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.

  • Letter
  • Published:

Mechanism of arginine sensing by CASTOR1 upstream of mTORC1

Abstract

The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids1,2,3. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7).Arginine activates mTORC1 upstream of the Rag family of GTPases8, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1)9,10,11,12. However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor.

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: Architecture of human CASTOR1.
Figure 2: The arginine-binding pocket of CASTOR1.
Figure 3: Arginine facilitates the intramolecular association of the ACT2 and ACT4 domains of CASTOR1.
Figure 4: The GATOR2 binding site of CASTOR1 is at the ACT2–ACT4 interface and is required for signalling arginine deprivation to mTORC1.
Figure 5: Insights into the evolution of arginine sensing by CASTOR1.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the x-ray crystal structure of CASTOR1 have been deposited in the Protein Data Bank (PDB) with accession code 5I2C.

References

  1. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012)

    Article  CAS  Google Scholar 

  2. Dibble, C. C. & Manning, B. D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat. Cell Biol. 15, 555–564 (2013)

    Article  CAS  Google Scholar 

  3. Jewell, J. L., Russell, R. C. & Guan, K. L. Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell Biol. 14, 133–139 (2013)

    Article  CAS  Google Scholar 

  4. Ban, H. et al. Arginine and Leucine regulate p70 S6 kinase and 4E-BP1 in intestinal epithelial cells. Int. J. Mol. Med. 13, 537–543 (2004)

    CAS  PubMed  Google Scholar 

  5. Bronte, V. & Zanovello, P. Regulation of immune responses by L-arginine metabolism. Nat. Rev. Immunol. 5, 641–654 (2005)

    Article  CAS  Google Scholar 

  6. Floyd, J. C., Jr, Fajans, S. S., Conn, J. W., Knopf, R. F. & Rull, J. Stimulation of insulin secretion by amino acids. J. Clin. Invest. 45, 1487–1502 (1966)

    Article  CAS  Google Scholar 

  7. Yao, K. et al. Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs. J. Nutr. 138, 867–872 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  9. 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  ADS  Google Scholar 

  10. Wang, S. et al. Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 347, 188–194 (2015)

    Article  CAS  ADS  Google Scholar 

  11. Rebsamen, M. et al. SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1. Nature 519, 477–481 (2015)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Aravind, L. & Koonin, E. V. Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. J. Mol. Biol. 287, 1023–1040 (1999)

    Article  CAS  Google Scholar 

  14. Grant, G. A. The ACT domain: a small molecule binding domain and its role as a common regulatory element. J. Biol. Chem. 281, 33825–33829 (2006)

    Article  CAS  Google Scholar 

  15. Chipman, D. M. & Shaanan, B. The ACT domain family. Curr. Opin. Struct. Biol. 11, 694–700 (2001)

    Article  CAS  Google Scholar 

  16. Chantranupong, L. et al. The Sestrins interact with GATOR2 to negatively regulate the amino-acid-sensing pathway upstream of mTORC1. Cell Reports 9, 1–8 (2014)

    Article  CAS  Google Scholar 

  17. Parmigiani, A. et al. Sestrins inhibit mTORC1 kinase activation through the GATOR complex. Cell Reports 9, 1281–1291 (2014)

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  19. Saxton, R. A. et al. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Science 351, 53–58 (2016)

    Article  CAS  ADS  Google Scholar 

  20. Kotaka, M., Ren, J., Lockyer, M., Hawkins, A. R. & Stammers, D. K. Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine. J. Biol. Chem. 281, 31544–31552 (2006)

    Article  CAS  Google Scholar 

  21. Robin, A. Y. et al. A new mode of dimerization of allosteric enzymes with ACT domains revealed by the crystal structure of the aspartate kinase from Cyanobacteria. J. Mol. Biol. 399, 283–293 (2010)

