SUMOylation attenuates the transcriptional activity of the NF-κB subunit RelB.

The NF-κB subunit RelB is known to act either as an activator or repressor of NF-κB-dependent gene expression. The RelB-p52 heterodimer, for instance, is the key element of the alternative NF-κB signaling pathway supporting the expression of a subset of NF-κB target genes. By contrast, RelB is crucial for the repression of important pro-inflammatory cytokines like TNFα or interleukin 1ß. Despite accumulating reports describing the functional variability of RelB, the molecular mechanisms underlying these divergent functions are still unknown. One potential explanation could be a functional reprogramming of RelB by different post-translational modifications. Here, we demonstrate that SUMOylation of RelB might be one of these post-translational modifications rendering the function of the NF-κB transcription factor RelB. In vivo SUMOylation analyses using either the UBC9-fusion-directed SUMOylation method or endogenous proteins from Namalwa B cells revealed that RelB is modified by either SUMO1 or SUMO2 attachment at various sites. Functional studies suggest that SUMOylation converts RelB into a transcriptional repressor. For instance, a SUMO1-RelB fusion protein mimicking RelB-SUMOylation displayed a reduced transcriptional activity in comparison to wild type RelB. Consistently, inactivation of specific SUMOylation sites in the central part of RelB augmented the transcription activity of the corresponding RelB mutant. Taken together, our data suggest that SUMOylation might be a potential molecular mechanism involved in reprogramming RelB, thus contributing to its functional diversity.

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.

Phosphorylation of serine 68 in the IkappaB kinase (IKK)-binding domain of NEMO interferes with the structure of the IKK complex and tumor necrosis factor-alpha-induced NF-kappaB activity.

The IkappaB-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the IkappaB proteins and thus controls the activity of the transcription factor NF-kappaB. Besides the two kinases IKKalpha and IKKbeta, the IKK complex contains NEMO/IKKgamma, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKKbeta-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKKbeta phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser(68) phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKKbeta-NEMO interaction. In contrast, the NEMO-IKKalpha interaction was only mildly affected by the phosphorylation of Ser(68). However, functional analysis revealed that Ser(68) phosphorylation primarily affects the activity of IKKalpha. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF-alpha-induced NF-kappaB activity, thus emphasizing the inhibitory role of the Ser(68) phosphorylation on the signal-induced NF-kappaB activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser(68)-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex.