The oncoprotein BCL-3 is a nuclear transcription factor that activates NF-kappaB target genes through formation of heterocomplexes with p50 or p52. BCL-3 is phosphorylated in vivo, but specific BCL-3 kinases have not been identified so far. In this report, we show that BCL-3 is a substrate for the protein kinase GSK3 and that GSK3-mediated BCL-3 phosphorylation, which is inhibited by Akt activation, targets its degradation through the proteasome pathway. This phosphorylation modulates its association with HDAC1, -3, and -6 and attenuates its oncogenicity by selectively controlling the expression of a subset of newly identified target genes such as SLPI and Cxcl1. Our results therefore suggest that constitutive BCL-3 phosphorylation by GSK3 regulates BCL-3 turnover and transcriptional activity.

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKK⑀-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/I-TRAF is required for the TBK1-and IKK⑀-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of doublestranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKK⑀ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide-and TBK1-IKK⑀-mediated Lys 63 -linked polyubiquitination, a mechanism that does not require TBK1-IKK⑀ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKK⑀ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys 63 -linked polyubiquitination of their scaffold protein, TANK.

IB␣ is an inhibitory molecule that sequesters NF-B dimers in the cytoplasm of unstimulated cells. Upon stimulation, NF-B moves to the nucleus and induces the expression of a variety of genes including IB␣. This newly synthesized IB␣ also translocates to the nucleus, removes activated NF-B from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-B activation. We show here that IB␣ enhances the transactivation potential of several homeodomain-containing proteins such as HOXB7 and Pit-1 through a NF-B-independent association with histone deacetylase (HDAC) 1 and HDAC3 but not with HDAC2, -4, -5, and -6. IB␣ bound both HDAC proteins through its ankyrin repeats, and this interaction was disrupted by p65. Immunofluorescence experiments demonstrated further that IB␣ acts by partially redirecting HDAC3 to the cytoplasm. At the same time, an IB␣ mutant, which lacked a functional nuclear localization sequence, interacted very efficiently with HDAC1 and -3 and intensively enhanced the transactivation potential of Pit-1. Our results support the hypothesis that the NF-B inhibitor IB␣ regulates the transcriptional activity of homeodomain-containing proteins positively through cytoplasmic sequestration of HDAC1 and HDAC3, a mechanism that would assign a new and unexpected role to IB␣.The paradigm of NF-B activation is based on its cytoplasmic sequestration by IB␣ through the masking of its nuclear localization sequence (NLS) 1 in unstimulated cells (1). Recent studies have challenged this hypothesis suggesting that IB␣ constantly shuttles in and out of the nucleus via nuclear import (2) and export sequences (3, 4). Because IB␣ nuclear export is more efficient than its nuclear import process, the NF-B⅐IB␣ complex remains mainly in the cytoplasm of unstimulated cells. Upon stimulation by proinflammatory cytokines, viral infection, or bacterial pathogens, IB␣ is phosphorylated by the IB kinase complex and degraded through the proteasome pathway (5). Then NF-B enters into the nucleus and activates the expression of multiple genes including IB␣. This newly synthesized IB␣ protein moves to the nucleus, removes NF-B from its target genes, and brings it back to the cytoplasm to terminate the phase of NF-B activation. IB␣ is part of a family of ankyrin-containing proteins that also includes IB␤ and IB⑀ (6) as well as p100, p105, and BCL-3 (7). In contrast to the IB proteins, BCL-3 is mainly nuclear and harbors some transactivation potential through heterodimer formation with the NF-B proteins p50 or p52 (8) and recruitment of coactivators such as JAB1 and BARD1 (9).We previously demonstrated that IB␣ enhances the transactivation potential of HOXB7, a homeodomain-containing protein (10). In this study, we elucidated further the underlying mechanism and showed that cytoplasmic IB␣ enhances the transactivation potential of other homeodomain-containing proteins such as Pit-1 and PAX8 through binding to some but not all HDAC proteins, a family of proteins sharing the ability to deacetylate their substrates a...

This study has determined the intracellular transport route of Shiga-like toxin (Stx) and the highly related Shiga toxin in human glomerular microvascular endothelial cells (GMVECs) and mesangial cells. In addition, the effect of tumor necrosis factor-alpha (TNF-alpha), which contributes to the pathogenesis of hemolytic-uremic syndrome, was evaluated more profound. Establishing the transport route will provide better understanding of the cytotoxic effect of Stx on renal cells. For our studies, we used receptor-binding B-subunit (StxB), which is identical between Shiga toxin and Stx-1. The transport route of StxB was studied by immunofluorescence microscopy and biochemical assays that allow quantitative analysis of retrograde transport from plasma membrane to Golgi apparatus and endoplasmic reticulum (ER). In both cell types, StxB was detergent-resistant membrane associated and followed the retrograde route. TNF-alpha upregulated Gb3 expression in mesangial cells and GMVECs, without affecting the efficiency of StxB transport to the ER. In conclusion, our study shows that in human GMVECs and mesangial cells, StxB follows the retrograde route to the Golgi apparatus and the ER. TNF-alpha treatment increases the amount of cell-associated StxB, but not retrograde transport as such, making it likely that the strong TNF-alpha-induced sensitization of mesangial cells and GMVECs for the toxic action of Stx is not due to a direct effect on the intracellular trafficking of the toxin.