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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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Loss of RanBP3 by RNAi results in a naked cuticle phenotype in D. melanogaster. Shown are dark field images of cuticle preparations of control (β-galactosidase; A), D. melanogaster Daxin (B and C), and D. melanogaster RanBP3 dsRNA–injected embryos (D–F). Loss of Daxin and RanBP3 results in increased Wnt signaling and replacement of denticles by naked cuticle. Partially naked cuticles (B and D), nearly naked cuticles (E), and naked cuticles (C and F) are shown. All views are ventral, top is posterior. (G) Quantification of two representative experiments showing the frequency of the cuticle phenotype. P values are calculated as in Fig. 7 B. Note that the contribution of the completely naked phenotype in the RanBP3 RNAi embryos is relatively high (not depicted). (H) RT-PCR showing reduction in RanBP3 mRNA levels in RanBP3 dsRNA–injected embryos. Embryos were injected as in A, and RNA was extracted after 15 h of development. RT-PCRs specific for RanBP3 or control (ribosomal protein RP49) were performed using nothing (0) or a series of twofold dilutions of extracted RNA. (I) Loss of RanBP3 function by dsRNA injection results in increased expression of the wg target gene engrailed. Shown are an Engrailed antibody staining of a buffer-injected embryo (left), a Daxin dsRNA–injected embryo (middle), and an RanBP3 dsRNA–injected embryo (right). Note that the buffer-injected embryo developed until late stage 11, whereas the Daxin and RanBP3 RNAi embryos shown are stage 10 embryos, which explains the larger cells in the former embryo. The number of Engrailed-positive cell rows between stages 10 and 11 is identical. Ventral-lateral view is shown, posterior is left.
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fig9: Loss of RanBP3 by RNAi results in a naked cuticle phenotype in D. melanogaster. Shown are dark field images of cuticle preparations of control (β-galactosidase; A), D. melanogaster Daxin (B and C), and D. melanogaster RanBP3 dsRNA–injected embryos (D–F). Loss of Daxin and RanBP3 results in increased Wnt signaling and replacement of denticles by naked cuticle. Partially naked cuticles (B and D), nearly naked cuticles (E), and naked cuticles (C and F) are shown. All views are ventral, top is posterior. (G) Quantification of two representative experiments showing the frequency of the cuticle phenotype. P values are calculated as in Fig. 7 B. Note that the contribution of the completely naked phenotype in the RanBP3 RNAi embryos is relatively high (not depicted). (H) RT-PCR showing reduction in RanBP3 mRNA levels in RanBP3 dsRNA–injected embryos. Embryos were injected as in A, and RNA was extracted after 15 h of development. RT-PCRs specific for RanBP3 or control (ribosomal protein RP49) were performed using nothing (0) or a series of twofold dilutions of extracted RNA. (I) Loss of RanBP3 function by dsRNA injection results in increased expression of the wg target gene engrailed. Shown are an Engrailed antibody staining of a buffer-injected embryo (left), a Daxin dsRNA–injected embryo (middle), and an RanBP3 dsRNA–injected embryo (right). Note that the buffer-injected embryo developed until late stage 11, whereas the Daxin and RanBP3 RNAi embryos shown are stage 10 embryos, which explains the larger cells in the former embryo. The number of Engrailed-positive cell rows between stages 10 and 11 is identical. Ventral-lateral view is shown, posterior is left.

