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Involvement of linear polyubiquitylation of NEMO in NF-κB activation

Nature Cell Biology volume 11, pages 123–132 (2009)Cite this article

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Abstract

Nuclear factor-κB (NF-κB) is a key transcription factor in inflammatory, anti-apoptotic and immune processes. The ubiquitin pathway is crucial in regulating the NF-κB pathway. We have found that the LUBAC ligase complex, composed of the two RING finger proteins HOIL-1L and HOIP, conjugates a head-to-tail-linked linear polyubiquitin chain to substrates. Here, we demonstrate that LUBAC activates the canonical NF-κB pathway by binding to NEMO (NF-κB essential modulator, also called IKKγ) and conjugates linear polyubiquitin chains onto specific Lys residues in the CC2–LZ domain of NEMO in a Ubc13-independent manner. Moreover, in HOIL-1 knockout mice and cells derived from these mice, NF-κB signalling induced by pro-inflammatory cytokines such as TNF-α and IL-1β was suppressed, resulting in enhanced TNF-α–induced apoptosis in hepatocytes of HOIL-1 knockout mice. These results indicate that LUBAC is involved in the physiological regulation of the canonical NF-κB activation pathway through linear polyubiquitylation of NEMO.

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Figure 1: LUBAC activates the NF-κB pathway.
Figure 2: Linear polyubiquitylation activity of LUBAC is indispensable for canonical NF-κB activation.
Figure 3: LUBAC targets NEMO for NF-κB activation.
Figure 4: LUBAC conjugates a linear polyubiquitin chain to NEMO.
Figure 5: Lys 285 and Lys 309 in the CC2∼LZ domain of NEMO are acceptor sites for LUBAC-mediated linear polyubiquitylation.
Figure 6: Ubc13-independent NF-κB activation by LUBAC.
Figure 7: Impaired TNF-α signalling in HOIL-1 null cells and mice.

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Change history

  • 13 January 2009

    In the version of this article initially published online figure panels 3a, 3e and 7e were not clearly defined. Also (NEMOCC2~LZ) was corrected to (NEMO∆CC2~LZ). These errors have been corrected for the print, HTML and PDF versions of the article.

References

  1. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  Google Scholar 

  2. Kornitzer, D. & Ciechanover, A. Modes of regulation of ubiquitin-mediated protein degradation. J. Cell Physiol. 182, 1–11 (2000).

    Article  CAS  Google Scholar 

  3. Hicke, L. & Dunn, R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 (2003).

    Article  CAS  Google Scholar 

  4. Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).

    Article  CAS  Google Scholar 

  5. Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

    Article  CAS  Google Scholar 

  6. Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Nakamura, M., Tokunaga, F., Sakata, S. & Iwai, K. Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex. Biochem. Biophys. Res. Commun. 351, 340–347 (2006).

    Article  CAS  Google Scholar 

  9. Hayden, M. S. & Ghosh, S. Signaling to NF-κB. Genes Dev. 18, 2195–2224 (2004).

    Article  CAS  Google Scholar 

  10. Karin, M. & Greten, F. R. NF-κB: linking inflammation and immunity to cancer development and progression. Nature Rev. Immunol. 5, 749–759 (2005).

    Article  CAS  Google Scholar 

  11. Chen, Z. J. Ubiquitin signalling in the NF-κB pathway. Nature Cell Biol. 7, 758–765 (2005).

    Article  CAS  Google Scholar 

  12. Adhikari, A., Xu, M. & Chen, Z. J. Ubiquitin-mediated activation of TAK1 and IKK. Oncogene 26, 3214–3226 (2007).

    Article  CAS  Google Scholar 

  13. Chen, Z. J., Parent, L. & Maniatis, T. Site-specific phosphorylation of IκBα by a novel ubiquitination-dependent protein kinase activity. Cell 84, 853–862 (1996).

    Article  CAS  Google Scholar 

  14. Yamamoto, M. et al. Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nature Immunol. 7, 962–970 (2006).

    Article  CAS  Google Scholar 

  15. Cong, Y. S., Yao, Y. L., Yang, W. M., Kuzhandaivelu, N. & Seto, E. The hepatitis B virus X-associated protein, XAP3, is a protein kinase C-binding protein. J. Biol. Chem. 272, 16482–16489 (1997).

    Article  CAS  Google Scholar 

  16. Tokunaga, C. et al. Molecular cloning and characterization of a novel protein kinase C-interacting protein with structural motifs related to RBCC family proteins. Biochem. Biophys. Res. Commun. 244, 353–359 (1998).

    Article  CAS  Google Scholar 

  17. Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).

    Article  CAS  Google Scholar 

  18. Saito, N. et al. Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-κB through distinct signaling pathways in fibroblast cell lines. J. Biol. Chem. 278, 46565–46575 (2003).

    Article  CAS  Google Scholar 

  19. Bubici, C., Papa, S., Pham, C. G., Zazzeroni, F. & Franzoso, G. NF-κB and JNK: an intricate affair. Cell Cycle 3, 1524–1529 (2004).

    Article  CAS  Google Scholar 

  20. Papa, S. et al. The NF-κB-mediated control of the JNK cascade in the antagonism of programmed cell death in health and disease. Cell Death Differ. 13, 712–729 (2006).

    Article  CAS  Google Scholar 

  21. Johnson, E. S., Ma, P. C., Ota, I. M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 (1995).

    Article  CAS  Google Scholar 

  22. Chen, F., Bhatia, D., Chang, Q. & Castranova, V. Finding NEMO by K63-linked polyubiquitin chain. Cell Death Differ. 13, 1835–1838 (2006).

