NF-κB is crucial for innate immune defence against microbial infection1,2. Inhibition of NF-κB signalling has been observed with various bacterial infections3,4. The NF-κB pathway critically requires multiple ubiquitin-chain signals of different natures5,6. The question of whether ubiquitin-chain signalling and its specificity in NF-κB activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-κB signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-κB signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-κB activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hayden, M. S. & Ghosh, S. Shared principles in NF-κB signaling. Cell 132, 344–362 (2008)
Vallabhapurapu, S. & Karin, M. Regulation and function of NF-κB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733 (2009)
Bhavsar, A. P., Guttman, J. A. & Finlay, B. B. Manipulation of host-cell pathways by bacterial pathogens. Nature 449, 827–834 (2007)
Roy, C. R. & Mocarski, E. S. Pathogen subversion of cell-intrinsic innate immunity. Nature Immunol. 8, 1179–1187 (2007)
Terzic, J., Marinovic-Terzic, I., Ikeda, F. & Dikic, I. Ubiquitin signals in the NF-κB pathway. Biochem. Soc. Trans. 35, 942–945 (2007)
Skaug, B., Jiang, X. & Chen, Z. J. The role of ubiquitin in NF-κB regulatory pathways. Annu. Rev. Biochem. 78, 769–796 (2009)
Cui, J. & Shao, F. Biochemistry and cell signaling taught by bacterial effectors. Trends Biochem. Sci. 36, 532–540 (2011)
Nadler, C. et al. The type III secretion effector NleE inhibits NF-κB activation. PLoS Pathog. 6, e1000743 (2010)
Newton, H. J. et al. The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-κB p65. PLoS Pathog. 6, e1000898 (2010)
Vossenkämper, A. et al. Inhibition of NF-κB signaling in human dendritic cells by the enteropathogenic Escherichia coli effector protein NleE. J. Immunol. 185, 4118–4127 (2010)
Ishitani, T. et al. Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling. EMBO J. 22, 6277–6288 (2003)
Cheung, P. C., Nebreda, A. R. & Cohen, P. TAB3, a new binding partner of the protein kinase TAK1. Biochem. J. 378, 27–34 (2004)
Jin, G. et al. Identification of a human NF-κB-activating protein, TAB3. Proc. Natl Acad. Sci. USA 101, 2028–2033 (2004)
Singhirunnusorn, P., Suzuki, S., Kawasaki, N., Saiki, I. & Sakurai, H. Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-β-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2. J. Biol. Chem. 280, 7359–7368 (2005)
Kanayama, A. et al. TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Mol. Cell 15, 535–548 (2004)
Kulathu, Y., Akutsu, M., Bremm, A., Hofmann, K. & Komander, D. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nature Struct. Mol. Biol. 16, 1328–1330 (2009)
Sato, Y., Yoshikawa, A., Yamashita, M., Yamagata, A. & Fukai, S. Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by NZF domains of TAB2 and TAB3. EMBO J. 28, 3903–3909 (2009)
Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011)
Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011)
Tokunaga, F. et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011)
Schubert, H. L., Blumenthal, R. M. & Cheng, X. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28, 329–335 (2003)
Meyer, H. H., Wang, Y. & Warren, G. Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 21, 5645–5652 (2002)
Wang, B. et al. Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4. J. Biol. Chem. 278, 20225–20234 (2003)
Laplantine, E. et al. NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain. EMBO J. 28, 2885–2895 (2009)
Alam, S. L. et al. Ubiquitin interactions of NZF zinc fingers. EMBO J. 23, 1411–1421 (2004)
Sedgwick, B., Robins, P., Totty, N. & Lindahl, T. Functional domains and methyl acceptor sites of the Escherichia coli Ada protein. J. Biol. Chem. 263, 4430–4433 (1988)
He, C. et al. A methylation-dependent electrostatic switch controls DNA repair and transcriptional activation by E. coli Ada. Mol. Cell 20, 117–129 (2005)
Castro, C. et al. Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis. Biochemistry 43, 5341–5351 (2004)
Koutmos, M. et al. Metal active site elasticity linked to activation of homocysteine in methionine synthases. Proc. Natl Acad. Sci. USA 105, 3286–3291 (2008)
Matthews, R. G. & Goulding, C. W. Enzyme-catalyzed methyl transfers to thiols: the role of zinc. Curr. Opin. Chem. Biol. 1, 332–339 (1997)
Ge, J. et al. A Legionella type IV effector activates the NF-κB pathway by phosphorylating the IκB family of inhibitors. Proc. Natl Acad. Sci. USA 106, 13725–13730 (2009)
Li, H. et al. The phosphothreonine lyase activity of a bacterial type III effector family. Science 315, 1000–1003 (2007)
Gong, Y. N. et al. Chemical probing reveals insights into the signaling mechanism of inflammasome activation. Cell Res. 20, 1289–1305 (2010)
Cui, J. et al. Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218 (2010)
Reyes-Turcu, F. E., Shanks, J. R., Komander, D. & Wilkinson, K. D. Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T. J. Biol. Chem. 283, 19581–19592 (2008)
We thank I. Rosenshine for providing NleE deletion strains, K. Iwai for the HOIL-1L and HOIP expression plasmids, H. Sakurai for the phospho-TAK1 antibody, Z. Chen for TAB2/3 and Npl4-NZF chimera constructs, and S. Fukai for the NZF expression plasmid. We also thank members of the Shao laboratory for helpful discussions and technical assistance. This work was supported by the National Basic Research Program of China (973 Programs, 2010CB835400 and 2012CB518700).
The authors declare no competing financial interests.
About this article
Cite this article
Zhang, L., Ding, X., Cui, J. et al. Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation. Nature 481, 204–208 (2012). https://doi.org/10.1038/nature10690
Substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of bacterial ubiquitin ligase IpaH9.8
Communications Biology (2020)
Nature Chemical Biology (2020)
Dissecting ribosomal particles throughout the kingdoms of life using advanced hybrid mass spectrometry methods
Nature Communications (2018)
Nature Communications (2018)