Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

mTOR kinase structure, mechanism and regulation

Abstract

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12–rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin–FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of the mTORΔΝ–mLST8–ATPγS-Mg complex.
Figure 2: mTOR kinase domain and active site conformation.
Figure 3: The kinase active site is recessed at the bottom of a deep cleft.
Figure 4: The rapamycin-binding site of the FRB recruits S6K1 into the catalytic cleft.
Figure 5: mTOR-activating mutations map to structural elements involved in restricting active site access.
Figure 6: Structures of the Torin2, PP242 and PI-103 inhibitors bound to the mTOR catalytic cleft.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 4JSN (native structure), 4JSP (ATPcS-Mgcomplex), 4JSV (ADP-Mg-F complex), 4JSX (Torin2complex), 4JT5 (PP242 complex) and 4JT6 (PI-103 complex).

References

  1. Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to cancer, diabetes and ageing. Nature Rev. Mol. Cell Biol. 12, 21–35 (2011)

    Article  CAS  Google Scholar 

  2. Shaw, R. J. & Cantley, L. C. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424–430 (2006)

    Article  ADS  CAS  Google Scholar 

  3. Keith, C. T. & Schreiber, S. L. PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science 270, 50–51 (1995)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 (2002)

    Article  CAS  Google Scholar 

  7. Sarbassov, D. D. et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14, 1296–1302 (2004)

    Article  CAS  Google Scholar 

  8. Chen, E. J. & Kaiser, C. A. LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J. Cell Biol. 161, 333–347 (2003)

    Article  CAS  Google Scholar 

  9. Kim, D. H. 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)

    Article  CAS  Google Scholar 

  10. Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008)

    Article  ADS  CAS  Google Scholar 

  11. Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. & Guan, K. L. Regulation of TORC1 by Rag GTPases in nutrient response. Nature Cell Biol. 10, 935–945 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Long, X., Lin, Y., Ortiz-Vega, S., Yonezawa, K. & Avruch, J. Rheb binds and regulates the mTOR kinase. Curr. Biol. 15, 702–713 (2005)

    Article  CAS  Google Scholar 

  15. Sato, T., Nakashima, A., Guo, L. & Tamanoi, F. Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein. J. Biol. Chem. 284, 12783–12791 (2009)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Zhang, Y. et al. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nature Cell Biol. 5, 578–581 (2003)

    Article  CAS  Google Scholar 

  18. Ma, X. M. & Blenis, J. Molecular mechanisms of mTOR-mediated translational control. Nature Rev. Mol. Cell Biol. 10, 307–318 (2009)

    Article  Google Scholar 

  19. Schalm, S. S., Fingar, D. C., Sabatini, D. M. & Blenis, J. TOS motif-mediated raptor binding regulates 4E–BP1 multisite phosphorylation and function. Curr. Biol. 13, 797–806 (2003)

    Article  CAS  Google Scholar 

  20. Nojima, H. 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)

    Article  CAS  Google Scholar 

  21. Oshiro, N. 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)

    Article  CAS  Google Scholar 

  22. Fonseca, B. D., Smith, E. M., Lee, V. H., MacKintosh, C. & Proud, C. G. PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J. Biol. Chem. 282, 24514–24524 (2007)

    Article  CAS  Google Scholar 

  23. Jacinto, E. et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nature Cell Biol. 6, 1122–1128 (2004)

    Article  CAS  Google Scholar 

  24. Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242 (1996)

    Article  ADS  CAS  Google Scholar 

  25. Choo, A. Y. & Blenis, J. Not all substrates are treated equally: implications for mTOR, rapamycin-resistance and cancer therapy. Cell Cycle 8, 567–572 (2009)

    Article  CAS  Google Scholar 

  26. Wander, S. A., Hennessy, B. T. & Slingerland, J. M. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J. Clin. Invest. 121, 1231–1241 (2011)

    Article  CAS  Google Scholar 

  27. Lovejoy, C. A. & Cortez, D. Common mechanisms of PIKK regulation. DNA Repair (Amst.) 8, 1004–1008 (2009)

    Article  CAS  Google Scholar 

  28. Bosotti, R., Isacchi, A. & Sonnhammer, E. L. FAT: a novel domain in PIK-related kinases. Trends Biochem. Sci. 25, 225–227 (2000)

