Article

Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40

  • Nature volume 552, pages 368373 (21 December 2017)
  • doi:10.1038/nature25023
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Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, energy levels, and growth factors. It contains the atypical kinase mTOR and the RAPTOR subunit that binds to the Tor signalling sequence (TOS) motif of substrates and regulators. mTORC1 is activated by the small GTPase RHEB (Ras homologue enriched in brain) and inhibited by PRAS40. Here we present the 3.0 ångström cryo-electron microscopy structure of mTORC1 and the 3.4 ångström structure of activated RHEB–mTORC1. RHEB binds to mTOR distally from the kinase active site, yet causes a global conformational change that allosterically realigns active-site residues, accelerating catalysis. Cancer-associated hyperactivating mutations map to structural elements that maintain the inactive state, and we provide biochemical evidence that they mimic RHEB relieving auto-inhibition. We also present crystal structures of RAPTOR–TOS motif complexes that define the determinants of TOS recognition, of an mTOR FKBP12–rapamycin-binding (FRB) domain–substrate complex that establishes a second substrate-recruitment mechanism, and of a truncated mTOR–PRAS40 complex that reveals PRAS40 inhibits both substrate-recruitment sites. These findings help explain how mTORC1 selects its substrates, how its kinase activity is controlled, and how it is activated by cancer-associated mutations.

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References

  1. 1.

    & mTORC1 signaling and the metabolic control of cell growth. Curr. Opin. Cell Biol. 45, 72–82 (2017)

  2. 2.

    & Regulation of mTORC1 by amino acids. Trends Cell Biol. 24, 400–406 (2014)

  3. 3.

    & Regulation of mTORC1 by PI3K signaling. Trends Cell Biol. 25, 545–555 (2015)

  4. 4.

    & mTOR signaling in growth control and disease. Cell 149, 274–293 (2012)

  5. 5.

    et al. A diverse array of cancer-associated MTOR mutations are hyperactivating and can predict rapamycin sensitivity. Cancer Discov. 4, 554–563 (2014)

  6. 6.

    & PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50–51 (1995)

  7. 7.

    et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002)

  8. 8.

    et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002)

  9. 9.

    et al. GβL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904 (2003)

  10. 10.

    et al. Architecture of human mTOR complex 1. Science 351, 48–52 (2016)

  11. 11.

    et al. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5, 566–571 (2003)

  12. 12.

    et al. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5, 559–566 (2003)

  13. 13.

    et al. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol. Cell 25, 903–915 (2007)

  14. 14.

    , , , & Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9, 316–323 (2007)

  15. 15.

    , , & PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding. J. Biol. Chem. 282, 20036–20044 (2007)

  16. 16.

    et al. The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J. Biol. Chem. 282, 20329–20339 (2007)

  17. 17.

    ., ., ., & RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl Acad. Sci. USA 95, 1432–1437 (1998)

  18. 18.

    & Molecular mechanisms of mTOR-mediated translational control. Nat. Rev. Mol. Cell Biol. 10, 307–318 (2009)

  19. 19.

    et al. The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling. Science 332, 1317–1322 (2011)

  20. 20.

    et al. mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science 341, 1236566 (2013)

  21. 21.

    , , & TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13, 797–806 (2003)

  22. 22.

    . et al. The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J. Biol. Chem. 278, 15461–15464 (2003)

  23. 23.

    et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013)

  24. 24.

    et al. 4.4 Å resolution cryo-EM structure of human mTOR complex 1. Protein Cell 7, 878–887 (2016)

  25. 25.

    , , & Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242 (1996)

  26. 26.

    et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 332, 1322–1326 (2011)

  27. 27.

    et al. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci. Signal. 5, ra42 (2012)

  28. 28.

    MAF1: a new target of mTORC1. Biochem. Soc. Trans. 39, 487–491 (2011)

  29. 29.

    et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146, 408–420 (2011)

  30. 30.

    , , & Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G. Mol. Cell 3, 707–716 (1999)

  31. 31.

    , & Raptor protein contains a caspase-like domain. Trends Biochem. Sci. 29, 522–524 (2004)

  32. 32.

    & Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012)

  33. 33.

    Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016)

  34. 34.

    et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

  35. 35.

    , , , & Tor forms a dimer through an N-terminal helical solenoid with a complex topology. Nat. Commun. 7, 11016 (2016)

  36. 36.

    et al. Structural basis for the unique biological function of small GTPase RHEB. J. Biol. Chem. 280, 17093–17100 (2005)

  37. 37.

    , , , & Evaluating PI3 kinase isoforms using Transcreener ADP assays. J. Biomol. Screen. 13, 476–485 (2008)

  38. 38.

    et al. Activating mTOR mutations in a patient with an extraordinary response on a phase I trial of everolimus and pazopanib. Cancer Discov. 4, 546–553 (2014)

  39. 39.

    et al. Isolation of hyperactive mutants of mammalian target of rapamycin. J. Biol. Chem. 283, 31861–31870 (2008)

  40. 40.

    et al. Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc. Natl Acad. Sci. USA 104, 3514–3519 (2007)

  41. 41.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  42. 42.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  43. 43.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  44. 44.

    . et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  45. 45.

    et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005)

  46. 46.

    et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017)

  47. 47.

    & CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

  48. 48.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  49. 49.

    , , & Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016)

  50. 50.

    Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014)

  51. 51.

    et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  52. 52.

    , , & Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70, 2487–2499 (2014)

  53. 53.

    Nat. Protocols 1, 16–22 (2006)

  54. 54.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

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Acknowledgements

We thank the staff of the Advanced Photon Source, the New York Structural Biology Center Simons Electron Microscopy Center, the Howard Hughes Medical Institute Cryo-EM facility, the Memorial Sloan Kettering Cancer Center Cryo-EM facility, and Subangstrom for help with data collection. This work was supported by the Howard Hughes Medical Institute and National Institutes of Health grant CA008748.

Author information

Affiliations

  1. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Haijuan Yang
    • , Xiaolu Jiang
    • , Buren Li
    • , Hyo J. Yang
    • , Meredith Miller
    • , Angela Yang
    • , Ankita Dhar
    •  & Nikola P. Pavletich
  2. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Xiaolu Jiang
    • , Hyo J. Yang
    • , Meredith Miller
    • , Angela Yang
    •  & Nikola P. Pavletich

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Contributions

H.Y., B.L., and N.P.P. designed the experiments, determined the mTOR structures, and wrote the manuscript. N.P.P. and X.J. determined the Raptor crystal structures and biochemical constants. H.Y., H.J.Y., M.M., A.Y., and A.D. performed the mTOR enzyme assays and biochemical experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Nikola P. Pavletich.

Reviewer Information Nature thanks D. Barford, N. MacDonald and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Information

    This file contains the Supplementary Discussion sections about steady state kinetic constants, about other mTORC1 substrates in Figure 1g, about Raptor structure, about mTORC1 structure, and about apo-mTORC1 conformational flexibility. The file also contains Supplementary Figure 1 showing the source data (autoradiograms and immunoblots of gels) for the Main text and Extended Data figures.

Videos

  1. 1.

    A morph from the inactive to the active mTOR conformation.

    The roundtrip morph from the inactive (at video start) to the active (video middle) monomeric mTORC1 structures superimposed on the KD C lobes (mLST8 not shown). Orientation and coloring similar to Figure 4a.

  2. 2.

    Close-up view of the catalytic cleft in the morph from the inactive to active mTOR conformation.

    Close-up of Supplementary Video 1 focusing on the relative orientation of the kinase N and C lobes. View is similar to Figure 5d looking into the kinase catalytic cleft.

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