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Bcl10 activates the NF-κB pathway through ubiquitination of NEMO

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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Figure 1: Isolation of a Bcl10 signalling complex and selective requirement of paracaspase for NF-κB activation by Bcl10.
Figure 2: Paracaspase and UBC13 are required for Bcl10-induced ubiquitination and NF-κB activation.
Figure 3: NEMO is a substrate of the Bcl10-induced ubiquitination pathway.
Figure 4: Role of NEMO ubiquitination in NF-κB activation.

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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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Correspondence to Vishva M. Dixit.

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Supplementary information

Supplementary Figure 1: Isolation of a Bcl10 signalling complex. (JPG 35 kb)

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)

Supplementary Figure 4: NEMO K399R mutant does not inhibit NF-kB activation by NIK. (JPG 32 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)

Supplementary Figure Legends (RTF 5 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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