Hwang WY et al. (2022), Kap-β2/Transportin mediates β-catenin nuclear t...

XB-ART-59423

Elife 2022 Oct 27;11. doi: 10.7554/eLife.70495.

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Kap-β2/Transportin mediates β-catenin nuclear transport in Wnt signaling.

Hwang WY , Kostiuk V , González DP , Lusk CP , Khokha MK .

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Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer, highlighting its importance for disease pathogenesis. β-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of β-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that β-catenin accumulates in the nucleus in a Ran-dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the ortholog of Kap-β2/Transportin-1 (TNPO1), was required for β-catenin nuclear import. We further demonstrate direct binding between TNPO1 and β-catenin that is mediated by a conserved PY-NLS. Finally, using Xenopus secondary axis and TCF/LEF (T Cell factor/lymphoid enhancer factor family) reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the nuclear localization signal in β-catenin and its cognate NTR, our study suggests new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step toward therapeutics.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: napsa ntsr1 ran rcc1 tnpo1 tnpo2 wnt3a
GO keywords: embryo development [+]

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	Figure 1 β-catenin requires a functional Ran cycle to accumulate in the nucleus of Saccharomyces cerevisiae. (A) Representative deconvolved fluorescence images of xβ-catenin-GFP in a wildtype yeast strain that expresses Heh2-mCherry to label the nucleus (left). White arrows indicate the nuclear compartment. Plot showing the quantification of mean nuclear to cytosolic fluorescence intensity from 30 to 40 cells from three independent replicates (right). (B) Representative deconvolved fluorescence images of xβ-catenin-GFP in the RanGEF mutant (mtr1-1) strain at room temperature or 37°C that co-expresses Heh2-mCherry as a nuclear envelope marker (left). The ratio of mean nuclear to cytosolic fluorescence intensity was measured in the wildtype or mtr1-1 strain from 30 to 35 cells from three independent replicates (right). Scale bar is 5 μm in (A) and (B). Red bar indicates the mean value with the SD. p-Values are from unpaired two-tailed t-test where ns is p>0.05, and ****p<0.0001 for both (A) and (B). The data is uploaded as Figure 1—source data 1.
	Figure 2 with 3 supplements The C-terminus of β-catenin contains a nuclear localization signal (NLS). (A) Schematic of Xenopus β-catenin truncation constructs tested in this study. (B) Plot of the ratio of mean nuclear to cytosolic fluorescence intensity of Xenopus β-catenin GFP truncation constructs tested in a wildtype yeast strain from 30 to 40 cells from three independent replicates. Red bar indicates the mean value with the SD. (C) Deconvolved fluorescence images of the N-terminal deletion (141-782), ARM-repeats deletion (Δ141-664) and C-terminal deletion (1-664) of Xenopus β-catenin GFP in the wildtype strain. GFP and full-length Xenopus β-catenin-GFP were used as controls. (D) Deconvolved fluorescence images of the indicated fragments of Xenopus β-catenin GFP in the wildtype strain. White arrows indicate nuclear rim localization. (E) Deconvolved fluorescence images of indicated C-terminus fragments of Xenopus β-catenin GFP in the wildtype strain. Heh2-mCherry was co-expressed to label the nuclear envelope in (C), (D), and (E). Scale bar is 5 µm in (C), (D), and (E). The data is uploaded as Figure 2—source data 1.
	Figure 3 with 2 supplements Kap104 is specifically required for β-catenin nuclear accumulation in Saccharomyces cerevisiae. (A) Schematic of the Anchor-Away assay mediated by the rapamycin-induced dimerization of nuclear transport receptor (NTR)-FKBP-rapamycin binding (FRBP and Pma1-FKBP12). Pma1 is a plasma membrane ATPase. (B) Deconvolved fluorescence images of cells with indicated FRB fusions expressing Xenopus β-catenin (665-782)-GFP treated with DMSO (vehicle) or rapamycin for 15 min. Heh2-mCherry was used as a nuclear envelope marker. White arrows indicate the nucleus. Scale bar is 5 µm. (C) Plot showing the ratio of mean nuclear to cytosolic fluorescence intensity of Xenopus β-catenin (665-782)-GFP in the 10 NTR-FRB strains treated with DMSO or rapamycin from 30 to 40 cells from three independent replicates. Red bar indicates the mean value with the SD. Experiments were performed three times. p-Values are from unpaired two-tailed t-test where ns is p>0.05 and ****p<0.0001. The data is uploaded as Figure 3—source data 1.
