Kobayashi M et al. (2002), Nuclear localization of Duplin, a beta-catenin-...

XB-ART-7955

J Biol Chem 2002 Feb 22;2778:5816-22. doi: 10.1074/jbc.M108433200.

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Nuclear localization of Duplin, a beta-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway.

Kobayashi M , Kishida S , Fukui A , Michiue T , Miyamoto Y , Okamoto T , Yoneda Y , Asashima M , Kikuchi A .

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Duplin binds to beta-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the beta-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin alpha was isolated as a binding protein of Duplin. Importin alpha bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin(Delta 500-584)) retained Duplin in the cytoplasm. Duplin(Delta 500-584) bound to beta-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a beta-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed beta-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin alpha, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream beta-catenin target genes.

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Species referenced: Xenopus laevis
Genes referenced: chd8 ctnnb1 kpna1 mbp myc ruvbl1 sia1 sox17a sox17b.1

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	FIG. 1. Structure of Duplin. Schematic representations of Duplin constructs used in this study. The white and gray boxes indicate basic amino acid clusters and -catenin-binding region, respectively.
	FIG. 2. Complex formation of Duplin with importin . A, interaction of Duplin with importin in intact cells. The lysates (20 g of protein) of COS cells with (lanes 2, 6, and 10) or without (lanes 1, 5, and 9) expression of Myc-Duplin were probed with the anti-Myc (lanes 1, 2, 5, 6, 9, and 10) and anti-importin -P (lanes 1 and 2), anti-importin -Q (lanes 5 and 6), or anti-importin -S (lanes 9 and 10) antibodies. The lysates (200 g of protein) described above were immunoprecipitated with the anti-Myc antibody (lanes 3, 4, 7, 8, 11, and 12) and the precipitates were probed with the anti-Myc (lanes 3, 4, 7, 8, 11, and 12) and anti-importin -P (lanes 3 and 4), anti-importin -Q (lanes 7 and 8), or anti-importin -S (lanes 11 and 12) antibodies. IP, immunoprecipitation; Ab, antibody. B, interaction of Duplin mutants with importin in intact cells. The lysates (20 g of protein) of COS cells (lane 1), COS cells expressing Myc-Duplin (full-length) (lane 2), Myc-Duplin-(1–482) (lane 3), Myc-Duplin-(482–749) (lane 4), or Myc-Duplin500–584 (lane 5) were probed with the anti-Myc and anti-importin -P antibodies. The lysates (200 g of protein) prepared in lanes 1–5 were immunoprecipitated with the anti-Myc antibody and the precipitates were probed with the anti-Myc and anti-importin -P antibodies (lanes 6–10). Ig, immunoglobulin. C, direct interaction of Duplin with importin . Purified proteins (0.5 g of protein) of GST-GFP and GST-GFP-Duplin-(500– 584) were subjected to SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining (lanes 1 and 2). GST-GFP and GST-GFP-Duplin-(500–584) (1 M each) were incubated with MBPimportin -P (30 pmol) immobilized on amylose resin. MBP-importin -P was precipitated by centrifugation and the precipitates were probed with the anti-GST antibody (lanes 3 and 4) (upper panel). 10% of MBP-importin -P used in this assay is shown by the anti-MBP antibody (lower panel). 5818 A Novel
	FIG. 3. NLSs of Duplin. A, nuclear localization of Duplin. L cells expressing Myc-Duplin (full-length) (a) or Myc-Duplin500–584 (b) were stained with the anti-Myc antibody and viewed by confocal microscopy. B, active import of Duplin into the nuclei. Purified recombinant GSTGFP-Duplin-(500–584) (a), GST-GFP-Duplin-(500–565) (b), GST-GFPDuplin-(565–668) (c), GST-Duplin-(500–521)-GFP (d), GST-Duplin- (542–546)-GFP (e), GST-SV40NLS-GFP (f), and GST-GFP (g) were injected into the cytoplasm of L cells. After incubation for 30 min at 37 °C, the cells were fixed and the localization of GST-GFP-fused proteins was detected by direct fluorescence microscopy. The results shown are representative of three independent experiments.
