Abstract

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation1,2. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

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References

  1. 1.

    & Functional roles of ubiquitin-like domain (ULD) and ubiquitin-binding domain (UBD) containing proteins. Chem. Rev. 109, 1481–1494 (2009)

  2. 2.

    et al. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun. 8, 416–421 (2007)

  3. 3.

    & Shared principles in NF-κB signaling. Cell 132, 344–362 (2008)

  4. 4.

    & Ubiquitin-mediated regulation of TNFR1 signaling. Cytokine Growth Factor Rev. 19, 313–324 (2008)

  5. 5.

    & Atypical ubiquitin chains: new molecular signals. Review series ‘Protein modifications: beyond the usual suspects’. EMBO Rep. 9, 536–542 (2008)

  6. 6.

    & Linear polyubiquitination: a new regulator of NF-κΒ activation. EMBO Rep. 10, 706–713 (2009)

  7. 7.

    Regulation of tissue homeostasis by NF-κB signalling: implications for inflammatory diseases. Nature Rev. Immunol. 9, 778–788 (2009)

  8. 8.

    & Regulation and function of NF-κB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733 (2009)

  9. 9.

    et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006)

  10. 10.

    et al. Sharpin, a novel postsynaptic density protein that directly interacts with the shank family of proteins. Mol. Cell. Neurosci. 17, 385–397 (2001)

  11. 11.

    et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009)

  12. 12.

    et al. Ubiquitin binding mediates the NF-κB inhibitory potential of ABIN proteins. Oncogene 27, 3739–3745 (2008)

  13. 13.

    et al. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol. Cell 36, 831–844 (2009)

  14. 14.

    et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biol. 11, 123–132 (2009)

  15. 15.

    , , , & Ultrastructure of epidermis of mice with chronic proliferative dermatitis. Ultrastruct. Pathol. 19, 107–111 (1995)

  16. 16.

    & Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003)

  17. 17.

    , & Death receptor signal transducers: nodes of coordination in immune signaling networks. Nature Immunol. 10, 348–355 (2009)

  18. 18.

    et al. c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 29, 4198–4209 (2010)

  19. 19.

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

  20. 20.

    & Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21–36 (2005)

  21. 21.

    , , , & In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols 1, 2856–2860 (2007)

  22. 22.

    et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nature Methods 5, 459–460 (2008)

  23. 23.

    , & Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5, 988–994 (2006)

  24. 24.

    et al. Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. Genome Res. 20, 837–846 (2010)

  25. 25.

    et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021 (2005)

  26. 26.

    & MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)

  27. 27.

    , & Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols 2, 1896–1906 (2007)

Download references

Acknowledgements

We thank E. Kim, K. Rajalingham and H.-J. Kreienkampfor reagents used in this study, I. Matic for initial MS analysis of HOIP/HOIL-1L samples, S. Wahl for sample preparation, and V. Dötsch and members of the Dikic lab for discussions and comments. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DI 931/3-1), the Cluster of Excellence “Macromolecular Complexes” of the Goethe University Frankfurt (EXC115) to I.D., Landesstiftung Baden-Württemberg to B.M., the Medical Research Council UK to K.R. and B.S., JSPS Postdoctoral Fellowships for Research Abroad to F.I., EMBO long-term fellowship to S.S.S. and The National Institutes of Health (AR049288 to J.P.S.). V.N. was supported by the Unity Through Knowledge Fund, 3B Grant. C.G. acknowledges support from The International Human Frontier Science Program Organization.

Author information

Author notes

    • Yonathan Lissanu Deribe
    •  & Sigrid S. Skånland

    These authors contributed equally to this work.

Affiliations

  1. Frankfurt Institute for Molecular Life Sciences and Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, D-60590 Frankfurt (Main), Germany

    • Fumiyo Ikeda
    • , Yonathan Lissanu Deribe
    • , Sigrid S. Skånland
    • , Caroline Grabbe
    • , Sjoerd J. L. van Wijk
    • , Panchali Goswami
    •  & Ivan Dikic
  2. MRC-National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK

    • Benjamin Stieglitz
    •  & Katrin Rittinger
  3. Department of Molecular Biology, Umeå University, Building 6L, 901 87 Umeå, Sweden

    • Caroline Grabbe
  4. Proteome Center Tübingen, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany

    • Mirita Franz-Wachtel
    •  & Boris Macek
  5. IMBA-Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohrgasse 3, 1030 Vienna, Austria

    • Vanja Nagy
  6. School of Medicine, University of Split, Soltanska 2, Split, HR-21000, Croatia

    • Janos Terzic
    •  & Ivan Dikic
  7. Department of Biophysics and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan

    • Fuminori Tokunaga
    • , Tomoko Nakagawa
    •  & Kazuhiro Iwai
  8. Institute for Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Zülpicher Str. 47a, 50674 Cologne, Germany

    • Ariadne Androulidaki
    •  & Manolis Pasparakis
  9. Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan

    • Kazuhiro Iwai
  10. The Jackson Laboratory, Bar Harbor, Maine 04609, USA

    • John P. Sundberg
  11. Allgemeine Pharmakologie und Toxikologie, Division Nephropharmakologie, Klinikum der Goethe Universität, Theodor-Stern Kai 7, 60590 Frankfurt, Germany

    • Liliana Schaefer

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Contributions

F.I., Y.L.D., S.S.S., B.S., C.G., S.J.L.v.W., B.M., V.N., M.F.-W. and P.G. performed the experiments. F.T., A.A. and T.N. contributed with reagents used throughout the study. F.I., Y.L.D., S.S.S., C.G., M.P., J.T., K.I., J.P.S., L.F., B.M. and K.R. contributed to the project by co-ordination of experimental work and writing the manuscript. I.D. provided ideas, co-ordinated the entire project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ivan Dikic.

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https://doi.org/10.1038/nature09814

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