Itoh K et al. (2005), Nuclear localization is required for Dishevelle...

XB-ART-2287

J Biol 2005 Jan 01;41:3. doi: 10.1186/jbiol20.

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Nuclear localization is required for Dishevelled function in Wnt/beta-catenin signaling.

Itoh K , Brott BK , Bae GU , Ratcliffe MJ , Sokol SY .

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BACKGROUND: Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined. RESULTS: We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein. CONCLUSIONS: These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/beta-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus.

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Species referenced: Xenopus
Genes referenced: ctnnb1 dvl1 dvl2 fzd8 jun mapk8 myc sia1 wnt3a wnt8a

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	Figure 1. Nuclear export of Dsh is not critical for its activity. (a) The Dsh constructs used to analyze nuclear export. (b-d) RNAs encoding Dsh-GFP, Ds2 and DsNESm (0.5 ng each) were injected into two animal blastomeres of 4–8-cell embryos. Animal-cap explants were excised at stage 10, fixed and examined for GFP fluorescence. (b) Wild type Dsh-GFP localized in punctate structures of the cytoplasm, whereas (c) Ds2 and (d) DsNESm accumulated in the nucleus of animal pole cells. (e,f) One ventral vegetal blastomere of 8-cell embryos was injected with 1 ng Dsh-GFP or DsNESm RNA as indicated. Complete secondary axes were induced in both cases. (g) Uninjected sibling embryos.
	Figure 2. Accumulation of Dsh in the nucleus in the absence of nuclear export. (a-d) Dsh-GFP RNA (0.7 ng) was injected into two animal blastomeres of 4–8 cell embryos. Animal caps were excised at stage 10 and then left (a) untreated or (b) treated with 10 mM NEM or (c,d) 50 ng/ml leptomycin B (LMB), fixed and examined for GFP fluorescence. (a) Dsh-GFP is mainly localized to vesicular structures in the cytoplasm. In the presence of (b) NEM or (c) LMB, Dsh-GFP accumulates in the nucleus, as supported by (d) DAPI staining of nuclei in the same field as in (c). Nuclear staining is marked by arrowheads (c,d). (e,f) The Ds3 construct, lacking amino acids 334–381, remained in the cytoplasm in the (e) absence or (f) presence of NEM.
	Figure 3. Endogenous Dsh shuttles between the cytoplasm and nucleus. Immunofluorescent staining of HEK293 cells with anti-Dvl2 antibodies reveals different subcellular localization of Dvl2 (a) without or (b) with LMB treatment. (c) DAPI staining shows the location of nuclei in the same field as (b); the arrowheads indicate corresponding nuclei in (b) and (c). (d) Distribution of endogenous Dvl2 recognized by anti-Dvl2 antibodies in the nuclear and the cytoplasmic fractions of Rat-1 fibroblasts. In the absence of NEM, Dvl2 is localized mainly in the cytoplasm (C), while after NEM treatment Dvl2 is exclusively localized in the nuclei (N). W, whole cell lysate. Antibodies to lamin and GAPDH show the separation of the nuclear and cytoplasmic fractions.
	Figure 4. Mapping nuclear localization signals in Dsh. (a) The Dsh constructs used to study nuclear transport and their localization to the nucleus after NEM or LMB treatment; the DIX, PDZ and DEP domains are shown as in Figure 1a; B is the basic region and nd denotes not done. (b-i) Subcellular localization of Dsh-GFP constructs in the absence or presence of NEM or LMB. Embryos were injected with 0.5 ng of each mRNA, and GFP analysis was carried out as in Figure 1b-d. (b-d) Ds1, (e-g) DsNLSm, (h) Dsh, (i) DsSNLS. (b,e,i) no NEM treatment; (c,f) after NEM treatment; (d,g,h) after LMB treatment. (j) Comparison of conserved amino-acid sequences that are required for Dsh nuclear localization; X denotes the Xenopus protein, m the mouse and h the human. Amino-acid residues mutated in DsNLSm are indicated by asterisks.
	Figure 5. Activation of the Wnt/β-catenin pathway requires nuclear localization of Dsh. (a) Axis-inducing activity of Dsh constructs. One ventral vegetal blastomere of 8-cell embryos was injected with 1 ng Dsh-GFP, DsNLSm, or DsSNLS mRNA as indicated. Uninjected sibling embryos are also shown. (b) Activation of the Siamois reporter gene. The reporter -833pSia-Luc plasmid (20 pg) was coinjected with Dsh-GFP, DsNLSm or DsSNLS mRNA (0.5 ng each) into a single animal ventral blastomere of 8-cell embryos. Injected embryos were lysed at stage 10+ for luciferase activity determination. Results are shown in relative light units as the mean +/- standard deviation from triplicate samples. (c) Requirement for Dsh NLS for the stabilization of β-catenin. Flag-β-catenin mRNA (0.4 ng) was coinjected with Dsh, DsNLSm, DsSNLS or ΔRGS-Axin mRNA (2 ng each) into four animal blastomeres of 4–8-cell embryos. Levels of β-catenin and Dsh constructs were assessed in stage 10 embryo lysates with anti-Flag antibodies and anti-Xdsh antibodies; β-tubulin serves as a loading control. Dsh and DsSNLS, but not DsNLSm, are able to stabilize β-catenin. ΔRGS-Axin was used as a control for an activator of the Wnt pathway.
	Figure 6. Dominant inhibition of Wnt-dependent transcription by Dsh mutants. Eight-cell embryos were injected (a) in one animal ventral blastomere or (b) in one vegetal ventral blastomere with -833pSia-Luc DNA (20 pg), mRNAs encoding Xwnt3a (5 pg) or Xwnt8 (2 pg), and Dsh-GFP, DsNLSm, Ds3 or DsSNLS mRNA (0.5 ng) as indicated. Luciferase activity was measured as described in Figure 5b.
	Figure 7. Dsh mutants retain the ability to bind CK1ε and XARP. Four-cell embryos were injected in four sites in the animal hemisphere with CK1ε, HA-XARP, Myc-tagged Dsh, DsNLSm, Ds3 or DsSNLS RNA alone (2 ng each) or in combinations as indicated. The embryonic lysates were collected at stage 10.5 for immunoprecipitation with anti-Myc antibodies. Co-immunoprecipitated (a) CK1ε or (b) HA-XARP was probed with anti-CK1ε or anti-HA antibodies; β-tubulin served as a loading control.
	Figure 8. DsNLSm, defective in the β-catenin pathway, is active in noncanonical signaling. (a) Fz8-dependent recruitment of Dsh-GFP constructs to the cell membrane. Dsh-GFP or DsNLSm RNA (0.5 ng) was injected alone or with Fz8 RNA (1 ng) into two animal blastomeres at the 4–8-cell stage. GFP fluorescence was assessed in animal cap explants as in Figure 1b-d. Both Dsh and DsNLSm are efficiently recruited to the cell membrane by Fz8. Arrowheads point to cell membranes. (b) DsNLSm can rescue convergent extension defects caused by Xdd1. Four-cell embryos were injected with 0.6 ng Xdd1 RNA alone or together with 2 ng Dsh-GFP or DsNLSm RNA into two vegetal dorsal blastomeres. The injected embryos were allowed to develop until the sibling embryos reached stage 32. (c) Activation of JNK by the Dsh nuclear import mutant. Four animal blastomeres of four-cell embryos were each injected with 1 ng of RNAs encoding Dsh-GFP or DsNLSm. Embryonic lysates were collected at stage 10.5 for in vitro JNK activity assay using anti-phospho-specific c-Jun antibodies. Total GST-c-Jun levels were assessed with anti-GST antibodies. Dsh-GFP and DsNLSm were equally expressed, as monitored with anti-Dvl2 antibodies; β-tubulin served as a loading control.
	Figure 9. Nuclear translocation of Dvl2 upon Wnt3a treatment. (a) MCF7 cells were treated either with Wnt3a-conditioned or control medium for 6 h, fixed and immunostained with anti-Dvl2 antibodies. In control cells, cytoplasmic and perinuclear staining is visible. Wnt3a-conditioned, but not control, medium enhanced nuclear translocation of Dvl2. DAPI staining indicates the position of cell nuclei. Corresponding cells are shown by arrowheads. (b) Nuclear/cytoplasmic (N/C) ratios of fluorescence were calculated for each panel in (a) as the mean +/- standard deviation.

References [+] :

Axelrod, Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. 1998, Pubmed, Xenbase