Identification of lysine residues critical for the transcriptional activity and poly-ubiquitination of the NF-κB family member RelB
Résumé
RelB is the key component of the alternative NF-κB signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1-Daxx complex to NF-κB target genes. Importantly, the molecular mechanisms, which determine the functions of RelB are still largely unknown. Here, we aimed to analyze whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively poly-ubiquitinated in the B cell lines Namalwa and 70Z/3. While a PMA+Ionomycin-induced augmentation of RelB poly-ubiquitination was linked to its proteasomal degradation in B cells, the constitutive RelB poly-ubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB-poly-ubiquitination in 293 HEK cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA-binding was enhanced by RelB-poly-ubiquitination. Interestingly, basal RelB-poly-ubiquitination depends neither on Lys48- nor on Lys63-conjugates but might involve unconventional ubiquitin-conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys273/274 and Lys305/308 significantly decreased the basal RelB-activity and the ubiquitin-induced augmentation of the RelB-activity. Collectively, these data imply a dual role of RelB-poly-ubiquitination for the stability and activity of this transcription factor.
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