Article | Published:

Phosphorylation-dependent activity of the deubiquitinase DUBA

Nature Structural & Molecular Biology volume 19, pages 171175 (2012) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Referenced accessions

Protein Data Bank

References

  1. 1.

    , & Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu. Rev. Biochem. 78, 363–397 (2009).

  2. 2.

    , , & Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).

  3. 3.

    & The regulatory crosstalk between kinases and proteases in cancer. Nat. Rev. Cancer 10, 278–292 (2010).

  4. 4.

    & Caspases and kinases in a death grip. Cell 138, 838–854 (2009).

  5. 5.

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

  6. 6.

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

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    , & Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006).

  11. 11.

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

  12. 12.

    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).

  13. 13.

    & Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010).

  14. 14.

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

  15. 15.

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

  16. 16.

    , & Phosphate recognition in structural biology. Angew. Chem. Int. Ed. Engl. 46, 338–352 (2007).

  17. 17.

    & Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36, 65–77 (2011).

  18. 18.

    & RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

  19. 19.

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

  20. 20.

    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).

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

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

  25. 25.

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

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

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. Search for Oscar W Huang in:

  2. Search for Xiaolei Ma in:

  3. Search for JianPing Yin in:

  4. Search for Jeremy Flinders in:

  5. Search for Till Maurer in:

  6. Search for Nobuhiko Kayagaki in:

  7. Search for Qui Phung in:

  8. Search for Ivan Bosanac in:

  9. Search for David Arnott in:

  10. Search for Vishva M Dixit in:

  11. Search for Sarah G Hymowitz in:

  12. Search for Melissa A Starovasnik in:

  13. Search for Andrea G Cochran in:

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.

Competing interests

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

Corresponding author

Correspondence to Andrea G Cochran.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Methods

Videos

  1. 1.

    Supplementary Video 1

    DUBA apo structure “morphing” into the pS177 DUBA Ub-al complex structure.

Excel files

  1. 1.

    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.

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading