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Jul-Larsen A.,University of Bergen | Grudic A.,University of Bergen | Bjerkvig R.,University of Bergen | Bjerkvig R.,Center Recherche Public Sante Luxembourg | And 2 more authors.
BMC Molecular Biology | Year: 2010

Background: The promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein.Results: Expression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain.Conclusions: This study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering. © 2010 Jul-Larsen et al; licensee BioMed Central Ltd. Source

Waaler J.,University of Oslo | Waaler J.,Center for Molecular Biology and Neuroscience | Machon O.,University of Oslo | Machon O.,Academy of Sciences of the Czech Republic | And 12 more authors.
Cancer Research | Year: 2012

Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2. ©2012 AACR. Source

Solberg N.,University of Oslo | Solberg N.,Center for Molecular Biology and Neuroscience | MacHon O.,University of Oslo | MacHon O.,Center for Molecular Biology and Neuroscience | And 6 more authors.
Molecular and Cellular Biochemistry | Year: 2012

Tcf3 acts as a transcription factor controlling gene expression in canonical Wnt signaling. In this study we show that mouse Tcf3 represses canonical Wnt signaling in mouse neural stem cells and in human HEK 293 cells. We demonstrate that mouse Tcf3 mediates repression of both moderate and high levels of canonical Wnt signaling, by either competing with other members of the Tcf/Lef family for binding to β-catenin, or for binding to DNA. We observed that the repressor activity of mouse Tcf3 was only relieved effectively upon simultaneous disruption of both mechanisms. Immunofluorescence of transfected HEK 293 cells showed co-localization of β-catenin and Tcf3 in the nucleus of cells transfected with full-length Tcf3, but not in cells transfected with N-terminal deleted versions. A direct physical interaction between β-catenin and Tcf3 in the nucleus was confirmed by co-immunoprecipitation studies. The inhibitory β-catenin/Tcf3 interface was independent of the ability of Tcf3 to directly interact with DNA. © 2012 Springer Science+Business Media, LLC. Source

Ambur O.H.,Center for Molecular Biology and Neuroscience | Ambur O.H.,University of Oslo | Frye S.A.,Center for Molecular Biology and Neuroscience | Frye S.A.,University of Oslo | And 6 more authors.
PLoS ONE | Year: 2012

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. © 2012 Ambur et al. Source

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