Abstract
The NF-κB family of transcription factors is activated in response to many stimuli, including pro-inflammatory cytokines, environmental stresses and, in the case of B and T lymphocytes, by antigenic stimulation1,2. Bcl10 is essential for NF-κB activation by T- and B-cell receptors. T and B lymphocytes from Bcl10-deficient mice fail to activate NF-κB in response to antigen-receptor stimulation and, as a consequence, are unable to proliferate3. Bcl10 overexpression is sufficient to activate NF-κB, a process that requires the NF-κB essential modulator NEMO (also known as IKK-γ), which is the regulatory subunit of the IκB kinase complex4. However, the cellular mechanism by which Bcl10 activates the NF-κB pathway remains unclear. Here we show that Bcl10 targets NEMO for lysine-63-linked ubiquitination. Notably, a mutant form of NEMO that cannot be ubiquitinated inhibited Bcl10-induced NF-κB activation. Paracaspase and a ubiquitin-conjugating enzyme (UBC13) were both required for Bcl10-induced NEMO ubiquitination and subsequent NF-κB activation. Furthermore, short interfering RNAs that reduced the expression of paracaspase and UBC13 abrogated the effects of Bcl10. Thus, the adaptor protein Bcl10 promotes activation of NF-κB transcription factors through paracaspase- and UBC13-dependent ubiquitination of NEMO.
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Acknowledgements
We thank members of the Dixit laboratory for discussions and support; K. Newton for critical reading of the manuscript; S. Sun for the JM4.5.2 cell line; S. Yamaoka for the 5R cell line; R. Baker for Usp2; M. Yan for experimental advice; Z. Zhang for bioinformatics analysis; P. Andersen for antibody preparation; P. Yin for protein purification; and the Genentech Sequencing Core. I.E.W. is funded by a PSTP fellowship from the University of California at Davis.
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Supplementary information
41586_2004_BFnature02273_MOESM2_ESM.jpg
Supplementary Figure 2: a. Paracaspase C-terminus is associated with in vitro ubiquitin ligase activity. In vitro auto-ubiquitination with biotinylated ubiquitin was detected by anti-biotin immunoblotting. b. In vitro auto-ubiquitination of MBP-tagged paracaspase (312-582). Ubiquitination was detected as above. (JPG 44 kb)
41586_2004_BFnature02273_MOESM3_ESM.jpg
Supplementary Figure 3: K399 is conserved in NEMO from multiple species. Shown is an alignment between residues 385 and 419 of human NEMO. K399 is marked by an asterisk. (JPG 84 kb)
41586_2004_BFnature02273_MOESM5_ESM.jpg
Supplementary Figure 5: Human paracaspase mutant (C453A) does not inhibit NEMO ubiquitination induced by Bcl10. (JPG 23 kb)
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Zhou, H., Wertz, I., O'Rourke, K. et al. Bcl10 activates the NF-κB pathway through ubiquitination of NEMO. Nature 427, 167–171 (2004). https://doi.org/10.1038/nature02273
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DOI: https://doi.org/10.1038/nature02273
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