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:

Phosphorylation-dependent activity of the deubiquitinase DUBA

Nature Structural & Molecular Biology volume 19, pages 171–175 (2012)Cite this article

Subjects

Abstract

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: DUBA is a phosphorylation-dependent deubiquitinase.
Figure 2: Phosphorylation of DUBA Ser177 is required for accumulation of DUBA following LPS stimulation of macrophages.
Figure 3: Crystal structure of pSer177 DUBA–Ub-al complex.
Figure 4: DUBA helices α1 and α6 are highly dynamic in the apo protein and fold around ubiquitin in the complex.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Reyes-Turcu, F.E., Ventii, K.H. & Wilkinson, K.D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Sowa, M.E., Bennett, E.J., Gygi, S.P. & Harper, J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. López-Otín, C. & Hunter, T. The regulatory crosstalk between kinases and proteases in cancer. Nat. Rev. Cancer 10, 278–292 (2010).

    Article  PubMed  Google Scholar 

  4. Kurokawa, M. & Kornbluth, S. Caspases and kinases in a death grip. Cell 138, 838–854 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Dephoure, N. et al. A quantitative atlas of mitotic phosphorylation. Proc. Natl. Acad. Sci. USA 105, 10762–10767 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon production. Science 318, 1628–1632 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Edelmann, M.J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Komander, D. & Barford, D. Structure of the A20 OTU domain and mechanistic insights into deubiquitination. Biochem. J. 409, 77–85 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Lin, S.-C. et al. Molecular basis for the unique deubiquitinating activity of the NF-KB inhibitor A20. J. Mol. Biol. 376, 526–540 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Hurley, J.H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mayya, V. et al. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2, ra46 (2009).

    Article  PubMed  Google Scholar 

  12. Songyang, Z. et al. A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol. Cell Biol. 16, 6486–6493 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hu, M. et al. Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Hirsch, A.K.H., Fischer, F.R. & Diederich, F. Phosphate recognition in structural biology. Angew. Chem. Int. Ed. Engl. 46, 338–352 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Taylor, S.S. & Kornev, A.P. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36, 65–77 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Deshaies, R.J. & Joazeiro, C.A.P. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Komander, D. Mechanism, specificity, and structure of the deubiquitinases. Subcell. Biochem. 54, 69–87 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Bourhis, E. et al. Reconstitution of a Frizzled8-Wnt3a-LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6. J. Biol. Chem. 285, 9172–9179 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Dong, K.C. et al. Preparation of distinct ubiquitin chain reagents of high purity and yield. Structure 19, 1053–1063 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Jones, J.W. et al. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc. Natl. Acad. Sci. USA 107, 9771–9776 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang, G.G. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat. Methods 3, 287–293 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K.C. Dong (Genentech) for the gift of Ub-BEA, M.P. Kamps (University of California, San Diego) for the gift of ER-Hoxb8 retrovirus, T. Kitamura (University of Tokyo) for the gift of pMXs-puro, the Genentech baculovirus cloning and expression group for production of DUBA in insect cells, and the Genentech oligonucleotide synthesis and sequencing groups. Portions of this research were carried out at the Advanced Light Source, supported by the Director, Office of Science, Office of Basic Energy Sciences of the US Department of Energy, under Contract No. DE-AC02-05CH11231.

Author information

Authors and Affiliations

  1. Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA

    Oscar W Huang & Andrea G Cochran

  2. Department of Structural Biology, Genentech, South San Francisco, California, USA

    Xiaolei Ma, JianPing Yin, Jeremy Flinders, Till Maurer, Ivan Bosanac, Sarah G Hymowitz & Melissa A Starovasnik

  3. Department of Physiological Chemistry, Genentech, South San Francisco, California, USA

    Nobuhiko Kayagaki & Vishva M Dixit

  4. Department of Protein Chemistry, Genentech, South San Francisco, California, USA

    Qui Phung & David Arnott

Authors
  1. Oscar W Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaolei Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. JianPing Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeremy Flinders
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Till Maurer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nobuhiko Kayagaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qui Phung
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ivan Bosanac
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David Arnott
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Vishva M Dixit
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sarah G Hymowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Melissa A Starovasnik
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Andrea G Cochran
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Proteins and complexes were purified by O.W.H. and J.Y.; O.W.H. carried out all biochemical studies, with advice from A.G.C., and J.Y. crystallized proteins under advice from M.A.S. J.F. and T.M. conducted the NMR analysis, with advice from M.A.S. I.B. and X.M. solved the crystal structures of apo DUBA and the ubiquitin complex, respectively, with advice from S.G.H. Q.P. and D.A. provided mass spectral data. Evaluation of DUBA phosphorylation in macrophages was done by N.K. and V.M.D. The manuscript was written by A.G.C., with contributions from O.W.H., X.M., J.Y., T.M., N.K., Q.P., S.G.H. and M.A.S.

Corresponding author

Correspondence to Andrea G Cochran.

Ethics declarations

Competing interests

All authors are employees of Genentech, a member of the Roche Group.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Methods (PDF 19557 kb)

Supplementary Video 1

DUBA apo structure “morphing” into the pS177 DUBA Ub-al complex structure. (AVI 8518 kb)

Supplementary Data

Backbone 1H, 15N, and 13Cα assignments for apo DUBA OTU domain, pSer177 OTU domain, and the BEA-ubiquitin adduct of the pSer177 OTU domain. (XLS 42 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, O., Ma, X., Yin, J. et al. Phosphorylation-dependent activity of the deubiquitinase DUBA. Nat Struct Mol Biol 19, 171–175 (2012). https://doi.org/10.1038/nsmb.2206

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2206

This article is cited by

Search

Advanced 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