	Figure 4 β-catenin contains a PY-NLS that is required for nuclear import. (A) Conservation of amino acid sequences that conform to the PY-NLS consensus (top, from Soniat and Chook, 2015) in the C-terminus of β-catenin. (B) Deconvolved fluorescence images of wildtype yeast cells expressing MBP(x3)-GFP tagged with the Xenopus β-catenin nuclear localization signal (NLS) (665-703) and also an NLS that contains the PM to AA mutation (top). Untagged MBP(x3)-GFP was used as a control. Plot of the ratio of mean nuclear to cytoplasmic fluorescence intensity from a single experiment (bottom). Scale bar is 5 µm. (C) Representative fluorescence image of HeLa cells expressing human β-catenin (665-782) or the PM to AA mutant version (top). LaminB1 was labeled to locate the nuclear envelope. GFP alone was used as a control. Plot of the ratio of mean nuclear to cytoplasmic fluorescence intensity from three experiments (bottom). Scale bar is 15 µm. p-Values are from unpaired two-tailed t-test where ns is p>0.05 and ****p<0.0001 for both (B) and (C). The data is uploaded as Figure 4—source data 1.
	Figure 5 with 1 supplement Direct binding of β-catenin and TNPO1 is destabilized by Ran-GTP. (A) In vitro binding assay of purified recombinant TNPO1 and GST fusions of human β-catenin and human β-catenin containing the PM to AA mutations. GST alone was used as a negative control. (B) In vitro binding assay of purified recombinant TNPO1 to GST fusions of human β-catenin in the presence of GTP hydrolysis deficient Ran mutant loaded with GTP (RanQ69L). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue in (A) and (B). * indicates TNPO1 bound to GST-hβ-catenin.
	Figure 6 with 4 supplements TNPO1/2 and the β-catenin nuclear localization signal (NLS) are required for Wnt signaling in vivo. (A) Depletion of tnpo1 and tnpo2 using two different pairs of non-overlapping sgRNAs represses gfp expression in Xenopus tropicalis Tg(pbin7Lef-dGFP) embryos at stage 10. Key used to quantify embryos with whole mount in situ hybridization (WMISH) signal (blue – normal gfp signal nd red – reduced gfp signal). Uninjected control (UIC) embryos were used as a negative control. (B) siRNA mediated TNPO1 and/or TNPO2 knockdown reduces luciferase activity in mouse embryonic fibroblasts that harbor a stable integration of luciferase under the control of TCF/LEF promoters. Wnt signaling was activated by human β-catenin-GFP overexpression. Control siRNA and GFP were used as negative controls. Experiments were performed in triplicate. (C) Schematic diagram of three β-catenin constructs used in the double axis assay in Xenopus laevis. ** indicates P687A, M688A substitutions (top left). Dorsal views of X. laevis embryos with anterior to the top (bottom left). Dotted lines indicate the embryonic axis, and the white arrows indicate the head. Histogram of the percent of embryos with secondary axes from three independent replicates. p-Values are from Fisher’s exact test (A) and (C) and unpaired two-tailed t-test (B) where ns is p>0.05, p<0.05 (), 0.0021 (), 0.0002 (), and p<0.0001 (**).The data is uploaded as Figure 6—source data 1.
	Figure 7 with 1 supplement The M9M peptide inhibits Wnt signaling. Wnt signaling was activated by Wnt3a (A, left), human β-catenin-GFP overexpression (A, right and B), or cNLS-human β-catenin-GFP (B). No Wnt3a or GFP overexpression were used as negative controls. Experiments were performed in triplicate (A) or duplicate in two independent experiments (B). p-Values are from unpaired two-tailed t-test where ns is p>0.05, p<0.05 (), 0.0021 (), 0.0002 (), and p<0.0001 (**). The data is uploaded as Figure 7—source data 1.

References [+] :

Andrade, Comparison of ARM and HEAT protein repeats. 2001, Pubmed