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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 antagonizes Wnt/β-catenin transactivation in APC-mutated colon carcinoma cells. Luciferase assay showing that RanBP3 inhibits β-catenin–mediated transactivation in colon carcinoma cell lines DLD1 and COLO320. (A) APC type I truncated human colon carcinoma cell line DLD1 (APC 1–1417) was transfected with luciferase reporter constructs and increasing amounts of RanBP3 expression constructs as indicated. DLD1 cells express a truncated APC protein that lacks all its COOH-terminal NESs. (B) Luciferase reporter assay as in A, performed in the APC type I truncated human colon carcinoma cell line COLO320 (APC 1–811). These cells express a short APC protein that lacks all β-catenin binding and regulatory sites. Relative luciferase activity was measured 48 h after transfection. Error bars show SDs of a representative experiment.
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fig4: RanBP3 antagonizes Wnt/β-catenin transactivation in APC-mutated colon carcinoma cells. Luciferase assay showing that RanBP3 inhibits β-catenin–mediated transactivation in colon carcinoma cell lines DLD1 and COLO320. (A) APC type I truncated human colon carcinoma cell line DLD1 (APC 1–1417) was transfected with luciferase reporter constructs and increasing amounts of RanBP3 expression constructs as indicated. DLD1 cells express a truncated APC protein that lacks all its COOH-terminal NESs. (B) Luciferase reporter assay as in A, performed in the APC type I truncated human colon carcinoma cell line COLO320 (APC 1–811). These cells express a short APC protein that lacks all β-catenin binding and regulatory sites. Relative luciferase activity was measured 48 h after transfection. Error bars show SDs of a representative experiment.

Mentions: To further address the question of whether RanBP3 represses β-catenin transcriptional activation by stimulating export of β-catenin via the APC–CRM1 pathway, we expressed RanBP3 in human colorectal cancer cell lines that express COOH-terminal truncations of APC. First, we tested DLD1 cells, which express APC1–1417, which retains some β-catenin binding sites but lacks all COOH-terminal nuclear export signals (NESs), the most highly conserved APC NESs in evolution. As shown in Fig. 4 A, β-catenin/TCF activity is already high in these cells. Expression of a RanBP3 wt or wv mutant could still dose-dependently down-regulate transcriptional activity, with the mutant again being a less potent inhibitor (Fig. 4 A). Because APC in DLD1 cells can still bind to β-catenin and NESs have also been reported in the NH2 terminus of APC, we repeated the experiment in COLO320 cells. These cells express a very short APC truncation (1–811) that lacks all β-catenin binding sites. β-catenin/TCF activity was much higher in these cells than in DLD1 cells, a finding that correlates with the severity of the APC mutation (Fig. 4 B; Rosin-Arbesfeld et al., 2003). Nevertheless, transfection of the RanBP3 expression constructs caused a significant down-regulation of transcription (Fig. 4 B). Therefore, the mechanism by which RanBP3 inhibits β-catenin is independent of a nuclear export function of APC.


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 antagonizes Wnt/β-catenin transactivation in APC-mutated colon carcinoma cells. Luciferase assay showing that RanBP3 inhibits β-catenin–mediated transactivation in colon carcinoma cell lines DLD1 and COLO320. (A) APC type I truncated human colon carcinoma cell line DLD1 (APC 1–1417) was transfected with luciferase reporter constructs and increasing amounts of RanBP3 expression constructs as indicated. DLD1 cells express a truncated APC protein that lacks all its COOH-terminal NESs. (B) Luciferase reporter assay as in A, performed in the APC type I truncated human colon carcinoma cell line COLO320 (APC 1–811). These cells express a short APC protein that lacks all β-catenin binding and regulatory sites. Relative luciferase activity was measured 48 h after transfection. Error bars show SDs of a representative experiment.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: RanBP3 antagonizes Wnt/β-catenin transactivation in APC-mutated colon carcinoma cells. Luciferase assay showing that RanBP3 inhibits β-catenin–mediated transactivation in colon carcinoma cell lines DLD1 and COLO320. (A) APC type I truncated human colon carcinoma cell line DLD1 (APC 1–1417) was transfected with luciferase reporter constructs and increasing amounts of RanBP3 expression constructs as indicated. DLD1 cells express a truncated APC protein that lacks all its COOH-terminal NESs. (B) Luciferase reporter assay as in A, performed in the APC type I truncated human colon carcinoma cell line COLO320 (APC 1–811). These cells express a short APC protein that lacks all β-catenin binding and regulatory sites. Relative luciferase activity was measured 48 h after transfection. Error bars show SDs of a representative experiment.
Mentions: To further address the question of whether RanBP3 represses β-catenin transcriptional activation by stimulating export of β-catenin via the APC–CRM1 pathway, we expressed RanBP3 in human colorectal cancer cell lines that express COOH-terminal truncations of APC. First, we tested DLD1 cells, which express APC1–1417, which retains some β-catenin binding sites but lacks all COOH-terminal nuclear export signals (NESs), the most highly conserved APC NESs in evolution. As shown in Fig. 4 A, β-catenin/TCF activity is already high in these cells. Expression of a RanBP3 wt or wv mutant could still dose-dependently down-regulate transcriptional activity, with the mutant again being a less potent inhibitor (Fig. 4 A). Because APC in DLD1 cells can still bind to β-catenin and NESs have also been reported in the NH2 terminus of APC, we repeated the experiment in COLO320 cells. These cells express a very short APC truncation (1–811) that lacks all β-catenin binding sites. β-catenin/TCF activity was much higher in these cells than in DLD1 cells, a finding that correlates with the severity of the APC mutation (Fig. 4 B; Rosin-Arbesfeld et al., 2003). Nevertheless, transfection of the RanBP3 expression constructs caused a significant down-regulation of transcription (Fig. 4 B). Therefore, the mechanism by which RanBP3 inhibits β-catenin is independent of a nuclear export function of APC.

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