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First published online 11 May 2004
doi: 10.1242/jcs.01101

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NLS-dependent nuclear localization of p120^ctn is necessary to relieve Kaiso-mediated transcriptional repression

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

View larger version (61K): [in a new window]   Fig. 1. Identification of an NLS in the p120ctn Armadillo domain. (A) A cross-species comparison of the p120ctn amino acid sequence revealed the presence of a highly conserved putative NLS. Key positively charged residues are boxed. p120ctn-NLSmut denotes the point mutations generated for NLS functionality experiments in D and E. (B,C) Direct fusion of the putative p120ctn NLS (a.a. 622-629, KKGKGKKP) to ß-galactosidase and GFP directs the heterologous fusion protein to the nuclei of HeLa and NIH 3T3 cells, respectively (ß-gal-NLS-GFP). (D,E) The functionality of the putative NLS was validated by the failure of a ß-gal-NLSmut fusion protein to localize to the nuclei of HeLa and NIH 3T3 cells, respectively. This mutant possessed a double point mutation within the p120ctn NLS as indicated. The images shown are representative of at least 100 cells scored for each construct. Bars, 20 µm.

View larger version (52K): [in a new window]   Fig. 2. Deletion mutagenesis mapping of the p120ctn NLS. (A) p120ctn deletion constructs for NLS analysis. Each p120ctn fragment analyzed was fused N-terminally to ß-gal and C-terminally to GFP. (B) Using HeLa cells, nuclear localization of our p120ctn deletion mutants was scored relative to the ß-gal-GFP fusion negative control and the SV40-LTAg-NLS-ß-gal-GFP positive control. (C) Forced expression of full-length p120ctn fused to ß-gal and GFP resulted in a nuclear and cytoplasmic localization of the fusion protein. (D) Of our five p120ctn deletion mutants, one mutant (p120ctn-2) showed nuclear localization in HeLa cells. However, this deletion mutant also localized to distinct cytosolic aggregates. (E) A putative NLS, encoded by amino acids 306-320 of p120ctn, does not show NLS activity as it was insufficient to target a ß-gal-GFP fusion to HeLa nuclei. The images shown are representative of at least 100 cells observed for each construct. Bars, 10 µm.

View larger version (85K): [in a new window]   Fig. 3. Leptomycin B insensitivity of p120ctn deletion mutants. NIH 3T3 cells were transfected with our panel of p120ctn-deletion mutants and treated with LMB for 18 hours before fixation and imaging. Wild-type GFP-fused p120ctn displayed marked LMB sensitivity (A), but none of the p120ctn deletion mutants used for NLS analyses were LMB sensitive (B). Bars, 20 µm.

View larger version (59K): [in a new window]   Fig. 4. An intact NLS in p120ctn is integral for p120ctn nuclear localization and the p120ctn-induced branching phenotype. (A) Expression of wild-type eGFP-fused p120ctn (p120ctn-WT) causes the characteristic branching phenotype in NIH 3T3 cells (Ai) and HeLa cells (Bi). Expression of NLS-defective p120ctn (p120ctn-NLSmut) is largely restricted to the cytosol and unable to induce the branching phenotype (Aii and Bii). (C) An 18 hour LMB treatment caused the nuclear retention of both p120ctn-WT (Cii) and p120ctn-NLSmut (Dii). Results are indicative of at least 100 cells observed for each construct. Bars, 10 µm.

View larger version (21K): [in a new window]   Fig. 5. NLS-defective p120ctn does not inhibit Kaiso-mediated transcriptional repression. (A) Artificial promoter assays in HeLa cells revealed that endogenous Kaiso repressed luciferase expression from the 4x KBS-luciferase construct approximately twofold (compare pGL3 Control to 4x KBS). Overexpression of wild-type Kaiso (4x KBS + Kaiso) repressed luciferase expression an additional twofold. Overexpression of wild-type p120ctn inhibited Kaiso-mediated repression of the luciferase reporter, whereas the NLS-defective p120ctn mutant (NLS Mut) failed to inhibit Kaiso-mediated transcriptional repression despite similar expression levels to wild-type p120ctn, as shown in (B). Expression levels of transfected p120ctn were detected by immunoprecipitation with p120ctn-specific antibody 8D11, which recognizes exogenous mouse, but not endogenous human, p120ctn. Inhibition of Kaiso-mediated transcriptional repression by p120ctn was fully rescued by fusing the SV40-LTAg NLS to the carboxy-terminus of NLS-defective p120ctn (compare 4x KBS + NLS Mut with 4x KBS + NLS Mut + SV40 NLS). Migration of p120ctn fused C-terminally to the SV40-LTAg NLS was impeded slightly on SDS-PAGE due to the addition of the heterologous NLS. Similar results were also obtained in Cos-1 cells (data not shown).

View larger version (68K): [in a new window]   Fig. 6. p120ctn nuclear translocation does not cause the branching phenotype. To address whether the nuclear translocation of p120ctn stimulates the branching phenotype, we directed ectopic p120ctn into the nucleus of HeLa cells via a direct C-terminal fusion to the SV40-LTag NLS. In contrast to wild-type p120ctn (Bi-iii), which strongly induces branching, nuclear-targeted p120ctn is unable to induce branching (Ci-iii). NLS-defective p120ctn localizes to the cytosol and does not induce branching (Di-iii). Direct fusion of NLS-defective p120ctn to the SV40-LTAg NLS is insufficient to rescue branching (Ei-iii), indicating that an intact NLS in p120ctn is required for this phenomenon. Endogenous and exogenous p120ctn are detected by pan-specific p120ctn antibody pp120ctn (Aii, Bii, Cii, Dii, Eii) to highlight the cell boundaries. Empty vector expressing GFP alone (empty) has no effect on branching and freely diffuses into the nucleus (Ai-iii). Similar results were obtained in NIH 3T3 cells. Bars, 20 µm.