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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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Expression of RanBP3 inhibits β-catenin/TCF–mediated transcriptional activation. (A) Wt and wv mutant RanBP3 are expressed at equal levels. HEK293 cells were transfected with the indicated constructs (ng), and lysates were analyzed 48 h after transfection by Western blot with the indicated antibodies. (B) RanBP3 represses Wnt1-induced β-catenin/TCF–mediated transcriptional activation dose dependently. HEK293 cells were transfected with TOP (black bars) or the control FOP (gray bars), Wnt1, and decreasing amounts of RanBP3 wt or wv mutant as indicated (ng), and luciferase activity was measured after 48 h. (C and D) RanBP3 represses transcriptional activation induced by wt β-catenin (C) or ΔGSK3–β-catenin (D). HEK293 cells were transfected with the indicated constructs, and luciferase activity was measured 48 h after transfection. In all experiments, normalized relative luciferase values are shown as corrected with pRL-CMV Renilla. Bars represent SEMs of independent experiments. (E) RanBP3 inhibits the expression of the endogenous Wnt target c-Myc. HCT116 colon carcinoma cells expressing Δ45–β-catenin were transfected with GFP and β-galactosidase, RanBP3 wt, or mutant plasmids. 2 d after transfection, GFP-positive cells were sorted using flow cytometry, lysed in sample buffer, and analyzed by Western blot using the indicated antibodies.
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fig2: Expression of RanBP3 inhibits β-catenin/TCF–mediated transcriptional activation. (A) Wt and wv mutant RanBP3 are expressed at equal levels. HEK293 cells were transfected with the indicated constructs (ng), and lysates were analyzed 48 h after transfection by Western blot with the indicated antibodies. (B) RanBP3 represses Wnt1-induced β-catenin/TCF–mediated transcriptional activation dose dependently. HEK293 cells were transfected with TOP (black bars) or the control FOP (gray bars), Wnt1, and decreasing amounts of RanBP3 wt or wv mutant as indicated (ng), and luciferase activity was measured after 48 h. (C and D) RanBP3 represses transcriptional activation induced by wt β-catenin (C) or ΔGSK3–β-catenin (D). HEK293 cells were transfected with the indicated constructs, and luciferase activity was measured 48 h after transfection. In all experiments, normalized relative luciferase values are shown as corrected with pRL-CMV Renilla. Bars represent SEMs of independent experiments. (E) RanBP3 inhibits the expression of the endogenous Wnt target c-Myc. HCT116 colon carcinoma cells expressing Δ45–β-catenin were transfected with GFP and β-galactosidase, RanBP3 wt, or mutant plasmids. 2 d after transfection, GFP-positive cells were sorted using flow cytometry, lysed in sample buffer, and analyzed by Western blot using the indicated antibodies.

Mentions: Wnt signaling ultimately results in the stabilization of β-catenin, which forms active transcriptional regulation complexes with transcription factors of the TCF/LEF family. A well-established functional readout of Wnt signaling makes use of TCF-responsive luciferase reporter constructs (Korinek et al., 1997). To test the functional relevance of the interaction between β-catenin and RanBP3, we transfected human embryonic kidney (HEK) 293 cells with reporter constructs that contain either three optimal TCF binding sites (TCF optimal promoter [TOP]) or three mutated binding sites (fake optimal promoter [FOP]). Transfection of a Wnt1 plasmid resulted in a strong activation of the TOP reporter but not of the FOP control (Fig. 2 B). Cotransfection of increasing amounts of RanBP3 repressed Wnt1/β-catenin transactivation dose dependently (Fig. 2 B). A mutant of RanBP3 that cannot interact with RanGTP and binds β-catenin with less affinity (Fig. 1 C) was less active than wild-type (wt) RanBP3 (Fig. 2 B). To investigate whether RanBP3 inhibits Wnt signaling downstream or upstream of β-catenin, we mimicked Wnt signaling in HEK293 cells by expressing β-catenin. RanBP3 could still specifically inhibit activation of the TOP reporter (Fig. 2 C), whereas the RanBP3 wv mutant was less effective. These experiments show that RanBP3 inhibits TCF-dependent transcription by acting on either β-catenin itself or regulators downstream of β-catenin. We confirmed that the expression levels of our wt and wv mutant RanBP3 constructs were equal by analyzing cell lysates from transfected HEK293 cells on Western blot (Fig. 2 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)

