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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 enhances nuclear export of active β-catenin independently of CRM1. (A and C) Effect of RanBP3 on mRFP–ΔGSK–β-catenin nucleocytoplasmic distribution in HEK293 cells in the presence or absence of 50 nM LMB for 3 h. (A) Box plot showing the distribution of nuclear/cytoplasmic ratios of mRFP–ΔGSK–β-catenin of two independent experiments. P values are according to Mann-Whitney tests. Representative mRFP fluorescence images are shown in C. Highlighted nuclear borders are drawn on the basis of accompanying phase-contrast images. (B) Functionality of mRFP–ΔGSK3–β-catenin. NCI-H28 cells (lacking endogenous β-catenin) were transfected with indicated constructs, and 48 h after transfection, luciferase activity was measured. Relative luciferase levels as corrected for transfection efficiency (Renilla luciferase activity) are shown. Error bars represent SDs. (D) Representative fluorescence images of HEK293 cells expressing GFP-Rev(1.4)-NES in the presence or absence of 50 nM LMB for 3 h. (E and F) Endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3. HEK293 cells were transfected with Wnt and RanBP3 as indicated together with TOP-TK-luc and Renilla transcription reporter plasmids and fractionated after 48 h as in Fig. 5. Localization of active β-catenin was monitored using anti–active β-catenin antibody. Amounts of protein loaded were normalized on transfection efficiency (Renilla luciferase activity). Normalized β-catenin/TCF–dependent luciferase activity is depicted in F.
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fig7: RanBP3 enhances nuclear export of active β-catenin independently of CRM1. (A and C) Effect of RanBP3 on mRFP–ΔGSK–β-catenin nucleocytoplasmic distribution in HEK293 cells in the presence or absence of 50 nM LMB for 3 h. (A) Box plot showing the distribution of nuclear/cytoplasmic ratios of mRFP–ΔGSK–β-catenin of two independent experiments. P values are according to Mann-Whitney tests. Representative mRFP fluorescence images are shown in C. Highlighted nuclear borders are drawn on the basis of accompanying phase-contrast images. (B) Functionality of mRFP–ΔGSK3–β-catenin. NCI-H28 cells (lacking endogenous β-catenin) were transfected with indicated constructs, and 48 h after transfection, luciferase activity was measured. Relative luciferase levels as corrected for transfection efficiency (Renilla luciferase activity) are shown. Error bars represent SDs. (D) Representative fluorescence images of HEK293 cells expressing GFP-Rev(1.4)-NES in the presence or absence of 50 nM LMB for 3 h. (E and F) Endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3. HEK293 cells were transfected with Wnt and RanBP3 as indicated together with TOP-TK-luc and Renilla transcription reporter plasmids and fractionated after 48 h as in Fig. 5. Localization of active β-catenin was monitored using anti–active β-catenin antibody. Amounts of protein loaded were normalized on transfection efficiency (Renilla luciferase activity). Normalized β-catenin/TCF–dependent luciferase activity is depicted in F.

Mentions: Reduction of active nuclear β-catenin by RanBP3 in SW480 cells was not accompanied by an increase in cytoplasmic signal, raising the question of whether RanBP3 induces enhanced nuclear export of active β-catenin or its increased phosphorylation. However, enhanced nuclear export would result in dilution in a cytoplasmic volume that is ∼10-fold larger than that of the nucleus, precluding detection by the anti–dephosphorylated β-catenin antibody. To discriminate between the two possibilities, we mimicked the active state of β-catenin using a monomeric red fluorescent protein (mRFP)–tagged, constitutively active form of β-catenin, the previously used β-cateninΔGSK3β. To determine whether this fusion protein was biologically active, we performed a TCF reporter assay in the malignant mesothelioma cell line NCI-H28, which carries a homozygous deletion of the β-catenin gene (Calvo et al., 2000). This prevented possible activating effects of this mutant on endogenous β-catenin. mRFP–β-cateninΔGSK3β activated the very low endogenous TCF activity of these cells to a great extent (Fig. 7 B). We next compared the subcellular localization of this protein in the presence or absence of exogenous RanBP3 (Fig. 7 A). Care was taken to record cells of similarly low expression levels (Fig. 7 C). In control cells, more mRFP–β-cateninΔGSK3β was present in the nuclei than in the cytoplasm (median nuclear to cytoplasmic ratio of 1.38, n = 37). In contrast, cells expressing exogenous RanBP3 showed higher cytoplasmic than nuclear mRFP–β-cateninΔGSK3β levels (median nuclear to cytoplasmic ratio of 0.77, n = 41). Importantly, addition of 50 mM of the CRM1 inhibitor leptomycin B (LMB; Wolff et al., 1997) did not significantly change the effect of RanBP3 (median nuclear to cytoplasmic ratio of 0.80, n = 52), even though photobleaching experiments show that mRFP–β-cateninΔGSK3β rapidly shuttles between the nucleus and cytoplasm (unpublished data). Identical LMB treatment dramatically relocalized the NES-containing reporter protein Rev(1.4)-NES-GFP (Henderson and Eleftheriou, 2000) to the nucleus (Fig. 6 D). We conclude that RanBP3 enhances nuclear export of active β-catenin and that this export is independent of CRM1. To confirm that endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3 in HEK293 cells, we transfected these cells with Wnt1 and RanBP3. Indeed, we observed increased active β-catenin levels in both nuclear and cytoplasmic fractions, with the nuclear pool being more sensitive than the cytoplasmic pool to RanBP3 overexpression (Fig. 7 E). The decrease in cytoplasmic active β-catenin is consistent with increased nuclear export of β-catenin and subsequent degradation in the cytoplasm.