Mentions: Wnt signaling is highly conserved between different species. We identified the D. melanogaster RanBP3 homologue and used RNAi to study its role in D. melanogaster development. At the end of embryogenesis, the ventral epidermis is covered by a cuticle that is built up by a repeating pattern of naked cuticle and denticles (Fig. 9 A). Wingless (Wg; D. melanogaster Wnt) signaling increases levels of ARM (β-catenin) that specifies the fate of epidermal cells responsible for secreting naked cuticle. Therefore, loss of wg expression results in an embryo that is covered with denticles lacking naked cuticle (Nusslein-Volhard and Wieschaus, 1980) and overexpression of wg results in a naked cuticle embryo (Noordermeer et al., 1992). Likewise, loss of an inhibitor of Wnt signaling also results in naked cuticle embryos as shown by RNAi against Daxin (Willert et al., 1999). As a control, we injected embryos with β-galactosidase double-stranded RNA (dsRNA) and observed that the majority (97%) developed into larvae that were indistinguishable from noninjected wt larvae (Fig. 9 A). 3% of these control embryos showed some very weak effects on denticle belt formation (Fig. 9 G). RNAi against Daxin resulted in a significant increase in naked cuticle phenotype in 24% of the Daxin dsRNA–injected embryos (Fig. 9 G), with phenotypes varying from partial loss of denticles to completely naked embryos (Fig. 9, B and C). Injection of dsRNA against the D. melanogaster RanBP3 caused a partial or complete transformation of denticles into naked cuticle in 14% of the embryos (Fig. 9, D–F). The most severe phenotypes of the RanBP3 RNAi embryos showed deformation of both the head and spiracles (Fig. 9 F), resembling Daxin RNAi (Fig. 9 C). In addition, almost all RanBP3 RNAi embryos showing a strong naked cuticle phenotype were shorter than the embryos injected with Daxin dsRNA. To confirm that the RanBP3 dsRNA injections resulted in decreased RanBP3 levels, we performed RT-PCR on buffer and RanBP3 dsRNA–injected embryos. Fig. 9 H shows that RanBP3 mRNA levels were indeed decreased in RanBP3 dsRNA–injected embryos, whereas RP49 control mRNA levels remained unaffected. We then assayed the effects of RanBP3 dsRNA injection on wg target gene induction. For this, stage 10 RanBP3 or Daxin dsRNA–injected embryos were stained with anti-Engrailed antibody. Normal engrailed expression is present in segmental stripes that are two cells wide (Fig. 9 I, left). Removal of the Wnt signaling inhibitor Daxin by dsRNA injection resulted in a broader Engrailed expression pattern that extended from two to four rows of cells (Fig. 9 I, middle). In RanBP3 dsRNA–injected embryos, Engrailed expression expanded by one row of cells (Fig. 9 I, right). These in vivo data show that removal of RanBP3 leads to a phenotype that is associated with Wnt signaling activation, suggesting that RanBP3 also acts as negative regulator of Wnt signaling in D. melanogaster.


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)

Loss of RanBP3 by RNAi results in a naked cuticle phenotype in D. melanogaster. Shown are dark field images of cuticle preparations of control (β-galactosidase; A), D. melanogaster Daxin (B and C), and D. melanogaster RanBP3 dsRNA–injected embryos (D–F). Loss of Daxin and RanBP3 results in increased Wnt signaling and replacement of denticles by naked cuticle. Partially naked cuticles (B and D), nearly naked cuticles (E), and naked cuticles (C and F) are shown. All views are ventral, top is posterior. (G) Quantification of two representative experiments showing the frequency of the cuticle phenotype. P values are calculated as in Fig. 7 B. Note that the contribution of the completely naked phenotype in the RanBP3 RNAi embryos is relatively high (not depicted). (H) RT-PCR showing reduction in RanBP3 mRNA levels in RanBP3 dsRNA–injected embryos. Embryos were injected as in A, and RNA was extracted after 15 h of development. RT-PCRs specific for RanBP3 or control (ribosomal protein RP49) were performed using nothing (0) or a series of twofold dilutions of extracted RNA. (I) Loss of RanBP3 function by dsRNA injection results in increased expression of the wg target gene engrailed. Shown are an Engrailed antibody staining of a buffer-injected embryo (left), a Daxin dsRNA–injected embryo (middle), and an RanBP3 dsRNA–injected embryo (right). Note that the buffer-injected embryo developed until late stage 11, whereas the Daxin and RanBP3 RNAi embryos shown are stage 10 embryos, which explains the larger cells in the former embryo. The number of Engrailed-positive cell rows between stages 10 and 11 is identical. Ventral-lateral view is shown, posterior is left.
