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:

Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis

Nature Immunology volume 14, pages 927–936 (2013)Cite this article

Subjects

Abstract

Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.

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: Pellino3 deficiency diminishes MDP-induced expression of proinflammatory cytokines in BMDMs.
Figure 2: Peli3−/− mice have less induction of the expression of proinflammatory cytokines in response to MDP.
Figure 3: Peli3−/− mice have impaired clearance of C. rodentium and less colonic inflammation.
Figure 4: Pellino3 interacts directly with RIP2 in a FHA domain–dependent manner.
Figure 5: Pellino3 catalyzes K63-linked polyubiquitination of RIP2.
Figure 6: Pellino3 mediates MDP-induced K63-linked polyubiquitination of RIP2 and downstream signaling in an IAP-independent manner.
Figure 7: The FHA and RING domains of Pellino3 are both needed to mediate MDP-induced ubiquitination of RIP2 and downstream signaling.
Figure 8: Pellino3 expression is much lower in colons from patients with Crohn's disease.

Similar content being viewed by others

References

  1. Newton, K. & Dixit, V.M. Signaling in innate immunity and inflammation. Cold Spring Harb. Perspect. Biol. 4, 1–19 (2012).

    Article  CAS  Google Scholar 

  2. Kawai, T. & Akira, S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Rubino, S.J., Selvanantham, T., Girardin, S.E. & Philpott, D.J. Nod-like receptors in the control of intestinal inflammation. Curr. Opin. Immunol. 24, 398–404 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Hugot, J.P. et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411, 599–603 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Ogura, Y. et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411, 603–606 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Hampe, J. et al. Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet 357, 1925–1928 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Miceli-Richard, C. et al. CARD15 mutations in Blau syndrome. Nat. Genet. 29, 19–20 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Kanazawa, N. et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome. Blood 105, 1195–1197 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Inohara, N. et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J. Biol. Chem. 278, 5509–5512 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Girardin, S.E. et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Tanabe, T. et al. Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition. EMBO J. 23, 1587–1597 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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  PubMed  Google Scholar 

  13. Ogura, Y. et al. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-κB. J. Biol. Chem. 276, 4812–4818 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Abbott, D.W. et al. Coordinated regulation of Toll-like receptor and NOD2 signaling by K63-linked polyubiquitin chains. Mol. Cell Biol. 27, 6012–6025 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yang, Y. et al. NOD2 pathway activation by MDP or Mycobacterium tuberculosis infection involves the stable polyubiquitination of Rip2. J. Biol. Chem. 282, 36223–36229 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Hasegawa, M. et al. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-κB activation. EMBO J. 27, 373–383 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. McCarthy, J.V., Ni, J. & Dixit, V.M. RIP2 is a novel NF-κB-activating and cell death-inducing kinase. J. Biol. Chem. 273, 16968–16975 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Windheim, M., Lang, C., Peggie, M., Plater, L.A. & Cohen, P. Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide. Biochem. J. 404, 179–190 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kanayama, A. et al. TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Mol. Cell 15, 535–548 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Moynagh, P.N. TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaB pathway. Trends Immunol. 26, 469–476 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Traenckner, E.B. et al. Phosphorylation of human IκB-α on serines 32 and 36 controls IκB-α proteolysis and NF-κB activation in response to diverse stimuli. EMBO J. 14, 2876–2883 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Moynagh, P.N. The NF-κB pathway. J. Cell Sci. 118, 4589–4592 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Damgaard, R.B. et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol. Cell 46, 746–758 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Bertrand, M.J. et al. Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2. Immunity 30, 789–801 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Tao, M. et al. ITCH K63-ubiquitinates the NOD2 binding protein, RIP2, to influence inflammatory signaling pathways. Curr. Biol. 19, 1255–1263 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moynagh, P.N. The Pellino family: IRAK E3 ligases with emerging roles in innate immune signalling. Trends Immunol. 30, 33–42 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Schauvliege, R., Janssens, S. & Beyaert, R. Pellino proteins: novel players in TLR and IL-1R signalling. J. Cell Mol. Med. 11, 453–461 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lin, C.C., Huoh, Y.S., Schmitz, K.R., Jensen, L.E. & Ferguson, K.M. Pellino proteins contain a cryptic FHA domain that mediates interaction with phosphorylated IRAK1. Structure 16, 1806–1816 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schauvliege, R., Janssens, S. & Beyaert, R. Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases. FEBS Lett. 580, 4697–4702 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Butler, M.P., Hanly, J.A. & Moynagh, P.N. Kinase-active interleukin-1 receptor-associated kinases promote polyubiquitination and degradation of the Pellino family: direct evidence for PELLINO proteins being ubiquitin-protein isopeptide ligases. J. Biol. Chem. 282, 29729–29737 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Ordureau, A. et al. The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1. Biochem. J. 409, 43–52 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Smith, H. et al. Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4. Proc. Natl. Acad. Sci. USA 106, 4584–4590 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Smith, H. et al. The role of TBK1 and IKKɛ in the expression and activation of Pellino 1. Biochem. J. 434, 537–548 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Goh, E.T. et al. Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system. Biochem. J. 441, 339–346 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Strelow, A., Kollewe, C. & Wesche, H. Characterization of Pellino2, a substrate of IRAK1 and IRAK4. FEBS Lett. 547, 157–161 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Chang, M., Jin, W. & Sun, S.C. Peli1 facilitates TRIF-dependent Toll-like receptor signaling and proinflammatory cytokine production. Nat. Immunol. 10, 1089–1095 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chang, M. et al. The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity. Nat. Immunol. 12, 1002–1009 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Moynagh, P.N. Peli1 (rel)ieves autoimmunity. Nat. Immunol. 12, 927–929 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Siednienko, J. et al. Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons. Nat. Immunol. 13, 1055–1062 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Kim, Y.G. et al. The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes. Immunity 34, 769–780 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Murphy, C.T. et al. The sphingosine-1-phosphate analogue FTY720 impairs mucosal immunity and clearance of the enteric pathogen Citrobacter rodentium. Infect. Immun. 80, 2712–2723 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Watanabe, T. et al. Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis. J. Clin. Invest. 118, 545–559 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hitotsumatsu, O. et al. The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals. Immunity 28, 381–390 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Inohara, N., del Peso, L., Koseki, T., Chen, S. & Nunez, G. RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis. J. Biol. Chem. 273, 12296–12300 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Bertrand, M.J. et al. cIAP1/2 are direct E3 ligases conjugating diverse types of ubiquitin chains to receptor interacting proteins kinases 1 to 4 (RIP1–4). PLoS ONE 6, e22356 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Haas, T.L. 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).