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 enhances nuclear export of active β-catenin independently of CRM1. (A and C) Effect of RanBP3 on mRFP–ΔGSK–β-catenin nucleocytoplasmic distribution in HEK293 cells in the presence or absence of 50 nM LMB for 3 h. (A) Box plot showing the distribution of nuclear/cytoplasmic ratios of mRFP–ΔGSK–β-catenin of two independent experiments. P values are according to Mann-Whitney tests. Representative mRFP fluorescence images are shown in C. Highlighted nuclear borders are drawn on the basis of accompanying phase-contrast images. (B) Functionality of mRFP–ΔGSK3–β-catenin. NCI-H28 cells (lacking endogenous β-catenin) were transfected with indicated constructs, and 48 h after transfection, luciferase activity was measured. Relative luciferase levels as corrected for transfection efficiency (Renilla luciferase activity) are shown. Error bars represent SDs. (D) Representative fluorescence images of HEK293 cells expressing GFP-Rev(1.4)-NES in the presence or absence of 50 nM LMB for 3 h. (E and F) Endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3. HEK293 cells were transfected with Wnt and RanBP3 as indicated together with TOP-TK-luc and Renilla transcription reporter plasmids and fractionated after 48 h as in Fig. 5. Localization of active β-catenin was monitored using anti–active β-catenin antibody. Amounts of protein loaded were normalized on transfection efficiency (Renilla luciferase activity). Normalized β-catenin/TCF–dependent luciferase activity is depicted in F.
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fig7: RanBP3 enhances nuclear export of active β-catenin independently of CRM1. (A and C) Effect of RanBP3 on mRFP–ΔGSK–β-catenin nucleocytoplasmic distribution in HEK293 cells in the presence or absence of 50 nM LMB for 3 h. (A) Box plot showing the distribution of nuclear/cytoplasmic ratios of mRFP–ΔGSK–β-catenin of two independent experiments. P values are according to Mann-Whitney tests. Representative mRFP fluorescence images are shown in C. Highlighted nuclear borders are drawn on the basis of accompanying phase-contrast images. (B) Functionality of mRFP–ΔGSK3–β-catenin. NCI-H28 cells (lacking endogenous β-catenin) were transfected with indicated constructs, and 48 h after transfection, luciferase activity was measured. Relative luciferase levels as corrected for transfection efficiency (Renilla luciferase activity) are shown. Error bars represent SDs. (D) Representative fluorescence images of HEK293 cells expressing GFP-Rev(1.4)-NES in the presence or absence of 50 nM LMB for 3 h. (E and F) Endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3. HEK293 cells were transfected with Wnt and RanBP3 as indicated together with TOP-TK-luc and Renilla transcription reporter plasmids and fractionated after 48 h as in Fig. 5. Localization of active β-catenin was monitored using anti–active β-catenin antibody. Amounts of protein loaded were normalized on transfection efficiency (Renilla luciferase activity). Normalized β-catenin/TCF–dependent luciferase activity is depicted in F.
Mentions: Reduction of active nuclear β-catenin by RanBP3 in SW480 cells was not accompanied by an increase in cytoplasmic signal, raising the question of whether RanBP3 induces enhanced nuclear export of active β-catenin or its increased phosphorylation. However, enhanced nuclear export would result in dilution in a cytoplasmic volume that is ∼10-fold larger than that of the nucleus, precluding detection by the anti–dephosphorylated β-catenin antibody. To discriminate between the two possibilities, we mimicked the active state of β-catenin using a monomeric red fluorescent protein (mRFP)–tagged, constitutively active form of β-catenin, the previously used β-cateninΔGSK3β. To determine whether this fusion protein was biologically active, we performed a TCF reporter assay in the malignant mesothelioma cell line NCI-H28, which carries a homozygous deletion of the β-catenin gene (Calvo et al., 2000). This prevented possible activating effects of this mutant on endogenous β-catenin. mRFP–β-cateninΔGSK3β activated the very low endogenous TCF activity of these cells to a great extent (Fig. 7 B). We next compared the subcellular localization of this protein in the presence or absence of exogenous RanBP3 (Fig. 7 A). Care was taken to record cells of similarly low expression levels (Fig. 7 C). In control cells, more mRFP–β-cateninΔGSK3β was present in the nuclei than in the cytoplasm (median nuclear to cytoplasmic ratio of 1.38, n = 37). In contrast, cells expressing exogenous RanBP3 showed higher cytoplasmic than nuclear mRFP–β-cateninΔGSK3β levels (median nuclear to cytoplasmic ratio of 0.77, n = 41). Importantly, addition of 50 mM of the CRM1 inhibitor leptomycin B (LMB; Wolff et al., 1997) did not significantly change the effect of RanBP3 (median nuclear to cytoplasmic ratio of 0.80, n = 52), even though photobleaching experiments show that mRFP–β-cateninΔGSK3β rapidly shuttles between the nucleus and cytoplasm (unpublished data). Identical LMB treatment dramatically relocalized the NES-containing reporter protein Rev(1.4)-NES-GFP (Henderson and Eleftheriou, 2000) to the nucleus (Fig. 6 D). We conclude that RanBP3 enhances nuclear export of active β-catenin and that this export is independent of CRM1. To confirm that endogenous activated β-catenin relocalizes from the nucleus to the cytoplasm upon overexpression of RanBP3 in HEK293 cells, we transfected these cells with Wnt1 and RanBP3. Indeed, we observed increased active β-catenin levels in both nuclear and cytoplasmic fractions, with the nuclear pool being more sensitive than the cytoplasmic pool to RanBP3 overexpression (Fig. 7 E). The decrease in cytoplasmic active β-catenin is consistent with increased nuclear export of β-catenin and subsequent degradation in the cytoplasm.

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