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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 rescues β-catenin–induced double axis formation in X. laevis embryos. (A) X. laevis embryos were injected ventrally at the four-cell stage with β-catenin mRNA in the presence or absence of control β-galactosidase or X. laevis RanBP3-b mRNA. (top) Wt noninjected embryos. (middle) Double axis phenotype as induced by the injection of β-catenin mRNA. (bottom) Embryos that are rescued from the double axis phenotype by coexpression of RanBP3 and β-catenin mRNA. (B) Quantification of the different phenotypes of two independent experiments in four categories: complete secondary axis (with cement gland), partial secondary axis (i.e., any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented line), and normal (only one axis). P values are according to Pearson's χ2 test for count data. (C) Dorsal injection of RanBP3 results in ventralization of X. laevis embryos. Four-cell stage embryos were injected dorsally with RanBP3 or control (β-galactosidase) mRNA and analyzed 3 d later for ventralization using the standardized DAI. This scale runs from 0 (complete ventralization) to 5 (normal development). Frequencies are derived from three independent experiments. P values as in B. (D) The β-catenin downstream target siamois is significantly down-regulated in RanBP3-injected embryos. Embryos were injected as in C and analyzed for siamois or ornithine decarboxylase (ODC) mRNA using RT-PCR. Amplified ethidium bromide–stained DNA of four experiments was quantified and normalized to mean signals from β-galactosidase–injected embryos and represented in a box plot. P values are according to Mann-Whitney tests. (E) Representative signals from RT-PCR reactions visualized by ethidium bromide staining.
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fig8: RanBP3 rescues β-catenin–induced double axis formation in X. laevis embryos. (A) X. laevis embryos were injected ventrally at the four-cell stage with β-catenin mRNA in the presence or absence of control β-galactosidase or X. laevis RanBP3-b mRNA. (top) Wt noninjected embryos. (middle) Double axis phenotype as induced by the injection of β-catenin mRNA. (bottom) Embryos that are rescued from the double axis phenotype by coexpression of RanBP3 and β-catenin mRNA. (B) Quantification of the different phenotypes of two independent experiments in four categories: complete secondary axis (with cement gland), partial secondary axis (i.e., any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented line), and normal (only one axis). P values are according to Pearson's χ2 test for count data. (C) Dorsal injection of RanBP3 results in ventralization of X. laevis embryos. Four-cell stage embryos were injected dorsally with RanBP3 or control (β-galactosidase) mRNA and analyzed 3 d later for ventralization using the standardized DAI. This scale runs from 0 (complete ventralization) to 5 (normal development). Frequencies are derived from three independent experiments. P values as in B. (D) The β-catenin downstream target siamois is significantly down-regulated in RanBP3-injected embryos. Embryos were injected as in C and analyzed for siamois or ornithine decarboxylase (ODC) mRNA using RT-PCR. Amplified ethidium bromide–stained DNA of four experiments was quantified and normalized to mean signals from β-galactosidase–injected embryos and represented in a box plot. P values are according to Mann-Whitney tests. (E) Representative signals from RT-PCR reactions visualized by ethidium bromide staining.

Mentions: To study the role of RanBP3 in Wnt signaling in a physiological context, we used an X. laevis axis duplication assay. During X. laevis embryonic development, Wnt signaling determines patterning along the dorsal-ventral axis. Ectopic ventral injection of β-catenin mRNA in four-cell embryos resulted in clear axis duplication (Fig. 8, A and B). The majority (75%) of the embryos showed a complete duplication of the dorsal-ventral axis. 22% of the embryos showed a partial duplication, i.e., a secondary axis without duplicated cement gland. However, coinjection of β-catenin mRNA with RanBP3 mRNA resulted in a strong suppression of the double axis phenotype in the majority (63%) of the embryos. Few partial or very partial secondary axis phenotypes (24 and 13%, respectively) were observed in these embryos (Fig. 6 B). We also coinjected β-catenin mRNA with mRNA of the RanBP3 wv mutant that is defective in RanGTP binding. This mutant suppressed the double axis phenotype but was not as potent of an inhibitor as the wt RanBP3 (Fig. 8, A and B; P = 4e-8). This data correlates with our findings that this RanBP3 mutant binds β-catenin with less affinity (Fig. 1) and that it is less active in repressing the transcriptional activity of a TCF reporter gene in human cell lines (Figs. 2 and 4). If RanBP3 is an inhibitor of nuclear β-catenin function, dorsal injection of RanBP3 mRNA is expected to result in ventralization of the embryo. We therefore injected four-cell embryos dorsally with either RanBP3 or control mRNA and scored ventralization after 3 d of development using the dorsoanterior index (DAI; Kao and Elinson, 1988). Mild to severe ventralization was observed (DAI 1–4) in 80% of RanBP3-injected embryos (Fig. 8 C), whereas <10% of control-injected embryos showed these phenotypes. Complete ventralization (DAI 0) was not observed. An important direct downstream target of dorsal nuclear β-catenin activity is the early Wnt-inducible homeobox gene Siamois (Brannon et al., 1997). We therefore tested to determine whether expression levels of this gene were reduced in the RanBP3-injected embryos by RT-PCR. In four independent experiments, we detected an approximately twofold decrease in Siamois levels in late stage 9 embryos (Fig. 8, D and E). This decrease is rather mild, consistent with the incomplete ventralization phenotypes observed. Based on these findings, we conclude that RanBP3 not only is a repressor of Wnt signaling in human cell lines but also functions as an antagonist of Wnt signaling in X. laevis embryos.


