Abstract

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & The tuberous sclerosis complex. New Engl. J. Med. 355, 1345–1356 (2006).

  2. 2.

    , & Tuberous Sclerosis Complex 3rd edn (Oxford Univ. Press, 1999).

  3. 3.

    & The Rheb family of GTP-binding proteins. Cell Signal. 16, 1105–1112 (2004).

  4. 4.

    & The TSC1–TSC2 complex: a molecular switchboard controlling cell growth. Biochem. J. 412, 179–190 (2008).

  5. 5.

    & Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).

  6. 6.

    et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).

  7. 7.

    et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).

  8. 8.

    , , & AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).

  9. 9.

    et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).

  10. 10.

    , & Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

  11. 11.

    et al. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416 (2013).

  12. 12.

    et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010).

  13. 13.

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

  14. 14.

    , & Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol. Cell 40, 310–322 (2010).

  15. 15.

    , & Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell Biol. 14, 133–139 (2013).

  16. 16.

    , , , & The late endosome is essential for mTORC1 signaling. Mol. Biol. Cell 21, 833–841 (2010).

  17. 17.

    et al. Lysosomal positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453–460 (2011).

  18. 18.

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

  19. 19.

    et al. ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS. Proc. Natl Acad. Sci. USA 107, 4153–4158 (2010).

  20. 20.

    & Peroxisomes and oxidative stress. Biochim. Biophys. Acta 1763, 1755–1766 (2006).

  21. 21.

    & Biochemistry of mammalian peroxisomes revisited. Annu. Rev. Biochem. 75, 295–332 (2006).

  22. 22.

    , & Peroxisome assembly: matrix and membrane protein biogenesis. J. Cell Biol. 193, 7–16 (2011).

  23. 23.

    et al. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1. Mol. Cell 47, 535–546 (2012).

  24. 24.

    et al. Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning. J. Cell Biol. 173, 279–289 (2006).

  25. 25.

    & Tuberous sclerosis complex, implication from a raregenetic disease to common cancer treatment. Hum. Mol. Genet. 18, R94–R100 (2009).

  26. 26.

    , & Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res. 58, 4766–4770 (1998).

  27. 27.

    , & Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ. 16, 1040–1052 (2009).

  28. 28.

    et al. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 26, 1749–1760 (2007).

  29. 29.

    et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).

  30. 30.

    , , & Peroxisome proliferation and beta-oxidation in Fao and MH1C1 rat hepatoma cells, HepG2 human hepatoblastoma cells and cultured human hepatocytes: effect of ciprofibrate. Eur. J. Cell Biol. 72, 314–323 (1997).

  31. 31.

    , , & Comparative effects of clofibrate on peroxisomal enzymes of human (Hep EBNA2) and rat (FaO) hepatoma cell lines. Eur. J. Cell Biol. 66, 375–381 (1995).

  32. 32.

    , & Peroxisome biogenesis disorders. Annu. Rev. Genom. Hum. Genet. 4, 165–211 (2003).

  33. 33.

    , & Restraining PI3K: mTOR signalling goes back to the membrane. Trends Biochem. Sci. 30, 35–42 (2005).

  34. 34.

    , , , & PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome. Nucleic Acids Res. 38, D800–D805 (2010).

  35. 35.

    et al. PEX5 protein binds monomeric catalase blocking its tetramerization and releases it upon binding the N-terminal domain of PEX14. J. Biol. Chem. 286, 40509–40519 (2011).

  36. 36.

    & Dynamic architecture of the peroxisomal import receptor Pex5p. Biochim. Biophys. Acta 1763, 1592–1598 (2006).

  37. 37.

    & PTS1-independent sorting of peroxisomal matrix proteins by Pex5p. Biochim. Biophys. Acta 1763, 1794–1800 (2006).

  38. 38.

    , , , & Multicompartmental distribution of the tuberous sclerosis gene products, hamartin and tuberin. Arch. Biochem. Biophys. 404, 210–217 (2002).

  39. 39.

    et al. Pathological mutations in TSC1 and TSC2 disrupt theinteraction between hamartin and tuberin. Hum. Mol. Genet. 10, 2899–2905 (2001).

  40. 40.

    et al. Tuberous sclerosis complex proteins control axon formation. Genes Dev. 22, 2485–2495 (2008).

  41. 41.

    et al. A reliable cell-based assay for testing unclassified TSC2 gene variants. Eur. J. Hum. Genet. 17, 301–310 (2009).

  42. 42.

    et al. Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305–1315 (1999).

  43. 43.

    , , & A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependentisocitrate dehydrogenase in Aspergillus nidulans. J. Biol. Chem. 276, 37722–37729 (2001).

  44. 44.

    , , & Efficient targeting of polyhydroxybutyrate biosynthetic enzymes to plant peroxisomes requires more than three amino acids in the carboxyl-terminal signal. J. Plant Physiol. 167, 329–332 (2010).

