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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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Reduction of RanBP3 by RNAi results in increased β-catenin/TCF–mediated transcription activation. (A) Western blot showing that different shRNAs against RanBP3 reduce RanBP3 protein levels in HEK293 cells. Cells were transfected with shRNAs, and pHA262-PUR was cotransfected to introduce puromycin resistance. 24 h after transfection, cells were grown on puromycin medium for 48 h and cell lysates were prepared and analyzed on Western blot with the indicated antibodies. (B) RNAi against RanBP3 increases Wnt1-induced β-catenin/TCF–mediated transcription. HEK293 cells were transfected with the indicated constructs, and activity of TOP (black bars) and FOP (gray bars) was measured 72 h after transfection. Error bars represent SDs of technical replicates of a representative experiment. (C) RNAi against RanBP3 increases β-catenin/TCF–driven transcription in HEK293 cells that transiently express an active form of β-catenin (ΔGSK3–β-catenin). Cells were transfected with indicated constructs, and luciferase activity was measured after 72 h. (D) Coexpression of CRM1 with RanBP3 shRNA constructs does not affect β-catenin/TCF–mediated transcription in Wnt1-transfected cells. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection luciferase activity was measured. For all experiments, relative luciferase levels are shown as corrected with CMV-Renilla-luc. Error bars in C and D represent SEMs of independent experiments.
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fig3: Reduction of RanBP3 by RNAi results in increased β-catenin/TCF–mediated transcription activation. (A) Western blot showing that different shRNAs against RanBP3 reduce RanBP3 protein levels in HEK293 cells. Cells were transfected with shRNAs, and pHA262-PUR was cotransfected to introduce puromycin resistance. 24 h after transfection, cells were grown on puromycin medium for 48 h and cell lysates were prepared and analyzed on Western blot with the indicated antibodies. (B) RNAi against RanBP3 increases Wnt1-induced β-catenin/TCF–mediated transcription. HEK293 cells were transfected with the indicated constructs, and activity of TOP (black bars) and FOP (gray bars) was measured 72 h after transfection. Error bars represent SDs of technical replicates of a representative experiment. (C) RNAi against RanBP3 increases β-catenin/TCF–driven transcription in HEK293 cells that transiently express an active form of β-catenin (ΔGSK3–β-catenin). Cells were transfected with indicated constructs, and luciferase activity was measured after 72 h. (D) Coexpression of CRM1 with RanBP3 shRNA constructs does not affect β-catenin/TCF–mediated transcription in Wnt1-transfected cells. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection luciferase activity was measured. For all experiments, relative luciferase levels are shown as corrected with CMV-Renilla-luc. Error bars in C and D represent SEMs of independent experiments.

Mentions: In addition to studying the effects of RanBP3 overexpression, we studied the effects of RanBP3 depletion. We expressed short hairpin RNAs (shRNAs) directed against unique parts of RanBP3 that are present in all isoforms of RanBP3. We obtained several shRNA RanBP3 constructs that down-regulate RanBP3 protein levels in HEK293 cells (Fig. 3 A).


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)

Reduction of RanBP3 by RNAi results in increased β-catenin/TCF–mediated transcription activation. (A) Western blot showing that different shRNAs against RanBP3 reduce RanBP3 protein levels in HEK293 cells. Cells were transfected with shRNAs, and pHA262-PUR was cotransfected to introduce puromycin resistance. 24 h after transfection, cells were grown on puromycin medium for 48 h and cell lysates were prepared and analyzed on Western blot with the indicated antibodies. (B) RNAi against RanBP3 increases Wnt1-induced β-catenin/TCF–mediated transcription. HEK293 cells were transfected with the indicated constructs, and activity of TOP (black bars) and FOP (gray bars) was measured 72 h after transfection. Error bars represent SDs of technical replicates of a representative experiment. (C) RNAi against RanBP3 increases β-catenin/TCF–driven transcription in HEK293 cells that transiently express an active form of β-catenin (ΔGSK3–β-catenin). Cells were transfected with indicated constructs, and luciferase activity was measured after 72 h. (D) Coexpression of CRM1 with RanBP3 shRNA constructs does not affect β-catenin/TCF–mediated transcription in Wnt1-transfected cells. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection luciferase activity was measured. For all experiments, relative luciferase levels are shown as corrected with CMV-Renilla-luc. Error bars in C and D represent SEMs of independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Reduction of RanBP3 by RNAi results in increased β-catenin/TCF–mediated transcription activation. (A) Western blot showing that different shRNAs against RanBP3 reduce RanBP3 protein levels in HEK293 cells. Cells were transfected with shRNAs, and pHA262-PUR was cotransfected to introduce puromycin resistance. 24 h after transfection, cells were grown on puromycin medium for 48 h and cell lysates were prepared and analyzed on Western blot with the indicated antibodies. (B) RNAi against RanBP3 increases Wnt1-induced β-catenin/TCF–mediated transcription. HEK293 cells were transfected with the indicated constructs, and activity of TOP (black bars) and FOP (gray bars) was measured 72 h after transfection. Error bars represent SDs of technical replicates of a representative experiment. (C) RNAi against RanBP3 increases β-catenin/TCF–driven transcription in HEK293 cells that transiently express an active form of β-catenin (ΔGSK3–β-catenin). Cells were transfected with indicated constructs, and luciferase activity was measured after 72 h. (D) Coexpression of CRM1 with RanBP3 shRNA constructs does not affect β-catenin/TCF–mediated transcription in Wnt1-transfected cells. HEK293 cells were transfected with the indicated constructs, and 72 h after transfection luciferase activity was measured. For all experiments, relative luciferase levels are shown as corrected with CMV-Renilla-luc. Error bars in C and D represent SEMs of independent experiments.
Mentions: In addition to studying the effects of RanBP3 overexpression, we studied the effects of RanBP3 depletion. We expressed short hairpin RNAs (shRNAs) directed against unique parts of RanBP3 that are present in all isoforms of RanBP3. We obtained several shRNA RanBP3 constructs that down-regulate RanBP3 protein levels in HEK293 cells (Fig. 3 A).

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