    Article  CAS  Google Scholar 

  22. Dumas, R., Cobessi, D., Robin, A. Y., Ferrer, J.-L. & Curien, G. The many faces of aspartate kinases. Arch. Biochem. Biophys. 519, 186–193 (2012)

    Article  CAS  Google Scholar 

  23. Bridgham, J. T., Carroll, S. M. & Thornton, J. W. Evolution of hormone-receptor complexity by molecular exploitation. Science 312, 97–101 (2006)

    Article  CAS  ADS  Google Scholar 

  24. Coyle, S. M., Flores, J. & Lim, W. A. Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation. Cell 154, 875–887 (2013)

    Article  CAS  Google Scholar 

  25. Peisajovich, S. G., Garbarino, J. E., Wei, P. & Lim, W. A. Rapid diversification of cell signaling phenotypes by modular domain recombination. Science 328, 368–372 (2010)

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  28. Andersen, K. R., Leksa, N. C. & Schwartz, T. U. Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81, 1857–1861 (2013)

    Article  CAS  Google Scholar 

  29. Brohawn, S. G., Leksa, N. C., Spear, E. D., Rajashankar, K. R. & Schwartz, T. U. Structural evidence for common ancestry of the nuclear pore complex and vesicle coats. Science 322, 1369–1373 (2008)

    Article  CAS  ADS  Google Scholar 

  30. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  34. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    Article  CAS  Google Scholar 

  35. Gibrat, J. F., Madej, T. & Bryant, S. H. Surprising similarities in structure comparison. Curr. Opin. Struct. Biol. 6, 377–385 (1996)

    Article  CAS  Google Scholar 

  36. Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009)

    Article  CAS  Google Scholar 

  37. Notredame, C., Higgins, D. G. & Heringa, J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 (2000)

    Article  CAS  Google Scholar 

  38. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    Article  CAS  Google Scholar 

  39. Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.3r1 (2010)

  40. 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 

  41. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92, 7297–7301 (1995)

    Article  CAS  ADS  Google Scholar 

  42. Tsun, Z.-Y. et al. The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol. Cell 52, 495–505 (2013)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank all members of the Sabatini and Schwartz laboratories for helpful insights. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work has been supported by grants from NIH (R01CA103866 and AI47389) and the US Department of Defense (W81XWH-07- 0448) to D.M.S. Fellowship support was provided by NIH to L.C. (F31 CA180271). D.M.S. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

R.A.S., T.U.S., and D.M.S. designed the research plan. R.A.S. performed the experiments with assistance from L.C. and K.E.K. on experimental design and interpretation. R.A.S., T.U.S., and D.M.S. wrote the manuscript and all authors edited it.

Corresponding authors

Correspondence to Thomas U. Schwartz or David M. Sabatini.

Ethics declarations

Competing interests

D.M.S. is a founder and member of the Scientific Advisory Board, a paid consultant, and a shareholder of Navitor Pharmaceuticals, which is targeting for therapeutic benefit the amino-acid-sensing pathway upstream of mTORC1.

Additional information

Reviewer Information Nature thanks L. Tong and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Multiple sequence alignment of CASTOR1 homologues.

a, Expanded Multiple Sequence Alignment of CASTOR1 homologues from various organisms. Positions are coloured white to blue according to increasing sequence identity. Secondary structure features are labelled and coloured by ACT domain as in Fig. 1a.

Extended Data Figure 2 Dimerization-deficient CASTOR1 mutants bind arginine but fail to inhibit mTORC1 in cells.

a, The dimerization-deficient CASTOR1 Y207S and I202E mutants bind arginine in vitro. FLAG-immunoprecipitates prepared from HEK-293T cells transiently expressing indicated FLAG-tagged proteins were used in binding assays with [3H]Arginine as described in the Methods. Unlabelled arginine was included as a competitor where indicated. Values are mean ± s.d. for three technical replicates from one representative experiment. b, Dimerization-deficient CASTOR1 Y207S and I202E mutants fail to inhibit mTORC1. HEK-293T cells transiently expressing FLAG–S6K1 and HA-tagged wild-type, Y207S, or I202E CASTOR1 were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. FLAG- immunoprecipitates were prepared from lysates and analysed as in Fig. 1c. Phospho-S6K1 was used as an indicator of mTORC1 activity.