	FIG. 4. Effect of nuclear localization of Duplin on -catenin signaling. A, complex formation of Duplin mutants with -catenin. The lysates (20 g of protein) of COS cells expressing Myc-Duplin (fulllength) (lane 2) or Myc-Duplin500–584 (lane 3) were probed with the anti-Myc and anti--catenin antibodies. The lysates prepared in lanes 2 and 3 were immunoprecipitated with the anti-Myc antibody and the precipitates were probed with the anti-Myc and anti--catenin antibodies (lanes 5 and 6). COS cells transfected with the empty vector were used as a control (lanes 1 and 4). B, Wnt-3a induced accumulation of -catenin in L cells. L cells constitutively expressing HA-Duplin (fulllength) (lanes 3 and 4) or HA-Duplin500–584 (lanes 5 and 6) were treated with Wnt-3a conditioned medium (lanes 4 and 6) or control medium (lanes 3 and 5). L cells transfected with empty vectors were used as a control (lanes 1 and 2). The lysates were probed with the anti-HA (upper panel) and anti--catenin (lower panel) antibodies. C, NLS-dependent inhibition of Tcf activation by Duplin. L cells used in Fig. 4B were transfected with pEF-BOS-HA/hTcf-4E and pTOPFLASH. Luciferase activity was assayed and expressed as fold increase compared with the level observed in L cells without Wnt-3a. Black bar, treatment with Wnt-3a conditioned medium; white bar, treatment with control medium. D, NLS-dependent ventralization activity of Duplin. Embryos were injected dorsally with mRNAs of GFP (1 ng) (a) or Duplin500–584 (1 ng) (c) and ventrally with GFP (1 ng) (b) or Duplin500–584 (1 ng) (d).
	FIG. 5. Functions of Duplin without -catenin binding activity. A, complex formation of Duplin mutants with -catenin. The lysates (20 g of protein) of COS cells expressing Myc-Duplin (lane 2) or MycDuplin-(1–668) (lane 3) were probed with the anti-Myc and anti-- catenin antibodies. The lysates (200 g of protein) prepared in lanes 2 and 3 were immunoprecipitated with the anti-Myc antibody, and the precipitates were probed with the anti-Myc and anti--catenin antibodies (lanes 5 and 6). COS cells transfected with the empty vector were used as a control (lanes 1 and 4). B, effects of Duplin-(1–668) on Wnt-dependent Tcf activation. Upper panel, after L cells constitutively expressing HA-Duplin (full-length) (lanes 3 and 4) or HA-Duplin-(1– 668) (lanes 5 and 6) were transfected with pEF-BOS-HA/hTcf-4E and pTOPFLASH, they were treated with Wnt-3a conditioned medium (lanes 4 and 6) or control medium (lanes 3 and 5). L cells transfected with empty vectors were used as a control (lanes 1 and 2). Luciferase activity was assayed and expressed as fold increase compared with the level observed in L cells without Wnt-3a. Black bar, treatment with Wnt-3a conditioned medium; white bar, treatment with control medium. Lower panel, the lysates were probed with the anti--catenin antibody.
	FIG. 6. Effect of Duplin without -catenin binding activity on axis formation of Xenopus embryos. A, effect of Duplin without -catenin binding activity on axis formation. Embryos were injected dorsally with mRNAs of GFP (1.6 ng) (a), Duplin (full-length) (1.6 ng) (b), or Duplin-(1–668) (1.6 ng) (c), and co-injected ventrally with mRNAs of siamois (50 pg) and GFP (800 pg) (d), siamois (50 pg) and Duplin (full-length) (800 pg) (e), or siamois (50 pg) and Duplin-(1–668) (800 pg) (f). B, average DAI (dorso-anterior index) of expressing Duplin (full-length) and Duplin-(1–668). Embryos were injected dorsally with the indicated amounts of mRNAs of Duplin (lanes 1, 3, 5, 7, and 9), Duplin-(1–668) (lanes 2, 4, 6, 8, and 10), and GFP (lane 11). White bar, Duplin (full-length); black bar, Duplin-(1–668); gray bar, GFP for negative control. C, the frequency of axis duplication. Embryos were injected with ventrally with mRNAs of siamois (50 pg) and GFP (800 pg) (lane 1), siamois (50 pg) and Duplin (full-length) (800 pg) (lane 2), or siamois (50 pg) and Duplin-(1–668) (800 pg) (lane 3). Black and white bars indicate the complete and incomplete axes, respectively.