suppresses mTORC1 kinase activity and leads to embryonic lethality in vivo.

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Data availability

All data that support the findings of this study are available in the figures or Extended Data figures. Mass spectrometry fragmentation spectra were searched against the concatenated decoy human protein database v.20210315 (UniProt). Source data are provided with this paper.

References

  1. DeBerardinis, R. J., Lum, J. J., Hatzivassiliou, G. & Thompson, C. B. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 7, 11–20 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rabinowitz, J. D. & White, E. Autophagy and metabolism. Science 330, 1344–1348 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kim, J. & Guan, K. L. mTOR as a central hub of nutrient signalling and cell growth. Nat. Cell Biol. 21, 63–71 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wolfson, R. L. & Sabatini, D. M. The dawn of the age of amino acid sensors for the mTORC1 pathway. Cell Metab. 26, 301–309 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 351, 43–48 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 165, 153–164 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, J. et al. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature 596, 281–284 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Orozco, J. M. et al. Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nat. Metab. 2, 893–901 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Herzig, S. & Shaw, R. J. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat. Rev. Mol. Cell Biol. 19, 121–135 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Lin, S.-C. & Hardie, D. G. AMPK: sensing glucose as well as cellular energy status. Cell Metab. 27, 299–313 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Inoki, K., Zhu, T. & Guan, K.-L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Van Nostrand, J. L. et al. AMPK regulation of Raptor and TSC2 mediate metformin effects on transcriptional control of anabolism and inflammation. Genes Dev. 34, 1330–1344 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gwinn, D. M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Efeyan, A. et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature 493, 679–683 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shaw, R. J. et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6, 91–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Cool, B. et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 3, 403–416 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Shen, K. et al. Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes. Nature 556, 64–69 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hardie, D. G., Schaffer, B. E. & Brunet, A. AMPK: an energy-sensing pathway with multiple inputs and outputs. Trends Cell Biol. 26, 190–201 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108, 1167–1174 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Xiao, B. et al. Structural basis of AMPK regulation by small molecule activators. Nat. Commun. 4, 3017 (2013).

    Article  PubMed  Google Scholar 

  27. Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Peng, M., Yin, N. & Li, M. O. SZT2 dictates GATOR control of mTORC1 signalling. Nature 543, 433–437 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bridges, D. & Moorhead, G. B. G. 14-3-3 proteins: a number of functions for a numbered protein. Sci. STKE 2005, re10 (2005).

    Article  PubMed  Google Scholar 

  30. Valenstein, M. L. et al. Structure of the nutrient-sensing hub GATOR2. Nature 607, 610–616 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Efeyan, A. et al. RagA, but not RagB, is essential for embryonic development and adult mice. Dev. Cell 29, 321–329 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gonzalez, A., Hall, M. N., Lin, S.-C. & Hardie, D. G. AMPK and TOR: the Yin and Yang of cellular nutrient sensing and growth control. Cell Metab. 31, 472–492 (2020).

    Article  CAS  PubMed  Google Scholar 

  33. Leprivier, G. & Rotblat, B. How does mTOR sense glucose starvation? AMPK is the usual suspect. Cell Death Discov. 6, 27 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lee, M. N. et al. Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. Mol. Cell. Biol. 29, 3991–4001 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Roberts, D. J., Tan-Sah, V. P., Ding, E. Y., Smith, J. M. & Miyamoto, S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol. Cell 53, 521–533 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, C. S. et al. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 548, 112–116 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Almacellas, E. et al. Phosphofructokinases axis controls glucose-dependent mTORC1 activation driven by E2F1. iScience 20, 434–448 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gan, W. et al. LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control. Nat. Cell Biol. 22, 246–256 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dai, X. et al. Energy status dictates PD-L1 protein abundance and anti-tumor immunity to enable checkpoint blockade. Mol. Cell 81, 2317–2331 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, P. et al. Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis. Nat. Cell Biol. 15, 1340–1350 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J. Liu, F. Dang and other Wei laboratory members for critical reading of the manuscript, as well as members of the Wei and Guo laboratories for helpful discussions. This work was supported in part by National Institutes of Health grant nos. R01CA177910 and R35CA253027 to W.W., no. 1K99CA259329 to X.D., no. P01CA120964 to J.A. and the China National Natural Science Foundation (nos. 31871410 and 32070767 to J.G. and no. 32100559 to Q.J.).

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Authors and Affiliations

Authors

Contributions

X.D. and C.J. designed and performed most of the experiments with assistance from Q.J., P.Y., F.C., T.Z., H.I. and J.M.A. Q.J., L.F., H.Y. and Jinhe Guo helped with mouse generation and phenotype analysis. P.W., Jianping Guo and W.W. guided and supervised the study. X.D., C.J., Jianping Guo and W.W. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Jianping Guo or Wenyi Wei.

