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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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Identification of RanBP3 as an interaction partner of β-catenin. (A) Pull-down experiment using immobilized GST (lane 2), GST-tagged β-catenin ARM repeats 1–12 (lane 3), and full-length β-catenin (lane 4) incubated with X. laevis egg extract (input; lane 1). Bound proteins were analyzed by Western blot using mAb414 recognizing a subset of nucleoporins. Two unknown proteins, p80 and p90, are marked with arrows. (B) Identification of p80 and p90 as the b and a isoforms of RanBP3. Pull-down experiment as in A, incubated with HeLa nuclear extracts and analyzed using RanBP3 antibody. (C) RanBP3 binds directly to β-catenin. GST-tagged full-length β-catenin (lanes 2–11) was incubated with 2 μM RanGTP and 0.2 μM (lanes 2 and 5), 0.5 μM (lanes 3, 6, and 8–11), or 2.0 μM (lanes 4 and 7) wt (lanes 2–4 and 8–11) or wv mutant (lanes 5–7) RanBP3-b. Bound proteins were eluted as indicated above the lanes and visualized with silver (lanes 1–7) or Coomassie (lanes 8–11) staining.
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fig1: Identification of RanBP3 as an interaction partner of β-catenin. (A) Pull-down experiment using immobilized GST (lane 2), GST-tagged β-catenin ARM repeats 1–12 (lane 3), and full-length β-catenin (lane 4) incubated with X. laevis egg extract (input; lane 1). Bound proteins were analyzed by Western blot using mAb414 recognizing a subset of nucleoporins. Two unknown proteins, p80 and p90, are marked with arrows. (B) Identification of p80 and p90 as the b and a isoforms of RanBP3. Pull-down experiment as in A, incubated with HeLa nuclear extracts and analyzed using RanBP3 antibody. (C) RanBP3 binds directly to β-catenin. GST-tagged full-length β-catenin (lanes 2–11) was incubated with 2 μM RanGTP and 0.2 μM (lanes 2 and 5), 0.5 μM (lanes 3, 6, and 8–11), or 2.0 μM (lanes 4 and 7) wt (lanes 2–4 and 8–11) or wv mutant (lanes 5–7) RanBP3-b. Bound proteins were eluted as indicated above the lanes and visualized with silver (lanes 1–7) or Coomassie (lanes 8–11) staining.

Mentions: To study the interaction between β-catenin and nuclear transport factors, we used GST-tagged β-catenin to pull down interacting proteins from X. laevis egg extracts. Interacting proteins were initially analyzed by Western blot using mAb414, which recognizes a phenylalanine glycine (FG)–rich epitope present in multiple nucleoporins. FG repeat–containing nucleoporins Nup62, Nup153, and Nup358 were specifically bound by full-length β-catenin and by the central armadillo (ARM) repeat region (unpublished data). Interestingly, we found a strong interaction between β-catenin and two unknown proteins of ∼80 and 90 kD that were recognized by mAb414 (Fig. 1 A, lanes 3 and 4). These proteins interacted with full-length β-catenin and to a lesser extent with the ARM repeats (ARM 1–12). The mAb414 reactivity indicated that these two proteins contained FG repeats. Two isoforms of RanBP3 stood out as possible candidates for these two unknown proteins because they contain FG repeats and have the correct sizes. Indeed, recombinant human RanBP3-a comigrated with the p90 protein and was recognized by mAb414 (Fig. 1 A, lane 5). To confirm that RanBP3 was one of these new β-catenin–interacting proteins, we repeated the pull-down experiment using HeLa nuclear extracts and an mAb recognizing human RanBP3. The b isoform of RanBP3 was more abundant in HeLa nuclear extracts and copurified with GST-tagged full-length and the ARM repeats of β-catenin (Fig. 1 B). To mimic nuclear conditions, 2 μM of a nonhydrolysable mutant of the small GTPase Ran (RanQ69L-GTP) was added, resulting in increased interaction between β-catenin and RanBP3 (Fig. 1 B, lanes 2 and 4). In the presence of RanQ69L-GTP, the less abundant a isoform of human RanBP3 also bound to full-length β-catenin (Fig. 1 B, lane 2).