  45. 45.

    , , , & Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p. J. Biol. Chem. 277, 25011–25019 (2002).

  46. 46.

    Viruses exploiting peroxisomes. Curr. Opin. Microbiol. 14, 458–469 (2011).

  47. 47.

    , & Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle. J. Virol. 76, 2543–2547 (2002).

  48. 48.

    , & Akt regulates nuclear/cytoplasmic localization of tuberin. Oncogene 26, 521–531 (2007).

  49. 49.

    et al. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet. 7, 1053–1057 (1998).

  50. 50.

    et al. Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus. Oncogene 13, 913–923 (1996).

  51. 51.

    , , , & Predominant nuclear localization of mammalian target of rapamycin in normal and malignant cells in culture. J. Biol. Chem. 277, 28127–28134 (2002).

  52. 52.

    et al. Rheb regulates mitophagy induced by mitochondrial energetic status. Cell Metab. 17, 719–730 (2013).

  53. 53.

    et al. Localization of a portion of extranuclear ATM to peroxisomes. J. Biol. Chem. 274, 34277–34282 (1999).

  54. 54.

    , , , & ATM activation by oxidative stress. Science 330, 517–521 (2010).

  55. 55.

    , , , & Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13, 1259–1268 (2003).

  56. 56.

    , , , & Retinoic acid isomers protect hippocampal neurons from amyloid-beta induced neurodegeneration. Neurotox Res. 7, 243–250 (2005).

Download references

Acknowledgements

We thank G. Mills and Y. Lu (University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA) for the MCF-7 cell line stably expressing GFP–LC3, and RIKEN BRC for providing the ATG5+/+ MEFs and ATG5−/− MEFs. We are also grateful for the assistance of K. Dunner in electron microscopy image acquisition and analysis and T. Berry, X. Tong and S. Hensley for technical assistance. This work was supported by National Institutes of Health (NIH) Grant R01 CA143811 to C.L.W., NIH R01CA157216 to M.B.K., and NIH R01NS058956, the John Merck Fund, and the Children’s Hospital Boston Translational Research Program to M.S. A.R.T. was supported by the Association for International Cancer Research Career Development Fellowship (No. 06-914/915).

Author information

Author notes

    • Jiangwei Zhang
    •  & Jinhee Kim

    These authors contributed equally to this work

Affiliations

  1. Center for Translational Cancer Research, Institute for Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas 77030, USA

    • Jiangwei Zhang
    • , Angela Alexander
    • , Shengli Cai
    • , Durga Nand Tripathi
    • , Ruhee Dere
    •  & Cheryl Lyn Walker
  2. Korea Institute of Oriental Medicine, Dajeon 305-811, South Korea

    • Jinhee Kim
  3. Institute of Medical Genetics, Cardiff University, Cardiff CF14 4XN, UK

    • Andrew R. Tee
    • , Elaine A. Dunlop
    •  & Kayleigh M. Dodd
  4. Departments of Pediatrics, Pharmacology and Cancer Biology, Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Jacqueline Tait-Mulder
    •  & Michael B. Kastan
  5. The F.M. Kirby Neurobiology Center, Department of Neurology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Alessia Di Nardo
    • , Juliette M. Han
    • , Erica Kwiatkowski
    •  & Mustafa Sahin
  6. Department of Pathology (Neuropathology), Brigham and Women’s Hospital, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Rebecca D. Folkerth
  7. Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA

    • Phyllis L. Faust

Authors

  1. Search for Jiangwei Zhang in:

  2. Search for Jinhee Kim in:

  3. Search for Angela Alexander in:

  4. Search for Shengli Cai in:

  5. Search for Durga Nand Tripathi in:

  6. Search for Ruhee Dere in:

  7. Search for Andrew R. Tee in:

  8. Search for Jacqueline Tait-Mulder in:

  9. Search for Alessia Di Nardo in:

  10. Search for Juliette M. Han in:

  11. Search for Erica Kwiatkowski in:

  12. Search for Elaine A. Dunlop in:

  13. Search for Kayleigh M. Dodd in:

  14. Search for Rebecca D. Folkerth in:

  15. Search for Phyllis L. Faust in:

  16. Search for Michael B. Kastan in:

  17. Search for Mustafa Sahin in:

  18. Search for Cheryl Lyn Walker in:

Contributions

J.Z., J.K. and C.L.W. designed research; J.Z., J.K., A.A., S.C., D.N.T., R.D., A.R.T., J.T-M., A.D.N., J.M.H., E.K., E.A.D. and K.M.D. performed research; J.Z., J.K., A.R.T., R.D.F., P.L.F., M.B.K., M.S. and C.L.W. analysed data; J.Z., J.K. and C.L.W. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Cheryl Lyn Walker.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb2822

Further reading