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Competing interests

W.W. is a co-founder and consultant for ReKindle Therapeutics. The other authors declare no competing interests.

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Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 GATOR1/2 complexes play an important role in mTORC1 glucose sensing.

a, WT and NPRL2 knockout (KO) HeLa cells were deprived of glucose (Glc) for 60 min and restimulated with glucose for 10 min as indicated. WCLs were analyzed via IB. b,c, WT and NPRL2 KO HEK 293 (b) or HeLa (c) cells were deprived of amino acids (AA) for 60 min and restimulated with amino acids for 10 min as indicated. d, IB analysis of WCLs derived from WT and WDR24 KO HeLa cells. The cells were treated as in (a). e,f, WT and WDR24 KO HEK 293 (e) or HeLa (f) cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated. g, WDR24 KO HEK 293 cells were re-introduced with or without WT WDR24, and the resulting cells were treated as in (d). h,i, WT and NPRL2 KO HeLa cells were deprived of glucose for 60 min and restimulated with glucose for 10 min before coimmunostaining for mTOR (red) and LAMP1 (green) (h). Scale bar, 10 μm. The imaging data were quantified (i). n = 10. P = 4.93E-07, 0.21. Data are the mean ± s.d., two-tailed t-test. NS, not significant, ***P < 0.001. j,k, WT and WDR24 KO HeLa cells were treated as in (h,i). The imaging data were quantified (k). Scale bar, 10 μm. n = 10. P = 1.12E-09, 0.90. Data are the mean ± s.d., two-tailed t-test. NS, not significant, ***P < 0.001. Representative image shown, a and d, n = 3; b, c, e, f and g, n = 2.

Source data

Extended Data Fig. 2 AMPK plays a pivotal role in mTORC1 glucose sensing.

a, IB analysis of WCLs derived from WT and AMPKα1/2 double knockout (DKO) HEK 293 cells. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. b, IB analysis of WCLs derived from WT and AMPKα1/2 DKO HEK 293 T cells. The cells were deprived of glucose or amino acids for 60 min and restimulated with glucose or amino acids for 10 min as indicated. c, IB analysis of WCLs derived from WT and Ampkα1/2 DKO MEFs. The cells were treated as in (a). d, IB analysis of WCLs derived from WT, AMPKα1/2 DKO, AMPKα1 and AMPKα2 reconstituted AMPKα1/2 DKO U2OS cells. The cells were treated as in (a). e,f, HEK 293 cells were treated with or without A-769662 (100 μM) for 60 min, and the co-localization of mTORC1 (Red) and LAMP1 (green) was analyzed via immunofluorescence (e). Scale bar, 10 μm. The imaging data were quantified (f). n = 17, 20. P = 0.00058. Data are the mean ± s.d., two-tailed t-test. NS, not significant, ***P < 0.001. Representative image shown, a, b and d, n = 2; c, n = 3.

Source data

Extended Data Fig. 3 AMPK interacts with and phosphorylates WDR24.

a, IB analysis of WCLs and anti-Flag IPs derived from WT and WDR24 Flag knockin (KI) HEK 293 cells. b,c, IB analysis of WCLs and anti-HA or anti-Flag IPs derived from 293 T cells transfected with the indicated constructs. d, IB analysis of WCLs and anti-HA IPs derived from 293 T cells transfected with the indicated constructs. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min before harvesting. e,f, IB analysis of WCLs and anti-HA IPs derived from WDR59 (e) or Mios (f) KO HEK 293 cells transfected with the indicated constructs. g, IB analysis of WCLs and GST-Pull down derived from WDR24 KO HEK 293 cells transfected with the indicated constructs. h, Bacterial purified GST-WDR24 fragments were incubated with full-length human AMPK (combination of A1/B1/G2 subunits) which was expressed by baculovirus in Sf9 insect cells using a C-terminal His tag (SignalChem) for 3 h at 4 °C. i, IB analysis of WCLs and GST-Pull down derived from WDR24 KO HEK 293 cells transfected with the indicated constructs. j, In vitro kinase assay of indicated proteins demonstrates that AMPK phosphorylates WDR24. GST-WDR24 and YAP1 proteins were bacterially purified as substrates, and recombinant active AMPK was used as the source of kinase. Anti-thiophosphate-ester antibody was used to detect phosphorylated proteins. GST-YAP1 as a positive control. Representative image shown, a, n = 3; b-j, n = 2.