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fig9: Loss of RanBP3 by RNAi results in a naked cuticle phenotype in D. melanogaster. Shown are dark field images of cuticle preparations of control (β-galactosidase; A), D. melanogaster Daxin (B and C), and D. melanogaster RanBP3 dsRNA–injected embryos (D–F). Loss of Daxin and RanBP3 results in increased Wnt signaling and replacement of denticles by naked cuticle. Partially naked cuticles (B and D), nearly naked cuticles (E), and naked cuticles (C and F) are shown. All views are ventral, top is posterior. (G) Quantification of two representative experiments showing the frequency of the cuticle phenotype. P values are calculated as in Fig. 7 B. Note that the contribution of the completely naked phenotype in the RanBP3 RNAi embryos is relatively high (not depicted). (H) RT-PCR showing reduction in RanBP3 mRNA levels in RanBP3 dsRNA–injected embryos. Embryos were injected as in A, and RNA was extracted after 15 h of development. RT-PCRs specific for RanBP3 or control (ribosomal protein RP49) were performed using nothing (0) or a series of twofold dilutions of extracted RNA. (I) Loss of RanBP3 function by dsRNA injection results in increased expression of the wg target gene engrailed. Shown are an Engrailed antibody staining of a buffer-injected embryo (left), a Daxin dsRNA–injected embryo (middle), and an RanBP3 dsRNA–injected embryo (right). Note that the buffer-injected embryo developed until late stage 11, whereas the Daxin and RanBP3 RNAi embryos shown are stage 10 embryos, which explains the larger cells in the former embryo. The number of Engrailed-positive cell rows between stages 10 and 11 is identical. Ventral-lateral view is shown, posterior is left.
Mentions: Wnt signaling is highly conserved between different species. We identified the D. melanogaster RanBP3 homologue and used RNAi to study its role in D. melanogaster development. At the end of embryogenesis, the ventral epidermis is covered by a cuticle that is built up by a repeating pattern of naked cuticle and denticles (Fig. 9 A). Wingless (Wg; D. melanogaster Wnt) signaling increases levels of ARM (β-catenin) that specifies the fate of epidermal cells responsible for secreting naked cuticle. Therefore, loss of wg expression results in an embryo that is covered with denticles lacking naked cuticle (Nusslein-Volhard and Wieschaus, 1980) and overexpression of wg results in a naked cuticle embryo (Noordermeer et al., 1992). Likewise, loss of an inhibitor of Wnt signaling also results in naked cuticle embryos as shown by RNAi against Daxin (Willert et al., 1999). As a control, we injected embryos with β-galactosidase double-stranded RNA (dsRNA) and observed that the majority (97%) developed into larvae that were indistinguishable from noninjected wt larvae (Fig. 9 A). 3% of these control embryos showed some very weak effects on denticle belt formation (Fig. 9 G). RNAi against Daxin resulted in a significant increase in naked cuticle phenotype in 24% of the Daxin dsRNA–injected embryos (Fig. 9 G), with phenotypes varying from partial loss of denticles to completely naked embryos (Fig. 9, B and C). Injection of dsRNA against the D. melanogaster RanBP3 caused a partial or complete transformation of denticles into naked cuticle in 14% of the embryos (Fig. 9, D–F). The most severe phenotypes of the RanBP3 RNAi embryos showed deformation of both the head and spiracles (Fig. 9 F), resembling Daxin RNAi (Fig. 9 C). In addition, almost all RanBP3 RNAi embryos showing a strong naked cuticle phenotype were shorter than the embryos injected with Daxin dsRNA. To confirm that the RanBP3 dsRNA injections resulted in decreased RanBP3 levels, we performed RT-PCR on buffer and RanBP3 dsRNA–injected embryos. Fig. 9 H shows that RanBP3 mRNA levels were indeed decreased in RanBP3 dsRNA–injected embryos, whereas RP49 control mRNA levels remained unaffected. We then assayed the effects of RanBP3 dsRNA injection on wg target gene induction. For this, stage 10 RanBP3 or Daxin dsRNA–injected embryos were stained with anti-Engrailed antibody. Normal engrailed expression is present in segmental stripes that are two cells wide (Fig. 9 I, left). Removal of the Wnt signaling inhibitor Daxin by dsRNA injection resulted in a broader Engrailed expression pattern that extended from two to four rows of cells (Fig. 9 I, middle). In RanBP3 dsRNA–injected embryos, Engrailed expression expanded by one row of cells (Fig. 9 I, right). These in vivo data show that removal of RanBP3 leads to a phenotype that is associated with Wnt signaling activation, suggesting that RanBP3 also acts as negative regulator of Wnt signaling in D. melanogaster.

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