Letter | Published:

TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling

Nature volume 545, pages 365369 (18 May 2017) | Download Citation

Abstract

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways1. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer1. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients3,4,5,6 and growth factors7, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling8,9,10. Although both mTOR and GβL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GβL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GβL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GβL to promote GβL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GβL, by either K305R/K313R mutations or a melanoma-associated GβL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GβL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.

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References

  1. 1.

    , & mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35 (2011)

  2. 2.

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

  3. 3.

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

  4. 4.

    et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334, 678–683 (2011)

  5. 5.

    , , & Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150, 1196–1208 (2012)

  6. 6.

    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)

  7. 7.

    , , & Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003)

  8. 8.

    & Defining the role of mTOR in cancer. Cancer Cell 12, 9–22 (2007)

  9. 9.

    et al. Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis. Nat. Cell Biol. 15, 1340–1350 (2013)

  10. 10.

    et al. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discov. 5, 1194–1209 (2015)

  11. 11.

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

  12. 12.

    , , & Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev. 20, 2820–2832 (2006)

  13. 13.

    et al. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127, 125–137 (2006)

  14. 14.

    et al. mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr. Biol. 16, 1865–1870 (2006)

  15. 15.

    et al. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCα, but not S6K1. Dev. Cell 11, 859–871 (2006)

  16. 16.

    & The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012)

  17. 17.

    , & What determines the specificity and outcomes of ubiquitin signaling? Cell 143, 677–681 (2010)

  18. 18.

    et al. K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex in nutrient-activated cells. Mol. Cell 51, 283–296 (2013)

  19. 19.

    et al. Skp2-mediated RagA ubiquitination elicits a negative feedback to prevent amino-acid-dependent mTORC1 hyperactivation by recruiting GATOR1. Mol. Cell 58, 989–1000 (2015)

  20. 20.

    et al. The ubiquitination of rag A GTPase by RNF152 negatively regulates mTORC1 activation. Mol. Cell 58, 804–818 (2015)

  21. 21.

    , , , & TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657 (2002)

  22. 22.

    et al. mTOR complex 2 is required for the development of prostate cancer induced by Pten loss in mice. Cancer Cell 15, 148–159 (2009)

  23. 23.

    et al. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154, 169–184 (2013)

  24. 24.

    et al. Ubiquitin hydrolase UCH-L1 destabilizes mTOR complex 1 by antagonizing DDB1-CUL4-mediated ubiquitination of raptor. Mol. Cell. Biol. 33, 1188–1197 (2013)

  25. 25.

    et al. Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne. Nature 538, 402–405 (2016)

  26. 26.

    , & Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nat. Struct. Mol. Biol. 17, 939–947 (2010)

  27. 27.

    et al. Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell 129, 957–968 (2007)

  28. 28.

    et al. Requirement for interaction of PI3-kinase p110α with RAS in lung tumor maintenance. Cancer Cell 24, 617–630 (2013)

  29. 29.

    et al. OTUD7B controls non-canonical NF-κB activation through deubiquitination of TRAF3. Nature 494, 371–374 (2013)

  30. 30.

    et al. Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression. Oncogene 31, 4599–4608 (2012)

  31. 31.

    et al. Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis. Nat. Cell Biol. 17, 1169–1181 (2015)

  32. 32.

    , , , & Cezanne (OTUD7B) regulates HIF-1α homeostasis in a proteasome-independent manner. EMBO Rep. 15, 1268–1277 (2014)

  33. 33.

    , , , & Abolition of cyclin-dependent kinase inhibitor p16Ink4a and p21Cip1/Waf1 functions permits Ras-induced anchorage-independent growth in telomerase-immortalized human fibroblasts. Mol. Cell. Biol. 23, 2859–2870 (2003)

  34. 34.

    et al. SPOP promotes ubiquitination and degradation of the ERG oncoprotein to suppress prostate cancer progression. Mol. Cell 59, 917–930 (2015)

  35. 35.

    et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl Acad. Sci. USA 112, 6637–6642 (2015)

  36. 36.

    et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014)

  37. 37.

    et al. Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol. Cell. Proteomics. 10, M110.003756 (2011)

  38. 38.

    . et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010)

  39. 39.

    et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010)

  40. 40.

    , , , & A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006)

  41. 41.

    et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011)

  42. 42.

    et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protocols 8, 2281–2308 (2013)

  43. 43.

    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)

  44. 44.

    et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 410, 1111–1116 (2001)

  45. 45.

    , , & Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One 8, e82241 (2013)

Download references

Acknowledgements

We thank T. Jacks and the NCI Mouse Repository for providing the KrasLA2 mice. We thank P. P. Pandolfi (Harvard), B. D. Manning (Harvard), and A. Toker (Harvard) for their insightful suggestions and critiques during the preparation of this manuscript. We also thank all Wei laboratory members for critical reading of the manuscript. W.W. is a LLS research scholar. P.L. is supported by ROOCA181342. W.G. is supported by 1K99CA207867. A.O. was supported by an Edward R. and Anne G. Lefler Center postdoctoral fellowship. This work was supported by NIH grants (W.W., R01CA177910 and R01GM094777; S.-C.S., R37AI064639 and R01GM084459; J.W.H., AG011085 and GM095567) and the National Natural Science Foundation of China (B.W., 81472294, L.Z., 81521064).

Author information

Author notes

    • Bin Wang
    •  & Zuliang Jie

    These authors contributed equally to this work.

Affiliations

  1. Department of Gastroenterology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China

    • Bin Wang
  2. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Bin Wang
    • , Pengda Liu
    • , Wenjian Gan
    • , Jianping Guo
    • , Jinfang Zhang
    • , Brian J. North
    • , Xiangpeng Dai
    •  & Wenyi Wei
  3. Department of Immunology, The University of Texas MD Anderson Cancer Center, 7455 Fannin Street, Box 902, Houston, Texas 77030, USA

    • Zuliang Jie
    • , Donghyun Joo
    • , Xuhong Cheng
    •  & Shao-Cong Sun
  4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Alban Ordureau
    •  & J. Wade Harper
  5. Institute of Pathology and Southwest Cancer Center and Key Laboratory of Tumor Immunopathology, Ministry of Education of China, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

    • Xiuwu Bian
  6. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China

    • Lingqiang Zhang

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Contributions

B.W., Z.J., P.L., S.-C.S. and W.W. designed the research. B.W., Z.J., A.O., D.J., P.L., W.G., J.G., J.Z., B.J.N., X.D. and X.C. performed experiments and/or analysed data. S.-C.S. and W.W. supervised the study. X.B., L.Z. and J.W.H. provided critical reagents. B.W., Z.J., P.L., S.-C.S. and W.W. interpreted data and wrote the manuscript. All authors commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shao-Cong Sun or Wenyi Wei.

Reviewer Information Nature thanks D. Fruman 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

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    Supplementary Figure 1

    This file contains the original uncropped source images of western blot data for the main Figures and Extended Data Figures.

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DOI

https://doi.org/10.1038/nature22344

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