• A Corrigendum to this article was published on 30 April 2015

This article has been updated

Abstract

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 01 April 2015

    In the version of this Article originally published, incorrect western blot scans were provided for the actin panels in Figure 4h,i. These panels have been corrected online. All samples in 4i were collected and processed simultaneously, on the same or on parallel gels/blots.

References

  1. 1.

    & An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal. 20, 460–473 (2014).

  2. 2.

    Autophagy and cell growth–the yin and yang of nutrient responses. J. Cell Sci. 125, 2359–2368 (2012).

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

    et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008).

  7. 7.

    et al. Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5, 973–979 (2009).

  8. 8.

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

  9. 9.

    , , , & PP2A regulates autophagy in two alternative ways in Drosophila. Autophagy 8, 623–636 (2012).

  10. 10.

    , , & Methionine inhibits autophagy and promotes growth by inducing the SAM-responsive methylation of PP2A. Cell 154, 403–415 (2013).

  11. 11.

    et al. CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. J. Cell Biol. 204, 713–727 (2014).

  12. 12.

    , & Protein phosphatase 2A regulatory subunits and cancer. Biochim. Biophys. Acta 1795, 1–15 (2009).

  13. 13.

    & From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell 33, 537–545 (2009).

  14. 14.

    & Involvement of PP2A in viral and cellular transformation. Oncogene 24, 7746–7755 (2005).

  15. 15.

    & Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439 (2001).

  16. 16.

    , & PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34–41 (2005).

  17. 17.

    et al. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 14, 2501–2514 (2000).

  18. 18.

    et al. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat. Cell Biol. 6, 308–318 (2004).

  19. 19.

    & Mechanisms of MYC stabilization in human malignancies. Cell Cycle 7, 592–596 (2008).

  20. 20.

    et al. Ambra1 regulates autophagy and development of the nervous system. Nature 447, 1121–1125 (2007).

  21. 21.

    et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168 (2010).

  22. 22.

    & The role of autophagy in mammalian development: cell makeover rather than cell death. Dev. Cell 15, 344–357 (2008).

  23. 23.

    et al. Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis. Autophagy 9, 476–495 (2013).

  24. 24.

    & Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 9, 153–166 (2009).

  25. 25.

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

  26. 26.

    et al. B55alpha PP2A holoenzymes modulate the phosphorylation status of the retinoblastoma-related protein p107 and its activation. J. Biol. Chem. 285, 29863–29873 (2010).

  27. 27.

    et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233 (2003).

  28. 28.

    & Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 (2005).

  29. 29.

    , & Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains. Nature 372, 375–379 (1994).

  30. 30.

    et al. Physical association of GPR54 C-terminal with protein phosphatase 2A. Biochem. Biophys. Res. Commun. 377, 1067–1071 (2008).

  31. 31.

    et al. Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis. Hybridoma 15, 49–53 (1996).

  32. 32.

    Lung epithelial proliferation: a biomarker for chemoprevention trials? J. Natl Cancer Inst. 93, 1042–1043 (2001).

  33. 33.

    Cell proliferation in the mammalian lung. Int. Rev. Exp. Pathol. 22, 131–191 (1980).

  34. 34.

    & Proliferation and differentiation in mammalian airway epithelium. Eur. Respir. J. 1, 58–80 (1988).

  35. 35.

    Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012).

  36. 36.

    et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075 (2009).

  37. 37.

    et al. Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3. J. Biol. Chem. 282, 18573–18583 (2007).

  38. 38.

    et al. Autophagy-deficient mice develop multiple liver tumors. Genes Dev. 25, 795–800 (2011).

  39. 39.

    et al. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Biol. 193, 275–284 (2011).

  40. 40.

    Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal. 13, 7–16 (2001).

  41. 41.

    & PP1 and PP2A phosphatases–cooperating partners in modulating retinoblastoma protein activation. FEBS J. 280, 627–643 (2013).

  42. 42.

    , & Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24, 186–191 (1999).

  43. 43.

    & Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53–57 (2007).

  44. 44.

    et al. Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239–1251 (2006).

  45. 45.

    & Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr. Opin. Genet. Dev. 21, 113–119 (2011).

  46. 46.

    et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev. 27, 1447–1461 (2013).

  47. 47.

    et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 25, 460–470 (2011).

  48. 48.

    et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J. Clin. Invest 112, 1809–1820 (2003).

  49. 49.

    et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672–676 (1999).

  50. 50.

    , , & A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp. Cell Res. 316, 3368–3378 (2010).

  51. 51.

    , & Role of autophagy in cancer prevention, development and therapy. Essays Biochem. 55, 133–151 (2013).

  52. 52.

    & Myc transcription factors: key regulators behind establishment and maintenance of pluripotency. Regen. Med. 5, 947–959 (2010).

  53. 53.

    et al. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc. Natl Acad. Sci. USA 107, 12564–12569 (2010).

  54. 54.

    et al. CIP2A inhibits PP2A in human malignancies. Cell 130, 51–62 (2007).

  55. 55.

    & Zebrafish. A Practical Approach (Oxford Univ. Press, 2002).

Download references

Acknowledgements

We wish to thank M. Canney, V. Unterkircher, R. Laricchia and M. Salomé for excellent technical assistance, and M. Acuña Villa and M. W. Bennett for editorial and secretarial work. We also thank S. Campello for critical reading of the manuscript. We are indebted to R. Sears (Portland, Oregon, USA), A. C. Gingras (Toronto, Canada) and A. Teleman and K. Dimitriadis (Heidelberg, Germany) for providing us with V5–Flag–c-Myc and Flag–PR65A constructs and Tsc2 MEFs, respectively, and to S. Cannata (Rome) for his advice on histopathology. This work was supported by grants from KBVU (R72-A4408), Lundbeck Foundation (R167-2013-16100), Novo Nordisk Foundation (7559), The Bjarne Saxhof Foundation, AIRC (IG2010 and IG2012 to both F.C. and M.P.), and in part from FISM (2009), the Telethon Foundation (GGP10225), the Italian Ministry of University and Research (PRIN 2009 and FIRB Accordi di Programma 2011) and the Italian Ministry of Health (RF 2009). V.C. is supported by the Lundbeck Foundation (R165-2013-15982). Also, we are grateful to the Spanish Ministry of Economy and Competitiveness (MINECO) (PS09/01401; PI12/02248, FR2009-0052 and IT2009-0053) and to Fundación Mutua Madrileña (AP101042012) for funding the laboratory of G.V.

Author information

Author notes

    • Maria Salazar
    •  & Pier Federico Gherardini

    Present addresses: Cell Division and Cancer Group, Spanish National Cancer Research Centre, Madrid E-28029, Spain (M.S.); Department of Microbiology and Immunology, Centre for Clinical Science Research, Stanford University School of Medicine, 94305 Stanford, California, USA (P.F.G.).

Affiliations

  1. Unit of Cell Stress and Survival, Danish Cancer Society Research Center, 2100 Copenhagen, Denmark

    • Valentina Cianfanelli
    • , Daniela De Zio
    •  & Francesco Cecconi
  2. Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, 00143 Rome, Italy

    • Valentina Cianfanelli
    • , Francesca Nazio
    • , Matteo Bordi
    •  & Francesco Cecconi
  3. Department of Biology, University of Rome ‘Tor Vergata’, 00133 Rome, Italy

    • Claudia Fuoco
    • , Pier Federico Gherardini
    • , Manuela Antonioli
    • , Melania D’Orazio
    • , Manuela Helmer-Citterich
    • , Mauro Piacentini
    • , Sabrina Di Bartolomeo
    •  & Francesco Cecconi
  4. Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain

    • Mar Lorente
    • , Maria Salazar
    •  & Guillermo Velasco
  5. Instituto de Investigaciones Sanitarias San Carlos (IdISSC), 28040 Madrid, Spain

    • Mar Lorente
    • , Maria Salazar
    •  & Guillermo Velasco
  6. Skin and Extracellular Matrix Research Group, Anatomy NUI Galway, Ireland

    • Fabio Quondamatteo
  7. National Institute for Infectious Diseases IRCCS ‘L. Spallanzani’, 00149 Rome, Italy

    • Manuela Antonioli
    • , Gian Maria Fimia
    •  & Mauro Piacentini
  8. Department of Biology, University of Padua, 35131 Padua, Italy

    • Tatjana Skobo
    •  & Luisa Dalla Valle
  9. Unit of Cell Death and Metabolism, Danish Cancer Society Research Center, 2100 Copenhagen, Denmark

    • Mikkel Rohde
  10. Department of Dermatology, University Freiburg Medical Center, 79104 Freiburg, Germany

    • Christine Gretzmeier
    •  & Joern Dengjel
  11. ZBSA Center for Biological Systems Analysis, University of Freiburg, 79104 Freiburg, Germany

    • Christine Gretzmeier
    •  & Joern Dengjel
  12. Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce 73100, Italy

    • Gian Maria Fimia

Authors

  1. Search for Valentina Cianfanelli in:

  2. Search for Claudia Fuoco in:

  3. Search for Mar Lorente in:

  4. Search for Maria Salazar in:

  5. Search for Fabio Quondamatteo in:

  6. Search for Pier Federico Gherardini in:

  7. Search for Daniela De Zio in:

  8. Search for Francesca Nazio in:

  9. Search for Manuela Antonioli in:

  10. Search for Melania D’Orazio in:

  11. Search for Tatjana Skobo in:

  12. Search for Matteo Bordi in:

  13. Search for Mikkel Rohde in:

  14. Search for Luisa Dalla Valle in:

  15. Search for Manuela Helmer-Citterich in:

  16. Search for Christine Gretzmeier in:

  17. Search for Joern Dengjel in:

  18. Search for Gian Maria Fimia in:

  19. Search for Mauro Piacentini in:

  20. Search for Sabrina Di Bartolomeo in:

  21. Search for Guillermo Velasco in:

  22. Search for Francesco Cecconi in:

Contributions

V.C. performed most experiments with crucial help from: C.F., M.B. and F.Q. (immunohistochemistry analysis); M.L. and M.S. (xenograft assay); P.F.G., M.R. and M.H-C. (bioinformatic analysis); D.D.Z. (real-time PCR); F.N. and M.A. (mutagenesis and cloning); M.D’O. (gel-filtration assay); T.S. and L.D.V. (experiment in zebrafish); C.G. and J.D. (mass-spectrometry analysis). G.M.F. and G.V. provided critical reagents. S.D.B. discussed the results and commented on the manuscript; V.C. and F.C. wrote the manuscript, with suggestions from M.P., G.M.F. and G.V.; F.C. and V.C. conceived and designed the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Francesco Cecconi.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Information

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3072

Further reading