    Article  CAS  Google Scholar 

  23. Yamamoto, M. et al. Cutting Edge: Pivotal function of Ubc13 in thymocyte TCR signaling. J. Immunol. 177, 7520–7524 (2006).

    Article  CAS  Google Scholar 

  24. Abbott, D. W., Wilkins, A., Asara, J. M. & Cantley, L. C. The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr. Biol. 14, 2217–2227 (2004).

    Article  CAS  Google Scholar 

  25. Sebban-Benin, H. et al. Identification of TRAF6-dependent NEMO polyubiquitination sites through analysis of a new NEMO mutation causing incontinentia pigmenti. Hum. Mol. Genet. 16, 2805–2815 (2007).

    Article  CAS  Google Scholar 

  26. Huang, T. T., Wuerzberger-Davis, S. M., Wu, Z. H. & Miyamoto, S. Sequential modification of NEMO/IKKγ by SUMO-1 and ubiquitin mediates NF-κB activation by genotoxic stress. Cell 115, 565–576 (2003).

    Article  CAS  Google Scholar 

  27. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    Article  CAS  Google Scholar 

  28. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).

    Article  CAS  Google Scholar 

  29. Evans, P. C. et al. Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity. Biochem. J. 378, 727–734 (2004).

    Article  CAS  Google Scholar 

  30. Dworkin-Rastl, E., Shrutkowski, A. & Dworkin, M. B. Multiple ubiquitin mRNAs during Xenopus laevis development contain tandem repeats of the 76 amino acid coding sequence. Cell 39, 321–325 (1984).

    Article  CAS  Google Scholar 

  31. Einspanier, R., Sharma, H. S. & Scheit, K. H. Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells. Biochem. Biophys. Res. Commun. 147, 581–587 (1987).

    Article  CAS  Google Scholar 

  32. Kim, J. H., Park, K. C., Chung, S. S., Bang, O. & Chung, C. H. Deubiquitinating enzymes as cellular regulators. J. Biochem. 134, 9–18 (2003).

    Article  CAS  Google Scholar 

  33. Ventii, K. H. et al. Protein partners of deubiquitinating enzymes. Biochem. J. 414, 161–175 (2008).

    Article  CAS  Google Scholar 

  34. Karin, M. Nuclear factor-κB in cancer development and progression. Nature 441, 431–436 (2006).

    Article  CAS  Google Scholar 

  35. Braun, T. et al. Targeting NF-κB in hematologic malignancies. Cell Death Differ. 13, 748–758 (2006).

    Article  CAS  Google Scholar 

  36. Janssens, S. & Tschopp, J. Signals from within: the DNA-damage-induced NF-κB response. Cell Death Differ. 13, 773–784 (2006).

    Article  CAS  Google Scholar 

  37. Thompson, H. G., Harris, J. W., Lin, L. & Brody, J. P. Identification of the protein Zibra, its genomic organization, regulation, and expression in breast cancer cells. Exp. Cell Res. 295, 448–459 (2004).

    Article  CAS  Google Scholar 

  38. Swerdlow, P. S., Finley, D. & Varshavsky, A. Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal. Biochem. 156, 147–153 (1986).

    Article  CAS  Google Scholar 

  39. Iwai, K. et al. Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc. Natl Acad. Sci. USA 96, 12436–12441 (1999).

    Article  CAS  Google Scholar 

  40. Fernandez-de-Cossio, J. et al. Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry. Electrophoresis 21, 1694–1699 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank Nobuhito Goda and Natsuko Fujiki for instruction in mouse primary hepatocyte preparation; Ayumi Taya for supporting the LC and MS analyses; and Koichi Nakajima for reagents. This work was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (F.T., T.T. and K.I.), the Japan Health Sciences Foundation (F.T.) and the Naito Foundation (F.T.).

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Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Medicine and Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University, Suita, 565-0871, Osaka, Japan

    Fuminori Tokunaga, Shin-ichi Sakata, Kiyoko Kamei, Tomoko Nakagawa & Kazuhiro Iwai

  2. CREST, Japan Science Technology Corporation, Kawaguchi, 332-0012, Saitama, Japan

    Fuminori Tokunaga, Shin-ichi Sakata & Kazuhiro Iwai

  3. Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Abeno-ku, 545-8585, Osaka

    Fuminori Tokunaga, Shin-ichi Sakata, Takayoshi Kirisako, Kiyoko Kamei, Tomoko Nakagawa, Michiko Kato & Kazuhiro Iwai

  4. Laboratory of Frontier Science, Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, 113-8613, Tokyo, Japan

    Yasushi Saeki, Shigeo Murata & Keiji Tanaka

  5. Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, 565-0871, Osaka, Japan

    Yoshinori Satomi & Toshifumi Takao

  6. Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Shigeo Murata

  7. Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, 113-8519, Tokyo, Japan

    Shoji Yamaoka

  8. Department of Microbiology and Immunology, Laboratory of Immune Regulation, Graduate School of Medicine,

    Masahiro Yamamoto

  9. Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Suita, 565-0871, Osaka, Japan

    Shizuo Akira

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  1. Fuminori Tokunaga
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Contributions

F.T., S.-I.S, K.K., T.N. and M.K. performed experiments; Y.S., Y.S. and T.T. conducted mass spectrometric analyses. S.-I.S. and S.M. generated the HOIL-1 KO mice; T.K., S.Y., M.Y., S.A. and K.T. provided reagents and suggestions; F.T. and K.I. coordinated the study and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Kazuhiro Iwai.

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Tokunaga, F., Sakata, Si., Saeki, Y. et al. Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat Cell Biol 11, 123–132 (2009). https://doi.org/10.1038/ncb1821

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