    Article  CAS  Google Scholar 

  29. Walker, E. H., Perisic, O., Ried, C., Stephens, L. & Williams, R. L. Structural insights into phosphoinositide 3-kinase catalysis and signalling. Nature 402, 313–320 (1999)

    Article  ADS  CAS  Google Scholar 

  30. Sibanda, B. L., Chirgadze, D. Y. & Blundell, T. L. Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. Nature 463, 118–121 (2010)

    Article  ADS  CAS  Google Scholar 

  31. Nolen, B., Taylor, S. & Ghosh, G. Regulation of protein kinases; controlling activity through activation segment conformation. Mol. Cell 15, 661–675 (2004)

    Article  CAS  Google Scholar 

  32. Miller, S. et al. Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34. Science 327, 1638–1642 (2010)

    Article  ADS  CAS  Google Scholar 

  33. McMahon, L. P., Choi, K. M., Lin, T. A., Abraham, R. T. & Lawrence, J. C., Jr The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin. Mol. Cell. Biol. 22, 7428–7438 (2002)

    Article  CAS  Google Scholar 

  34. Sekulic, A. et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 60, 3504–3513 (2000)

    CAS  PubMed  Google Scholar 

  35. Edinger, A. L. & Thompson, C. B. An activated mTOR mutant supports growth factor-independent, nutrient-dependent cell survival. Oncogene 23, 5654–5663 (2004)

    Article  CAS  Google Scholar 

  36. Bao, Z. Q., Jacobsen, D. M. & Young, M. A. Briefly bound to activate: transient binding of a second catalytic magnesium activates the structure and dynamics of CDK2 kinase for catalysis. Structure 19, 675–690 (2011)

    Article  CAS  Google Scholar 

  37. Madhusudan, A. P. Xuong, N. H. & Taylor, S. S. Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase. Nature Struct. Biol. 9, 273–277 (2002)

    Article  CAS  Google Scholar 

  38. Brown, E. J. et al. Control of p70 s6 kinase by kinase activity of FRAP in vivo . Nature 377, 441–446 (1995)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  40. Vilella-Bach, M., Nuzzi, P., Fang, Y. & Chen, J. The FKBP12-rapamycin-binding domain is required for FKBP12-rapamycin-associated protein kinase activity and G1 progression. J. Biol. Chem. 274, 4266–4272 (1999)

    Article  CAS  Google Scholar 

  41. Shor, B. et al. A new pharmacologic action of CCI-779 involves FKBP12-independent inhibition of mTOR kinase activity and profound repression of global protein synthesis. Cancer Res. 68, 2934–2943 (2008)

    Article  CAS  Google Scholar 

  42. Rodríguez, A. et al. A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants. Mol. Cell 33, 616–626 (2009)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Reinke, A., Chen, J. C., Aronova, S. & Powers, T. Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p. J. Biol. Chem. 281, 31616–31626 (2006)

    Article  CAS  Google Scholar 

  45. Urano, J. 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)

    Article  ADS  CAS  Google Scholar 

  46. Liu, Q. et al. Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer. J. Med. Chem. 54, 1473–1480 (2011)

    Article  ADS  CAS  Google Scholar 

  47. Apsel, B. et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nature Chem. Biol. 4, 691–699 (2008)

    Article  CAS  Google Scholar 

  48. Knight, Z. A. et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell 125, 733–747 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

    Article  CAS  Google Scholar 

  51. Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  52. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. 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)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. King for mass spectroscopic analysis, H. Erdjument-Bromage for N-terminal sequencing, the staff of the Advanced Photon Source ID24 beamlines for help with data collection and D. Tzvetkova-Robev for help with protein expression in HEK293 cells. This work was supported by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

H.Y. and N.P.P. designed the experiments, solved the structures and wrote the manuscript. H.Y., D.G.R., J.D.K., B.M. and H.J.Y. carried out protein production, crystallization and biochemical experiments. H.Y. performed all other experiments.

Corresponding author

Correspondence to Nikola P. Pavletich.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3, Supplementary Figures 1-13 and Supplementary References. (PDF 7776 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, H., Rudge, D., Koos, J. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013). https://doi.org/10.1038/nature12122

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12122

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing