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Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation

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Abstract

Termination and resolution of inflammation are tightly linked to the inactivation of one of its strongest inducers, NF-κB. While canonical post-stimulus inactivation is achieved by upregulation of inhibitory molecules that relocate NF-κB complexes to the cytoplasm, termination of the NF-κB response can also be accomplished directly in the nucleus by posttranslational modifications, e.g., ubiquitination of the RelA subunit. Here we reveal a functional role for RelA monoubiquitination in regulating NF-κB activity. By employing serine-to-alanine mutants, we found that hypo-phosphorylated nuclear RelA is monoubiquitinated on multiple lysine residues. Ubiquitination was reversed by IκBα expression and was reduced when nuclear translocation was inhibited. RelA monoubiquitination decreased NF-κB transcriptional activity despite prolonged nuclear presence and independently of RelA degradation, possibly through decreased CREB-binding protein (CBP) co-activator binding. Polyubiquitin-triggered proteasomal degradation has been proposed as a model for RelA inactivation. However, here we show that proteasomal inhibition, similar to RelA hypo-phosphorylation, resulted in nuclear translocation and monoubiquitination of RelA. These findings indicate a degradation-independent mechanism for regulating the activity of nuclear RelA by ubiquitination.

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Acknowledgments

We are grateful to the following persons for providing materials used in this study: Drs. Amer A. Beg (RelA−/− 3T3), Dirk Bohmann (pMT107), Rainer De Martin (pKSII/ECI-6), Ivan Dikic (pcDNA3-HA-ubiquitin) and Alexander Hoffmann (IκBα−/− 3T3). We also thank Dr. Wei Chen for performing the MS analysis. This work was supported by a National Institutes of Health grant [HL077308 to J.A.] and American Heart Association Scientist Development grant [10SDG2600298 to K.H.].

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Authors and Affiliations

  1. Division of Neurobiology, Department of Neurology and Neuroscience, Weill Cornell Medical College, 407 East 61st Street, RR-409, New York, NY, 10065, USA

    Karin Hochrainer, Gianfranco Racchumi, Costantino Iadecola & Josef Anrather

  2. Institute of Biotechnology and Life Sciences Biotechnologies, Cornell University, Ithaca, NY, 14853, USA

    Sheng Zhang

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  1. Karin Hochrainer
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  2. Gianfranco Racchumi
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  5. Josef Anrather
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Correspondence to Josef Anrather.

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Hochrainer, K., Racchumi, G., Zhang, S. et al. Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation. Cell. Mol. Life Sci. 69, 2057–2073 (2012). https://doi.org/10.1007/s00018-011-0912-2

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