Extended Data Figure 3 Model of lysine-binding in CASTOR1.

a, Comparison of the arginine-bound pocket of human CASTOR1 with a model of the pocket with lysine in place of arginine. Arginine and lysine stick representations are shown in yellow and orange, respectively. The distances in the lysine-bound model, 3.8 Å and 5.0 Å, are beyond the range of standard hydrogen bonds and salt bridges, respectively. ACT domains are labelled as in Fig. 1a. b, Chemical structures of arginine analogues used in Fig. 2e. Differences relative to l-arginine are highlighted in oranges boxes.

Extended Data Figure 4 Differences in the arginine-binding capacities of CASTOR1 and CASTOR2.

a, Multiple sequence alignment of human CASTOR1 and CASTOR2, highlighting differences in amino acid sequence that are in close proximity to arginine-binding residues in CASTOR1. b, The CASTOR1 HHV108–110QNI mutant constitutively binds GATOR2 in cells. HEK-293T cells transiently expressing HA–metap2 or the indicated HA-tagged CASTOR1 constructs were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. HA-immunoprecipitates were prepared and analysed as in Fig. 1c. c, The CASTOR1 HHV108–110QNI mutant displays reduced arginine-binding capacity in vitro. Binding assays were performed with the indicated CASTOR1 or CASTOR2 constructs and immunoprecipitates analysed as in Fig. 2c. Values are mean ± s.d. for three technical replicates from one representative experiment. d, Comparison of the CASTOR1 HHV108–110QNI mutant and wild-type CASTOR2. HEK-293T cells transiently expressing HA–metap2 or the indicated HA-tagged CASTOR1 or CASTOR2 constructs were starved of arginine for 50 min and, where indicated, re-stimulated for 10 min. HA-immunoprecipitates were prepared and analysed as in Fig. 1c.

Extended Data Figure 5 GATOR2-binding-deficient CASTOR1 mutants still bind arginine and homodimerize.

a, The CASTOR1 YQ118–119AA, D121A, E261A and D292A mutants bind arginine in vitro. FLAG-immunoprecipitates prepared from HEK-293T cells transiently expressing indicated FLAG-tagged proteins were used in binding assays with [3H]arginine as described in the Methods. Unlabelled arginine was included as a competitor where indicated. Values are mean ± s.d. for three technical replicates from one representative experiment. b, The CASTOR1 YQ118–119AA, D121A, E261A and D292A mutants dimerize in cells. HA-immunoprecipitates prepared from HEK293T-cells transiently expressing CASTOR1–FLAG and HA–metap2 or the indicated HA-tagged CASTOR1 constructs were analysed as in Fig. 1c.

Extended Data Figure 6 Similarities between human CASTOR1 and prokaryotic aspartate kinases.

a, Ribbon diagram views of human CASTOR1, AKeco (PDB ID: 2J0x) and AKsyn (PDB ID: 3L76), highlighting the different modes of dimerization. Aspartate kinases can dimerize through an interlocked-ACT domain conformation (as in AKeco) or through their kinase domains (AKsyn), both of which are distinct from the side-by-side ACT-domain dimerization in CASTOR1. b, View of AKeco depicting positions of residues R305, E346, and V347, which correspond to the positions of the GATOR2-interacting residues of CASTOR1.

Extended Data Table 1 Data collection and refinement statistics (SAD)

Supplementary information

Supplementary Figure

This file contains the uncropped blots. (PDF 879 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Saxton, R., Chantranupong, L., Knockenhauer, K. et al. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 536, 229–233 (2016). https://doi.org/10.1038/nature19079

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature19079

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