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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01541

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Nuclear import of the BTB/POZ transcriptional regulator Kaiso

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

View larger version (50K): [in a new window]   Fig. 1. Kaiso NLS identification using deletion mutagenesis. (A) Schematic representation of the Kaiso deletion mutants used in the initial experiment for NLS identification. Each Kaiso deletion mutant was simultaneously fused N-terminally to ß-gal and C-terminally to GFP. (B) The positive control, a ß-gal/SV40-NLS/GFP fusion protein, localized strongly to nuclei, whereas the negative control ß-gal/GFP fusion protein localized to the cytosol following transient transfection of HeLa cells. (C) Full-length Kaiso fused to ß-gal/GFP localized to discrete nuclear dots. (D) Out of five initial Kaiso deletion mutants used for NLS analysis, only the Kaiso-4 deletion mutant, which encompassed Kaiso's zinc-finger domain, localized to the nucleus. This indicates the presence of an NLS within Kaiso amino acids 432-581. The images shown are representative of at least 100 cells observed for each construct in three independent trials. Bars, 20 µm.

View larger version (40K): [in a new window]   Fig. 2. The Kaiso NLS maps to ten amino acids upstream of the zinc-finger domain. (A) Schematic representation showing deletion mutants of the Kaiso-4 construct to delineate further the Kaiso NLS; these deletion mutants were called Kaiso-4A, Kaiso-4B, Kaiso-4A1 and Kaiso-4A2, as indicated. (B,C) Interestingly, the Kaiso-4A and Kaiso-4A2 fusion proteins both localized to nuclei in HeLa cells, whereas Kaiso-4B and Kaiso-4A1 localized to the cytosol. The Kaiso-4A2 construct encoded a stretch of basic amino acids (amino acids 469-478) with the potential to serve as an NLS. (D) BLAST analysis revealed that the putative Kaiso NLS is highly conserved across diverse species such as M. musculus, H. sapiens and X. laevis. (E) Amino acids 469-478 (PPNKRMKVKH) of Kaiso constitute a functional NLS that is capable of targeting a heterologous ß-gal/GFP fusion protein to HeLa-cell nuclei. (F) A point mutation at a key basic residue within the Kaiso NLS (K472A) abolishes its ability to target the ß-gal/GFP fusion protein to nuclei. The images shown are representative of at least 100 cells observed for each construct in three independent trials. Bars, 20 µm.

View larger version (48K): [in a new window]   Fig. 3. Kaiso deletion mutants are insensitive to the CRM-I-dependent nuclear export inhibitor leptomycin B. To verify that there were no additional NLS in Kaiso whose activity was masked by a stronger, out-competing NES, we transfected HeLa cells with the indicated constructs, and then treated HeLa cells with either vehicle alone (A) or 50 nM leptomycin B (B) for 16 hours before paraformaldehyde fixation and imaging. None of the Kaiso deletion mutants were sensitive to leptomycin B, indicating that none of these constructs encoded a dominant CRM-I-dependent NES that masked a weaker NLS. The images shown are representative of at least two independent trials. Bars, 20 µm.

View larger version (38K): [in a new window]   Fig. 4. NLS-dependent nuclear translocation of full-length Kaiso. (A) To validate the relevance of the identified Kaiso NLS in the context of full-length Kaiso, wild-type and NLS-defective Kaiso were fused N-terminally to eGFP. Consistent with the localization of endogenous Kaiso, eGFP/Kaiso was targeted predominantly to HeLa-cell and Va-2-cell nuclei, as determined by confocal microscopy. NLS-defective Kaiso with a K472A point mutation demonstrated a nearly exclusive cytosolic localization in all cell lines tested. (B) To verify that the eGFP/Kaiso fusion protein was intact and that the eGFP moiety had not been cleaved, we counterstained transfected cells with our Kaiso-specific antibodies (red) and visualized the stained cells on an Axiovert 200 inverted microscope. Our antibodies efficiently and specifically detected the eGFP/Kaiso fusion proteins, indicating that the nuclear fluorescence was due to intact eGFP/Kaiso fusion proteins. (C) To confirm the expression of intact eGFP/Kaiso and eGFP/Kaiso-NLSmut proteins of correct molecular weight, we performed immunoprecipitation and immunoblot analysis of whole-cell lysates from transfected cells, using specific anti-GFP antibodies. The eGFP/Kaiso and eGFP/Kaiso-NLSmut fusion proteins migrated at the expected molecular weight of 125 kDa, whereas eGFP alone migrated at 30 kDa. Bars, 20 µm.

View larger version (34K): [in a new window]   Fig. 5. Kaiso interacts directly with Importin-2. (A) Equivalent amounts of immobilized GST, GST/Importin- and GST/Importin- + 6x-His/Importin-ß were incubated with radioactively labeled, in vitro transcribed and translated (IVTT) wild-type Kaiso and NLS-mutated Kaiso, as indicated. Wild-type Kaiso but not NLS-mutated Kaiso was able to bind GST/Importin- in both the presence and absence of 6x-His/Importin-ß. The autoradiograph of samples after 7% SDS-PAGE is shown. SDS-PAGE of 2.0 µl IVTT proteins is shown as representing 20% total input. (B) To determine whether Kaiso and Importin-2 form a complex in vivo, we performed coimmunoprecipitation experiments from HCT116 cell lysates with Kaiso- and Importin-2-specific monoclonal antibodies. Importin-2 co-precipitated weakly with endogenous Kaiso (lane 1) but not with p120ctn (lane 2) or the irrelevant negative control anti-HA antibody 12CA5 (lane 4).

View larger version (24K): [in a new window]   Fig. 6. Sequence-specific Kaiso-mediated transcriptional repression is NLS dependent. (A) To determine the effects of the NLS point mutation on Kaiso transcriptional activity, wild-type and NLS-defective Kaiso effectors were cotransfected with the pGL3-4xKBS-luciferase reporter plasmid into HeLa cells. Endogenous Kaiso repressed transcription of the 4xKBS-luciferase reporter 1.6 times. Co-transfection of Kaiso further reduced luciferase activity fivefold (pGL3 + Kaiso), whereas NLS-defective Kaiso had a negligible effect (pGL3 + NLSmut) despite expression levels comparable to those of wild-type Kaiso, as determined by immunoprecipitation and immunoblot analysis in (B). The addition of one copy of the SV40-LTAg/NLS at the C-terminus of NLS-defective Kaiso fully restored Kaiso-mediated transcriptional repression of the luciferase reporter.

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