Source data

Extended Data Fig. 4 AMPK phosphorylates WDR24 on the S155 residue.

a, Scansite shows that WDR24 has a possible AMPK phosphorylation site. b, The MS/MS fragmentation spectrum showing fragment ions for the WDR24 peptide KDpSVSTFSGQSESV defining the WDR24-pS155 site. c, Titration of the indicated WDR24 peptides with or without S155 phosphorylation demonstrates that the generated WDR24-pS155 antibodies specifically recognize the pS155 epitope in the dot blot analysis. d, IB analysis of anti-Flag IPs derived from 293 T cells transfected with indicated constructs to demonstrate that mutating the Ser155 site in WDR24 abolished the ability of the generated WDR24-pS155 antibody (#3 and #5) to recognize WDR24 phosphorylated species in cells. e, In vitro kinase assays demonstrated that the WDR24-pS155 antibody recognizes the phosphorylated WDR24. GST-WDR24 protein was purified from E. coli, and recombinant active AMPK was used as the source of kinase. f, IB analysis of WCLs and anti-Flag IPs derived from WT and WDR24 Flag knock-in HEK 293 cells. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. g, WDR24 KO HEK 293 cells reconstituted with WT-WDR24 were starved of glucose and treated with Compound C (10 μM) for 1 h before harvesting for IB analysis. h, IB analysis of WCLs and anti-HA IPs derived from WT or AMPKα1/2 DKO HEK 293 T cells transfected with indicated constructs. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. i, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. Representative image shown, e-i, n = 2.

Source data

Extended Data Fig. 5 Phosphorylation of S155 on WDR24 inhibits the activation of mTORC1 induced by glucose.

a, WDR24 KO HeLa cells re-introduced with indicated constructs were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. b, Cell size histogram of WDR24 KO HeLa cells reconstituted with WT and WDR24 S155D by FACS. c, WDR24 KO HeLa cells reconstituted with indicated constructs were plated for 8 d for the colony-formation assays. Data are shown as the mean ± s.d. of n = 3 independent experiments (bottom). P = 0.0004. two-tailed t-test. ***P < 0.001. d, IB analysis of WCLs of HEK 293 cells starved of glucose or/and amino acids for 1 h, and restimulated with amino acids for 10 min. e, WDR24 KO HEK 293 cells re-introduced with indicated constructs were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated before IB analysis. f, WDR24 KO HeLa cells were re-introduced with indicated constructs. The cells were treated as in (e). g,h, WT and AMPKα1/2 DKO HEK 293 (g) or 293 T (h) cells were starved of amino acids or amino acids and glucose together for 60 min, then stimulated with amino acids for 10 min. i, Working model to show how AMPK regulates mTORC1 amino acid sensing under energy stress conditions. Representative image shown, a and d, n = 3; e, f, g and h, n = 2.

Source data

Extended Data Fig. 6 The WDR24-S155D mutation inhibits mTORC1 kinase activity.

a, Schematic representation of the amino sequence to generate WDR24-S155D CRISPR knock-in cells. b, Identification of the potential knock-in mutants. Genomic DNA containing WDR24-S155D mutation was amplified by PCR and digested with BslI. c, Confirmation of the correct mutation of Raptor-S606D or S606A by Sanger DNA sequencing. d, WT and WDR24-S155D knock-in 293 cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated. Representative image shown, n = 3. e, WT and WDR24-S155D knock-in 293 cells were deprived of glucose for 60 min and restimulated with glucose for 10 min before immunostaining for mTOR and LAMP1. The imaging data were quantified under each condition. n = 17, 16, 16, 15. P = 5.67E-06, 0.11. Data are the mean ± s.d., two-tailed t-test. NS, not significant, ***P < 0.001. See Fig. 4e for imaging data. f,g, WT and WDR24-S155D knock-in 293 cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min before coimmunostaining for mTOR (red) and LAMP1 (green) (f). Scale bar, 10 μm. The imaging data were quantified with 10-20 cells under each condition (g). n = 19, 18, 15, 18. P = 2.56E-13, 0.08. Data are the mean ± s.d., two-tailed t-test. NS, not significant. ***P < 0.001. h, IB analysis of WCLs and anti-HA IPs derived from WT and WDR24-S155D knock-in HEK 293 cells transfected with the indicated constructs. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min before harvesting. Representative image shown, n = 2.