Expression of RanBP3 inhibits β-catenin/TCF–mediated transcriptional activation. (A) Wt and wv mutant RanBP3 are expressed at equal levels. HEK293 cells were transfected with the indicated constructs (ng), and lysates were analyzed 48 h after transfection by Western blot with the indicated antibodies. (B) RanBP3 represses Wnt1-induced β-catenin/TCF–mediated transcriptional activation dose dependently. HEK293 cells were transfected with TOP (black bars) or the control FOP (gray bars), Wnt1, and decreasing amounts of RanBP3 wt or wv mutant as indicated (ng), and luciferase activity was measured after 48 h. (C and D) RanBP3 represses transcriptional activation induced by wt β-catenin (C) or ΔGSK3–β-catenin (D). HEK293 cells were transfected with the indicated constructs, and luciferase activity was measured 48 h after transfection. In all experiments, normalized relative luciferase values are shown as corrected with pRL-CMV Renilla. Bars represent SEMs of independent experiments. (E) RanBP3 inhibits the expression of the endogenous Wnt target c-Myc. HCT116 colon carcinoma cells expressing Δ45–β-catenin were transfected with GFP and β-galactosidase, RanBP3 wt, or mutant plasmids. 2 d after transfection, GFP-positive cells were sorted using flow cytometry, lysed in sample buffer, and analyzed by Western blot using the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171279&req=5

fig2: Expression of RanBP3 inhibits β-catenin/TCF–mediated transcriptional activation. (A) Wt and wv mutant RanBP3 are expressed at equal levels. HEK293 cells were transfected with the indicated constructs (ng), and lysates were analyzed 48 h after transfection by Western blot with the indicated antibodies. (B) RanBP3 represses Wnt1-induced β-catenin/TCF–mediated transcriptional activation dose dependently. HEK293 cells were transfected with TOP (black bars) or the control FOP (gray bars), Wnt1, and decreasing amounts of RanBP3 wt or wv mutant as indicated (ng), and luciferase activity was measured after 48 h. (C and D) RanBP3 represses transcriptional activation induced by wt β-catenin (C) or ΔGSK3–β-catenin (D). HEK293 cells were transfected with the indicated constructs, and luciferase activity was measured 48 h after transfection. In all experiments, normalized relative luciferase values are shown as corrected with pRL-CMV Renilla. Bars represent SEMs of independent experiments. (E) RanBP3 inhibits the expression of the endogenous Wnt target c-Myc. HCT116 colon carcinoma cells expressing Δ45–β-catenin were transfected with GFP and β-galactosidase, RanBP3 wt, or mutant plasmids. 2 d after transfection, GFP-positive cells were sorted using flow cytometry, lysed in sample buffer, and analyzed by Western blot using the indicated antibodies.
Mentions: Wnt signaling ultimately results in the stabilization of β-catenin, which forms active transcriptional regulation complexes with transcription factors of the TCF/LEF family. A well-established functional readout of Wnt signaling makes use of TCF-responsive luciferase reporter constructs (Korinek et al., 1997). To test the functional relevance of the interaction between β-catenin and RanBP3, we transfected human embryonic kidney (HEK) 293 cells with reporter constructs that contain either three optimal TCF binding sites (TCF optimal promoter [TOP]) or three mutated binding sites (fake optimal promoter [FOP]). Transfection of a Wnt1 plasmid resulted in a strong activation of the TOP reporter but not of the FOP control (Fig. 2 B). Cotransfection of increasing amounts of RanBP3 repressed Wnt1/β-catenin transactivation dose dependently (Fig. 2 B). A mutant of RanBP3 that cannot interact with RanGTP and binds β-catenin with less affinity (Fig. 1 C) was less active than wild-type (wt) RanBP3 (Fig. 2 B). To investigate whether RanBP3 inhibits Wnt signaling downstream or upstream of β-catenin, we mimicked Wnt signaling in HEK293 cells by expressing β-catenin. RanBP3 could still specifically inhibit activation of the TOP reporter (Fig. 2 C), whereas the RanBP3 wv mutant was less effective. These experiments show that RanBP3 inhibits TCF-dependent transcription by acting on either β-catenin itself or regulators downstream of β-catenin. We confirmed that the expression levels of our wt and wv mutant RanBP3 constructs were equal by analyzing cell lysates from transfected HEK293 cells on Western blot (Fig. 2 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