Abstract
NF-κB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IκBs1,2,3. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin–proteasome system to eliminate inhibitory regulators. Here we isolate the pIκBα–ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIκBα degradation motif as an F-box/WD-domainprotein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RSIκB (pIκBα-E3 receptor subunit), binds specifically to pIκBα and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RSIκB, which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RSIκB represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
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Acknowledgements
We thank I. Alkalay for her invaluable role in developing many of the assays used in this work, A. Ciechanover for his support and advice in the early phases of the project, and A. Mahler and A. Bar-Sinai for comments on the manuscript. This research was supported by grants from the Israel Science Foundation funded by the Israel Academy for Sciences and Humanities-Centers of Excellence Program, the German–Israel Foundation for Scientific Research and Development, and Signal Pharmaceuticals Inc. (San Diego).
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Yaron, A., Hatzubai, A., Davis, M. et al. Identification of the receptor component of the IκBα–ubiquitin ligase. Nature 396, 590–594 (1998). https://doi.org/10.1038/25159
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DOI: https://doi.org/10.1038/25159
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