Editors' ChoiceBiochemistry

Yet Another Ubiquitin Arrangement for NF-κB

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Science Signaling  10 Feb 2009:
Vol. 2, Issue 57, pp. ec51
DOI: 10.1126/scisignal.257ec51

Both degradative and nondegradative ubiquitination of proteins are involved in activating the transcription factor NF-κB (see Haas). However, in cells lacking an essential component of the ubiquitin ligase, Ubc13, that mediates the nondegradative Lys63-linked polyubiquitination, NF-κB activation is still robust. Tokunaga et al. show that NF-κB activation can be mediated by yet another form of nondegradative polyubiquitination, linear polyubiquitination, mediated by a ubiquitin ligase called LUBAC (linear ubiquitin chain assembly complex) that contains two RING finger proteins, HOIL-1L and HOIP. Forced expression of HOIL-1L and HOIP activated NF-κB in the absence of any stimulus, whereas knockdown of HIOL-1L and HOIP reduced basal NF-κB activity, as well as that stimulated by tumor necrosis factor–α (TNF-α). In cells transfected with HOIL-1L and HOIP and tagged forms of components of the IKK (inhibitor of κB kinase) complex, coimmunoprecipitation revealed that LUBAC interacted with NEMO (NF-κB essential modulator) and that in the presence of NEMO, an interaction with the IKKs was promoted. When mutant forms of NEMO or LUBAC that could not interact were introduced into cells, they failed to stimulate NF-κB activity, and neither could mutant LUBAC that lacked ubiquitin ligation activity. The formation of linear polyubiquitin chains on NEMO was confirmed with in vitro assays with mutant forms of ubiquitin or methylated ubiquitin and by mass spectrometry analysis in cultured cells transfected with LUBAC and tagged NEMO. Linear polyubiquitin chains were detected with an antibody specific for this form of ubiquitylation, and linear polyubiquitin was detected on endogenous NEMO in cultured cells after treatment with TNF-α. TNF-α–mediated NF-κB activation was not impaired in cells deficient for Ubc13; however, silencing of HOIP did impair NF-κB activation. Further confirmation of the importance of linear polyubiquitin modification was obtained from mice in which HOIL-1 was knocked out. Mouse embryo fibroblasts from these mice exhibited impaired activation of NF-κB in response to TNF-α but no reduction in activation of the Jun N-terminal kinase (JNK) pathway. Knockout mice exposed to TNF-α exhibited hepatocyte apoptosis, which suggests that in the absence of LUBAC activity, impairment of NF-κB survival signaling allows JNK-mediated cell death.

F. Tokunaga, S.-i. Sakata, Y. Saeki, Y. Satomi, T. Kirisako, K. Kamei, T. Nakagawa, M. Kato, S. Murata, S. Yamaoka, M. Yamamoto, S. Akira, T. Takao, K. Tanaka, K. Iwai, Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nat. Cell. Biol. 11, 123–132 (2009). [PubMed]

A. L. Haas, Linear polyubiquitylation: The missing link in NF-κB signalling. Nat. Cell. Biol. 11, 116–118 (2009). [PubMed]

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