beta-catenin can be transported into the nucleus in a Ran-unassisted manner

Abstract

The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion. These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner. We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.

Figure 1

Double axis formation induced by purified recombinant β-catenin. (A) SDS-PAGE profile of purified β-catenin. The purified FLAG-β-catenin was subjected to 10% SDS-PAGE and stained with Coomassie Brilliant Blue. (B) Right panel, a Xenopus embryo was injected at the four-cell stage with purified recombinant FLAG-β-catenin (∼25 ng protein/25 nl) in 50 mM potassium phosphate buffer, pH 7.2, and 50 mM NaCl. Tadpoles exhibit axis duplication, including two eye pairs and two cement glands. Left panel, control experiment demonstrating that the injection of BSA in the same buffer had no effect.

Figure 2

β-Catenin migrates into the nucleus of living cells in a temperature-dependent and WGA-sensitive manner. Purified recombinant mouse FLAG-β-catenin (1 mg/ml) was injected into the cytoplasm of MDBK cells with (e and f) or without (a–d) 2 mg/ml WGA. After incubation for 30 min at 37°C (a, b, e, and f) or on ice (c and d), cells were fixed with 3.7% formaldehyde in PBS. Injected FLAG-β-catenin was visualized by indirect immunofluorescence with anti-FLAG mouse mAb and CY3-labeled anti-mouse goat IgG. The localization of β-catenin was examined by Axiophoto microscopy (Zeiss).

Figure 3

β-Catenin migrates into the nucleus in the absence of exogenous soluble factors or ATP in the digitonin-permeabilized cell-free import assay. (A) Digitonin-permeabilized MDBK cells were incubated with 10 μl of testing solution containing 8 pmol of FLAG-β-catenin (a, c, and e) or 1 μg of allophycocyanin-NLS (b, d, and f) in the presence of 10 mg/ml cytosol (e and f) or 1 mg/ml cytosol (c and d) or in the absence of cytosol (a and b). All reaction mixtures contained ATP and GTP. After incubation for 20 min at 30°C, cells were fixed, and FLAG-β-catenin was visualized by indirect immunofluorescence with anti-FLAG mouse mAb and CY3-labeled anti-mouse goat IgG. Allophycocyanin-NLS was directly examined by Axiophoto microscopy (Zeiss) after fixation. (B) Digitonin-permeabilized MDBK cells were incubated with 10 μl of testing solution containing 8 pmol of FLAG-β-catenin (a, c, and e) without cytosol or 1 μg of allophycocyanin-NLS (b, d, and f) supplemented with 10 mg/ml cytosol in the absence (a, b, e, and f) or presence (c and d) of ATP and GTP. e and f show import reactions performed in the permeabilized cells, which were pretreated with apyrase (see MATERIALS AND METHODS). After incubation for 20 min at 30°C, cells were fixed, and the localization of β-catenin and allophycocyanin-NLS was examined as in A. (C) Digitonin-permeabilized MDBK cells were incubated with 8 pmol of FLAG-β-catenin for 20 min at 30°C (a and c) or on ice (b). c shows the import reaction performed in the permeabilized cells, which were pretreated with WGA (see MATERIALS AND METHODS). After incubation, cells were fixed, and localization of β-catenin was examined as in A. (D) Digitonin-permeabilized MDBK cells were incubated with 8 pmol of GFP-β-catenin in the absence of energy source as in B. After 20 min of incubation, distribution of GFP-β-catenin (a) was examined by confocal laser scanning microscope (Zeiss, LSM410) using a 40× objective with oil immersion, without washing and fixation of the cells. Cells were recorded simultaneously by interference contrast (b). A 1-μm section, corresponding to the equator of the nucleus, is shown. The final figure was produced using Adobe Photoshop (Adobe Systems, Mountain View, CA).

Figure 4

Nuclear accumulation of β-catenin is not inhibited by mutant Ran (G19V Ran)-GTP in living cells. A mixture of FLAG-β-catenin (0.8 mg/ml) and GST-NLS-GFP (0.5 mg/ml) (a–d) or GST-M9-GFP (1 mg/ml) (e–h) was coinjected with 5 mg/ml G19V Ran-GTP (a, b, e, and f) or alone (c, d, g, and h) into the cytoplasm of MDBK cells. After incubation for 30 min at 37°C, cells were fixed, and the localization of FLAG-β-catenin (b, d, f, and h) was examined as in Figure 2. The localization of GST-NLS-GFP (a and c) or GST-M9-GFP (e and g) shows that G19V Ran-GTP strongly inhibited the nuclear accumulation of these two substrates, whereas the nuclear accumulation of FLAG-β-catenin was not affected in the same cells.

Figure 5

Nuclear accumulation of β-catenin is insensitive to nuclear Ran-GTP depletion in living cells. A mixture of 0.8 mg/ml FLAG-β-catenin and 0.5 mg/ml GST-NLS-GFP (a and b) or 1 mg/ml GST-M9-GFP (c and d) was injected into the cytoplasm of tsBN2 cells cultured at nonpermissive temperature (upper panels). After further incubation for 30 min at nonpermissive temperature, cells were fixed, and localization of FLAG-β-catenin (b and d) was examined as in Figure 2. Localization of GST-NLS-GFP (a) or GST-M9-GFP (c) shows that nuclear accumulation of these two substrates declined in the tsBN2 cells incubated at nonpermissive temperature, whereas nuclear accumulation of FLAG-β-catenin was not affected in the same cells. Lower panels, control experiments showing that GST-NLS-GFP (a), GST-M9-GFP (b), and FLAG-β-catenin (c) all migrate normally in the nucleus of tsBN2 cells, which were incubated at permissive temperature within 30 min after cytoplasmic injection.

Figure 6

Nuclear accumulation of β-catenin does not require Ran in vitro. Upper panel, digitonin-permeabilized MDBK cells were incubated with a 10-μl testing solution containing 8 pmol of FLAG-β-catenin in the absence (a) or presence of 80 pmol of Ran-GDP (b) or 80 pmol of G19V Ran-GTP (c). After incubation for 30 min at 30°C, the cells were fixed, and the localization of FLAG-β-catenin was examined as in Figure 3. Lower panel, digitonin-permeabilized MDBK cells were incubated with 10 μl of testing solution containing 3 pmol GFP-importin β in the absence (a) or presence of 80 pmol of G19V Ran-GTP (b). After incubation for 20 min at 30°C, the cells were fixed, and the localization of GFP-importin β was examined by Axiophoto microscopy (Zeiss).

Figure 7

Nuclear accumulation of β-catenin is saturable and competitively inhibited by importin β family proteins. Digitonin-permeabilized MDBK cells were incubated with 10 μl of testing solution containing 4 pmol of GFP-β-catenin (a–d) or 4 pmol of GFP-importin β (e–h) in the absence (a and e) or presence of 80 pmol of importin β (b and f), 80 pmol of GST-transportin (c and g), or 80 pmol of FLAG-β-catenin (d and h). After incubation for 10 min at 30°C, cells were fixed, and localization of the GFP fusion proteins was examined by Axiophoto microscopy (Zeiss).

Figure 8

β-Catenin rapidly exits the nucleus in a temperature-dependent manner. A mixture of 1 mg/ml GFP-β-catenin and 0.5 mg/ml Texas Red–labeled BSA was injected into the nuclei of homokaryon of BHK21 cells. After incubation for 30 min at 37°C (upper panels) or on ice (lower panels), cells were fixed, and localization of GFP-β-catenin (middle panels) and Texas Red–labeled BSA (left panels) was examined by Axiophoto microscopy (Zeiss). The localization of Texas Red–labeled BSA shows an injection site.