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RanBP3 enhances nuclear export of active (beta)-catenin independently of CRM1.

Hendriksen J, Fagotto F, van der Velde H, van Schie M, Noordermeer J, Fornerod M - J. Cell Biol. (2005)

Bottom Line: beta-Catenin is the nuclear effector of the Wnt signaling cascade.Conversely, overexpression of RanBP3 leads to a shift of active beta-catenin toward the cytoplasm.We conclude that RanBP3 is a direct export enhancer for beta-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
beta-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of beta-catenin is regulated is not well defined. Therefore, we used the nuclear marker RanGTP to screen for novel nuclear beta-catenin binding proteins. We identified a cofactor of chromosome region maintenance 1 (CRM1)-mediated nuclear export, Ran binding protein 3 (RanBP3), as a novel beta-catenin-interacting protein that binds directly to beta-catenin in a RanGTP-stimulated manner. RanBP3 inhibits beta-catenin-mediated transcriptional activation in both Wnt1- and beta-catenin-stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with beta-catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated beta-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active beta-catenin toward the cytoplasm. Modulation of beta-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. We conclude that RanBP3 is a direct export enhancer for beta-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

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RanBP3 induces specific depletion of endogenous nuclear active β-catenin. SW480 (A and B) or DLD1 (C) colon carcinoma cells were transfected with RanBP3 expression plasmids and stained after 45 h for dephosphorylated β-catenin (A and C) or total β-catenin (B). RanBP3 expression was visualized in the same cells using a RanBP3 polyclonal (A and C) or mAb. (D) Luciferase reporter assay as in Figs. 2–4 measuring relative β-catenin activity. Cells were transfected as in A and C. Error bars represent SDs of technical replicates.
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fig6: RanBP3 induces specific depletion of endogenous nuclear active β-catenin. SW480 (A and B) or DLD1 (C) colon carcinoma cells were transfected with RanBP3 expression plasmids and stained after 45 h for dephosphorylated β-catenin (A and C) or total β-catenin (B). RanBP3 expression was visualized in the same cells using a RanBP3 polyclonal (A and C) or mAb. (D) Luciferase reporter assay as in Figs. 2–4 measuring relative β-catenin activity. Cells were transfected as in A and C. Error bars represent SDs of technical replicates.

Mentions: Nuclear/cytoplasmic fractionation data does not always reflect the subcellular localization in living cells because pools of proteins that are not tightly bound to nuclear or cytoplasmic structures and are relatively small may leak through nuclear pore complexes of permeabilized cells. We therefore assayed the effect of RanBP3 overexpression on active β-catenin in situ using the anti–active β-catenin antibody. In our hands, this antibody did not visualize endogenous dephosphorylated β-catenin in Wnt1-transfected HEK293 cells (unpublished data). We therefore tested two colon carcinoma cell lines (SW480 and DLD1) that have a constitutively activated β-catenin because of a mutation in APC (Rosin-Arbesfeld et al., 2003). In SW480, but not in DLD1, the anti–dephosphorylated β-catenin antibody recognizes a clear nuclear signal above background (Fig. 6, A and C). The presence of this signal correlates with the exceptionally high β-catenin activity as measured in luciferase assays (Fig. 6 D), i.e., ∼30-fold higher than in DLD1. Importantly, RanBP3 overexpression leads to a clear reduction of active β-catenin signal from the SW480 nuclei (Fig. 6 A) but has no influence on total β-catenin localization (Fig. 6 B). This indicates that, even in the extremely active SW480 cell line, only a very small proportion of total β-catenin is properly dephosphorylated and active, and that this is the pool RanBP3 acts on.


RanBP3 enhances nuclear export of active (beta)-catenin independently of CRM1.

Hendriksen J, Fagotto F, van der Velde H, van Schie M, Noordermeer J, Fornerod M - J. Cell Biol. (2005)

RanBP3 induces specific depletion of endogenous nuclear active β-catenin. SW480 (A and B) or DLD1 (C) colon carcinoma cells were transfected with RanBP3 expression plasmids and stained after 45 h for dephosphorylated β-catenin (A and C) or total β-catenin (B). RanBP3 expression was visualized in the same cells using a RanBP3 polyclonal (A and C) or mAb. (D) Luciferase reporter assay as in Figs. 2–4 measuring relative β-catenin activity. Cells were transfected as in A and C. Error bars represent SDs of technical replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171279&req=5

fig6: RanBP3 induces specific depletion of endogenous nuclear active β-catenin. SW480 (A and B) or DLD1 (C) colon carcinoma cells were transfected with RanBP3 expression plasmids and stained after 45 h for dephosphorylated β-catenin (A and C) or total β-catenin (B). RanBP3 expression was visualized in the same cells using a RanBP3 polyclonal (A and C) or mAb. (D) Luciferase reporter assay as in Figs. 2–4 measuring relative β-catenin activity. Cells were transfected as in A and C. Error bars represent SDs of technical replicates.
Mentions: Nuclear/cytoplasmic fractionation data does not always reflect the subcellular localization in living cells because pools of proteins that are not tightly bound to nuclear or cytoplasmic structures and are relatively small may leak through nuclear pore complexes of permeabilized cells. We therefore assayed the effect of RanBP3 overexpression on active β-catenin in situ using the anti–active β-catenin antibody. In our hands, this antibody did not visualize endogenous dephosphorylated β-catenin in Wnt1-transfected HEK293 cells (unpublished data). We therefore tested two colon carcinoma cell lines (SW480 and DLD1) that have a constitutively activated β-catenin because of a mutation in APC (Rosin-Arbesfeld et al., 2003). In SW480, but not in DLD1, the anti–dephosphorylated β-catenin antibody recognizes a clear nuclear signal above background (Fig. 6, A and C). The presence of this signal correlates with the exceptionally high β-catenin activity as measured in luciferase assays (Fig. 6 D), i.e., ∼30-fold higher than in DLD1. Importantly, RanBP3 overexpression leads to a clear reduction of active β-catenin signal from the SW480 nuclei (Fig. 6 A) but has no influence on total β-catenin localization (Fig. 6 B). This indicates that, even in the extremely active SW480 cell line, only a very small proportion of total β-catenin is properly dephosphorylated and active, and that this is the pool RanBP3 acts on.

Bottom Line: beta-Catenin is the nuclear effector of the Wnt signaling cascade.Conversely, overexpression of RanBP3 leads to a shift of active beta-catenin toward the cytoplasm.We conclude that RanBP3 is a direct export enhancer for beta-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
beta-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of beta-catenin is regulated is not well defined. Therefore, we used the nuclear marker RanGTP to screen for novel nuclear beta-catenin binding proteins. We identified a cofactor of chromosome region maintenance 1 (CRM1)-mediated nuclear export, Ran binding protein 3 (RanBP3), as a novel beta-catenin-interacting protein that binds directly to beta-catenin in a RanGTP-stimulated manner. RanBP3 inhibits beta-catenin-mediated transcriptional activation in both Wnt1- and beta-catenin-stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with beta-catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated beta-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active beta-catenin toward the cytoplasm. Modulation of beta-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. We conclude that RanBP3 is a direct export enhancer for beta-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

Show MeSH
Related in: MedlinePlus