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 rescues β-catenin–induced double axis formation in X. laevis embryos. (A) X. laevis embryos were injected ventrally at the four-cell stage with β-catenin mRNA in the presence or absence of control β-galactosidase or X. laevis RanBP3-b mRNA. (top) Wt noninjected embryos. (middle) Double axis phenotype as induced by the injection of β-catenin mRNA. (bottom) Embryos that are rescued from the double axis phenotype by coexpression of RanBP3 and β-catenin mRNA. (B) Quantification of the different phenotypes of two independent experiments in four categories: complete secondary axis (with cement gland), partial secondary axis (i.e., any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented line), and normal (only one axis). P values are according to Pearson's χ2 test for count data. (C) Dorsal injection of RanBP3 results in ventralization of X. laevis embryos. Four-cell stage embryos were injected dorsally with RanBP3 or control (β-galactosidase) mRNA and analyzed 3 d later for ventralization using the standardized DAI. This scale runs from 0 (complete ventralization) to 5 (normal development). Frequencies are derived from three independent experiments. P values as in B. (D) The β-catenin downstream target siamois is significantly down-regulated in RanBP3-injected embryos. Embryos were injected as in C and analyzed for siamois or ornithine decarboxylase (ODC) mRNA using RT-PCR. Amplified ethidium bromide–stained DNA of four experiments was quantified and normalized to mean signals from β-galactosidase–injected embryos and represented in a box plot. P values are according to Mann-Whitney tests. (E) Representative signals from RT-PCR reactions visualized by ethidium bromide staining.
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fig8: RanBP3 rescues β-catenin–induced double axis formation in X. laevis embryos. (A) X. laevis embryos were injected ventrally at the four-cell stage with β-catenin mRNA in the presence or absence of control β-galactosidase or X. laevis RanBP3-b mRNA. (top) Wt noninjected embryos. (middle) Double axis phenotype as induced by the injection of β-catenin mRNA. (bottom) Embryos that are rescued from the double axis phenotype by coexpression of RanBP3 and β-catenin mRNA. (B) Quantification of the different phenotypes of two independent experiments in four categories: complete secondary axis (with cement gland), partial secondary axis (i.e., any secondary axis lacking the cement gland), vestigial axis (very small posterior protrusion or pigmented line), and normal (only one axis). P values are according to Pearson's χ2 test for count data. (C) Dorsal injection of RanBP3 results in ventralization of X. laevis embryos. Four-cell stage embryos were injected dorsally with RanBP3 or control (β-galactosidase) mRNA and analyzed 3 d later for ventralization using the standardized DAI. This scale runs from 0 (complete ventralization) to 5 (normal development). Frequencies are derived from three independent experiments. P values as in B. (D) The β-catenin downstream target siamois is significantly down-regulated in RanBP3-injected embryos. Embryos were injected as in C and analyzed for siamois or ornithine decarboxylase (ODC) mRNA using RT-PCR. Amplified ethidium bromide–stained DNA of four experiments was quantified and normalized to mean signals from β-galactosidase–injected embryos and represented in a box plot. P values are according to Mann-Whitney tests. (E) Representative signals from RT-PCR reactions visualized by ethidium bromide staining.
Mentions: To study the role of RanBP3 in Wnt signaling in a physiological context, we used an X. laevis axis duplication assay. During X. laevis embryonic development, Wnt signaling determines patterning along the dorsal-ventral axis. Ectopic ventral injection of β-catenin mRNA in four-cell embryos resulted in clear axis duplication (Fig. 8, A and B). The majority (75%) of the embryos showed a complete duplication of the dorsal-ventral axis. 22% of the embryos showed a partial duplication, i.e., a secondary axis without duplicated cement gland. However, coinjection of β-catenin mRNA with RanBP3 mRNA resulted in a strong suppression of the double axis phenotype in the majority (63%) of the embryos. Few partial or very partial secondary axis phenotypes (24 and 13%, respectively) were observed in these embryos (Fig. 6 B). We also coinjected β-catenin mRNA with mRNA of the RanBP3 wv mutant that is defective in RanGTP binding. This mutant suppressed the double axis phenotype but was not as potent of an inhibitor as the wt RanBP3 (Fig. 8, A and B; P = 4e-8). This data correlates with our findings that this RanBP3 mutant binds β-catenin with less affinity (Fig. 1) and that it is less active in repressing the transcriptional activity of a TCF reporter gene in human cell lines (Figs. 2 and 4). If RanBP3 is an inhibitor of nuclear β-catenin function, dorsal injection of RanBP3 mRNA is expected to result in ventralization of the embryo. We therefore injected four-cell embryos dorsally with either RanBP3 or control mRNA and scored ventralization after 3 d of development using the dorsoanterior index (DAI; Kao and Elinson, 1988). Mild to severe ventralization was observed (DAI 1–4) in 80% of RanBP3-injected embryos (Fig. 8 C), whereas <10% of control-injected embryos showed these phenotypes. Complete ventralization (DAI 0) was not observed. An important direct downstream target of dorsal nuclear β-catenin activity is the early Wnt-inducible homeobox gene Siamois (Brannon et al., 1997). We therefore tested to determine whether expression levels of this gene were reduced in the RanBP3-injected embryos by RT-PCR. In four independent experiments, we detected an approximately twofold decrease in Siamois levels in late stage 9 embryos (Fig. 8, D and E). This decrease is rather mild, consistent with the incomplete ventralization phenotypes observed. Based on these findings, we conclude that RanBP3 not only is a repressor of Wnt signaling in human cell lines but also functions as an antagonist of Wnt signaling in X. laevis embryos.

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