Source data

Extended Data Fig. 7 Glucose deprivation regulates GATOR2 complex integrity through phosphorylating WDR24 on S155.

a, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. The cells were deprived of glucose for 60 min and restimulated with glucose for different time points as indicated. b, WCLs of HEK 293 cells deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated were run through a Superose 6 Increase 10/300 GL column. Elutes were collected for each fraction and analyzed by IB analysis. c, WCLs of HEK 293 cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated and analyzed as (b). d, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. The cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated. e, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. Cells were starved with amino acids or amino acids and glucose together for 60 min, then stimulated with amino acids for 10 min. f,g, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. The cells were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. h, IB analysis of WCLs and IPs derived from HEK 293 cells transfected with indicated plasmids. i, IB analysis of WCLs and anti-WDR24 IPs derived from WT and 14-3-3γ knockdown HEK 293 cells. The cells were treated as (f,g). j, IB analysis of WCLs and anti-Flag IPs derived from WDR24 KO HEK 293 cells reconstituted with indicated constructs. k,l, Wild-type (WT) and Sesn1/2/3 knockout MEFs were deprived of amino acids (AA) (k) or glucose (Glc) (l) for 60 min and restimulated with amino acids for 10 min as indicated. WCLs were analyzed via IB. Representative image shown, a-j and i, n = 2; k, n = 3.

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Extended Data Fig. 8 Generation of Wdr24S155D and Wdr24S155A knock-in mice by CRISPR-Cas9-mediated genome editing.

a, sgRNA sequence and part of ssODN sequence used for generating Wdr24S155A/D knock-in mice. b, Sanger sequencing results of hetero Wdr24S155D knock-in mouse genomic DNA. c, Sanger sequencing results of hetero Wdr24S155A knock-in mouse genomic DNA. d, Neonates from WDR24+/A parents were counted at birth.

Extended Data Fig. 9 Phospho-deficient Wdr24S155A knock-in mice show relatively high mTORC1 activity under fasting.

a, Representative images of hematoxylin and eosin (H&E) (scale bar, 50 μm), p-S6(S40/244) (scale bar, 100 μm) staining of the heart section (n = 3) of Wdr24+/+and Wdr24A/A littermates. The mice were fasted for 24 h, or fasted and refed for 2 h. b, IB analysis of WCLs derived from Wdr24+/+or Wdr24A/A mouse livers. The mice were fasted for 24 h, or fasted and refed for 2 h. n = 3 mice. c, Representative images of hematoxylin and eosin (H&E) (scale bar, 50 μm), p-S6(S240/244) staining (scale bar, 100 μm; insets magnification=1.5) of the kidney section (n = 3) of Wdr24+/+and Wdr24A/A littermates. The mice were fasted for 24 h, or fasted and refed for 2 h. d, IB analysis of WCLs derived from Wdr24+/+or Wdr24A/A mouse kidneys. The mice were fasted for 24 h or fasted and refed for 2 h. n = 3 mice. e,f, IF analysis of the kidney section of Wdr24+/+and Wdr24A/A littermates (e). Scale bar, 20 μm. The mice were fasted for 24 h, or fasted and refed for 2 h. The pS6 (240/244) intensity was quantified via Image J (f). n = 7, 6, 6, 6. P = 1.27E-06, 5.95E-07, 0.01. Data are mean ± s.d., two-tailed t-test. *P < 0.05, ***P < 0.001.

Source data

Extended Data Fig. 10 The WDR24-S155A mutation partially regulates mTORC1 lysosome localization under glucose starvation.

a, IB analysis of WCLs derived from Wdr24+/+or Wdr24A/A MEFs. The cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated. Representative image shown, n = 2. b, IF analysis of Wdr24+/+or Wdr24A/A MEFs. Indicated cells were deprived of glucose for 60 min and restimulated with glucose for 10 min as indicated. Scale bar, 10 μm. c, The imaging data in (b) were quantified. n = 14, 12, 12, 14. P = 4.74E-09, 1.57E-08, 0.003. Data are the mean ± s.d., two-tailed t-test. **P < 0.01, ***P < 0.001. d, IF analysis of Wdr24+/+or Wdr24A/A MEFs. Indicated cells were deprived of amino acids for 60 min and restimulated with amino acids for 10 min as indicated. Scale bar, 10 μm. e, The imaging data in (d) were quantified. n = 15, 14, 11, 13. P = 1.05E-07, 0.08, 1.72E-07. Data are the mean ± s.d., two-tailed t-test. NS, not significant. ***P < 0.001. f, A working model to show how glucose regulates mTORC1 kinase activity through AMPK-mediated WDR24, Raptor and TSC2 phosphorylation.

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Supplementary information

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Supplementary Tables 1–3

Supplementary Table 1: Information about antibodies. Supplementary Table 2: List of primers, sgRNAs and ssODNs. Supplementary Table 3: Quantification of western blots in the figures.

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Dai, X., Jiang, C., Jiang, Q. et al. AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation. Nat Metab 5, 265–276 (2023). https://doi.org/10.1038/s42255-022-00732-4

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