Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-kappaB family member RelB.
RelB is the key component of the alternative NF-kappaB (nuclear factor kappaB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)-Daxx (death domain-associated protein) complex to NF-kappaB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys(48) nor on Lys(63) 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 Lys(273/274) and Lys(305/308) significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.
Leidner
Julia
J
Institute of Physiological Chemistry, Ulm University, Ulm, Germany.
Palkowitsch
Lysann
L
Marienfeld
Uta
U
Fischer
Dietmar
D
Marienfeld
Ralf
R
D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000196:embryology; D008239:Lysine / Q000502:physiology*; D051841:Transcription Factor RelB / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D025801:Ubiquitin; D051841:Transcription Factor RelB; D008239:Lysine