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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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Related in: MedlinePlus

Depletion of RanBP3 results in nuclear accumulation of active β-catenin. (A) Depletion of RanBP3 does not alter the levels of both total and active dephosphorylated β-catenin. HEK293 cells were transfected with or without Wnt1 and shRNA constructs against GFP or RanBP3. 72 h after transfection, whole cell lysates were analyzed by Western blot with the indicated antibodies. (B) RNAi against RanBP3 results in increased levels of active β-catenin in the nucleus. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection, nuclear and cytoplasmic extracts were prepared and analyzed by Western blot. TCF4 and tubulin staining are shown as markers for purity of the nuclear and cytoplasmic fractions. As a loading control in the nuclear fractions, TCF4 and a nonspecific reaction of the antibody recognizing active β-catenin are shown.
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fig5: Depletion of RanBP3 results in nuclear accumulation of active β-catenin. (A) Depletion of RanBP3 does not alter the levels of both total and active dephosphorylated β-catenin. HEK293 cells were transfected with or without Wnt1 and shRNA constructs against GFP or RanBP3. 72 h after transfection, whole cell lysates were analyzed by Western blot with the indicated antibodies. (B) RNAi against RanBP3 results in increased levels of active β-catenin in the nucleus. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection, nuclear and cytoplasmic extracts were prepared and analyzed by Western blot. TCF4 and tubulin staining are shown as markers for purity of the nuclear and cytoplasmic fractions. As a loading control in the nuclear fractions, TCF4 and a nonspecific reaction of the antibody recognizing active β-catenin are shown.

Mentions: To study the mechanism by which RanBP3 inhibits Wnt signaling, we tested the possibility that RanBP3 influences the stability of β-catenin. We transfected HEK293 cells with or without Wnt1 in combination with shRNA constructs. Total β-catenin levels were virtually unchanged after expression of Wnt1 alone or in combination with shRNA against RanBP3 (Fig. 5 A). When the same blot was probed with anti–active β-catenin, recognizing NH2-terminally dephosphorylated β-catenin, we observed an increase in Wnt1-transfected cells but no effects on RanBP3 (Fig. 5 A). From this data, we conclude that RanBP3 depletion does not affect β-catenin degradation.


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)

Depletion of RanBP3 results in nuclear accumulation of active β-catenin. (A) Depletion of RanBP3 does not alter the levels of both total and active dephosphorylated β-catenin. HEK293 cells were transfected with or without Wnt1 and shRNA constructs against GFP or RanBP3. 72 h after transfection, whole cell lysates were analyzed by Western blot with the indicated antibodies. (B) RNAi against RanBP3 results in increased levels of active β-catenin in the nucleus. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection, nuclear and cytoplasmic extracts were prepared and analyzed by Western blot. TCF4 and tubulin staining are shown as markers for purity of the nuclear and cytoplasmic fractions. As a loading control in the nuclear fractions, TCF4 and a nonspecific reaction of the antibody recognizing active β-catenin are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Depletion of RanBP3 results in nuclear accumulation of active β-catenin. (A) Depletion of RanBP3 does not alter the levels of both total and active dephosphorylated β-catenin. HEK293 cells were transfected with or without Wnt1 and shRNA constructs against GFP or RanBP3. 72 h after transfection, whole cell lysates were analyzed by Western blot with the indicated antibodies. (B) RNAi against RanBP3 results in increased levels of active β-catenin in the nucleus. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection, nuclear and cytoplasmic extracts were prepared and analyzed by Western blot. TCF4 and tubulin staining are shown as markers for purity of the nuclear and cytoplasmic fractions. As a loading control in the nuclear fractions, TCF4 and a nonspecific reaction of the antibody recognizing active β-catenin are shown.
Mentions: To study the mechanism by which RanBP3 inhibits Wnt signaling, we tested the possibility that RanBP3 influences the stability of β-catenin. We transfected HEK293 cells with or without Wnt1 in combination with shRNA constructs. Total β-catenin levels were virtually unchanged after expression of Wnt1 alone or in combination with shRNA against RanBP3 (Fig. 5 A). When the same blot was probed with anti–active β-catenin, recognizing NH2-terminally dephosphorylated β-catenin, we observed an increase in Wnt1-transfected cells but no effects on RanBP3 (Fig. 5 A). From this data, we conclude that RanBP3 depletion does not affect β-catenin degradation.

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