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)

Identification of RanBP3 as an interaction partner of β-catenin. (A) Pull-down experiment using immobilized GST (lane 2), GST-tagged β-catenin ARM repeats 1–12 (lane 3), and full-length β-catenin (lane 4) incubated with X. laevis egg extract (input; lane 1). Bound proteins were analyzed by Western blot using mAb414 recognizing a subset of nucleoporins. Two unknown proteins, p80 and p90, are marked with arrows. (B) Identification of p80 and p90 as the b and a isoforms of RanBP3. Pull-down experiment as in A, incubated with HeLa nuclear extracts and analyzed using RanBP3 antibody. (C) RanBP3 binds directly to β-catenin. GST-tagged full-length β-catenin (lanes 2–11) was incubated with 2 μM RanGTP and 0.2 μM (lanes 2 and 5), 0.5 μM (lanes 3, 6, and 8–11), or 2.0 μM (lanes 4 and 7) wt (lanes 2–4 and 8–11) or wv mutant (lanes 5–7) RanBP3-b. Bound proteins were eluted as indicated above the lanes and visualized with silver (lanes 1–7) or Coomassie (lanes 8–11) staining.
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Related In: Results  -  Collection

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

fig1: Identification of RanBP3 as an interaction partner of β-catenin. (A) Pull-down experiment using immobilized GST (lane 2), GST-tagged β-catenin ARM repeats 1–12 (lane 3), and full-length β-catenin (lane 4) incubated with X. laevis egg extract (input; lane 1). Bound proteins were analyzed by Western blot using mAb414 recognizing a subset of nucleoporins. Two unknown proteins, p80 and p90, are marked with arrows. (B) Identification of p80 and p90 as the b and a isoforms of RanBP3. Pull-down experiment as in A, incubated with HeLa nuclear extracts and analyzed using RanBP3 antibody. (C) RanBP3 binds directly to β-catenin. GST-tagged full-length β-catenin (lanes 2–11) was incubated with 2 μM RanGTP and 0.2 μM (lanes 2 and 5), 0.5 μM (lanes 3, 6, and 8–11), or 2.0 μM (lanes 4 and 7) wt (lanes 2–4 and 8–11) or wv mutant (lanes 5–7) RanBP3-b. Bound proteins were eluted as indicated above the lanes and visualized with silver (lanes 1–7) or Coomassie (lanes 8–11) staining.
Mentions: To study the interaction between β-catenin and nuclear transport factors, we used GST-tagged β-catenin to pull down interacting proteins from X. laevis egg extracts. Interacting proteins were initially analyzed by Western blot using mAb414, which recognizes a phenylalanine glycine (FG)–rich epitope present in multiple nucleoporins. FG repeat–containing nucleoporins Nup62, Nup153, and Nup358 were specifically bound by full-length β-catenin and by the central armadillo (ARM) repeat region (unpublished data). Interestingly, we found a strong interaction between β-catenin and two unknown proteins of ∼80 and 90 kD that were recognized by mAb414 (Fig. 1 A, lanes 3 and 4). These proteins interacted with full-length β-catenin and to a lesser extent with the ARM repeats (ARM 1–12). The mAb414 reactivity indicated that these two proteins contained FG repeats. Two isoforms of RanBP3 stood out as possible candidates for these two unknown proteins because they contain FG repeats and have the correct sizes. Indeed, recombinant human RanBP3-a comigrated with the p90 protein and was recognized by mAb414 (Fig. 1 A, lane 5). To confirm that RanBP3 was one of these new β-catenin–interacting proteins, we repeated the pull-down experiment using HeLa nuclear extracts and an mAb recognizing human RanBP3. The b isoform of RanBP3 was more abundant in HeLa nuclear extracts and copurified with GST-tagged full-length and the ARM repeats of β-catenin (Fig. 1 B). To mimic nuclear conditions, 2 μM of a nonhydrolysable mutant of the small GTPase Ran (RanQ69L-GTP) was added, resulting in increased interaction between β-catenin and RanBP3 (Fig. 1 B, lanes 2 and 4). In the presence of RanQ69L-GTP, the less abundant a isoform of human RanBP3 also bound to full-length β-catenin (Fig. 1 B, lane 2).

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