    Article  CAS  PubMed  Google Scholar 

  47. Geddes, K. et al. Identification of an innate T helper type 17 response to intestinal bacterial pathogens. Nat. Med. 17, 837–844 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Smith, P. et al. Infection with a helminth parasite prevents experimental colitis via a macrophage-mediated mechanism. J. Immunol. 178, 4557–4566 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Wirtz, S., Neufert, C., Weigmann, B. & Neurath, M.F. Chemically induced mouse models of intestinal inflammation. Nat. Protoc. 2, 541–546 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Hall, L.J. et al. Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor. Mucosal Immunol. advance online publication, doi:10.1038/mi.2012.140 (23 January 2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank P.A. Barker and M. Saleh (McGill University) for Flag-tagged RIP2 and Nod2; M. Parkers and the Broad Institute for help and use of the Ricopili search tool; and G. Holleran for assistance in collecting biopsy samples from human subjects. Supported by Science Foundation Ireland (07/IN.1/B972 to P.N.M.; 10/IN.1/B3004 to P.G.F.; 02/CE/B124 and 07/CE/B1368 to F.S.; and funding for the Alimentary Pharmabiotic Centre as a Centre for Science, Engineering and Technology) and the Health Research Board of Ireland (PhD/2007/09).

Author information

Authors and Affiliations

  1. Department of Biology, Institute of Immunology, National University of Ireland Maynooth, Maynooth, Ireland

    Shuo Yang, Bingwei Wang, Fiachra Humphries, Ruaidhri Jackson, Marc E Healy, Ronan Bergin & Paul N Moynagh

  2. Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland

    Gabriella Aviello & Padraic G Fallon

  3. Department of Gastroenterology, Adelaide and Meath Hospital & Clinical Medicine, Trinity College Dublin, Dublin, Ireland

    Barry Hall & Deirdre McNamara

  4. Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

    Trevor Darby, Aoife Quinlan, Fergus Shanahan & Silvia Melgar

  5. Institute of Molecular Medicine, School of Medicine, Trinity College Dublin, Dublin, Ireland

    Padraic G Fallon

  6. National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland

    Padraic G Fallon

Authors
  1. Shuo Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bingwei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fiachra Humphries
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruaidhri Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marc E Healy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ronan Bergin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gabriella Aviello
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Barry Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Deirdre McNamara
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Trevor Darby
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Aoife Quinlan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Fergus Shanahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Silvia Melgar
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Padraic G Fallon
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Paul N Moynagh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.Y. developed the concept, designed and did experiments, analyzed data and prepared the figures; B.W. designed and did experiments and analyzed data; F.H. did the studies involving RIP2 mutants. R.J. and R.B. did the studies of colon explants and primary intestinal epithelial cells. M.E.H. and R.B. did the flow cytometry of cells from mesenteric lymph nodes; G.A. did the DSS and TNBS colitis studies and analyzed data. B.H. and D.M. obtained colon biopsy samples from adult humans and did clinical phenotyping of patients; T.D. and A.Q. did the C. rodentium studies; F.S. and S.M. designed, analyzed and supervised the C. rodentium studies; P.G.F. designed and supervised the DSS and TNBS colitis studies; and P.N.M. conceived of the study, supervised the overall project, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Paul N Moynagh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1761 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, S., Wang, B., Humphries, F. et al. Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis. Nat Immunol 14, 927–936 (2013). https://doi.org/10.1038/ni.2669

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2669

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