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First published online 12 September 2006
doi: 10.1242/jcs.03169

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Nucleo-cytoplasmic shuttling of human Kank protein accompanies intracellular translocation of ß-catenin

Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

View larger version (34K): [in a new window]   Fig. 1. Intracellular distribution of Kank protein and locations of potential NLS and NES motifs. (A) Intracellular distribution of Kank protein. The localization of endogenous Kank protein in OS-RC-2 (upper) and VMRC-RCW (lower) cells was examined by immunostaining with an FITC-conjugated antibody against Kank protein. The nuclei were stained with DAPI. LM, light microscopy. Bar, 20 µm. (B) The sequences of NLS (NLS1 and NLS2) and NES (NES1 to NES3) motifs predicted in Kank-L. The conserved sites are underlined (NLSs) or shadowed (NESs), which were substituted by alanines in the respective mutants. (C) The maps of Kank-L and Kank-S.

View larger version (57K): [in a new window]   Fig. 2. Identification of NLSs. (A) Schematic structures of FLAG-tagged GST (FLAG-GST) or GST constructs of FLAG-tagged N-terminal Kank with wild-type (Nt-WT-GST) or mutant NLS1 (NLSm-GST). (B) Representative images of NIH3T3 cells transiently transfected with the constructs shown in (A). The localization of FLAG-tagged GST-fused proteins was monitored by immunostaining with an FITC-conjugated antibody and the nucleus was stained with DAPI. LM, light microscopy. (C) Quantification of the cells according to the localization of GST-fused proteins. The cells with FITC signals predominantly in the nucleus or in the cytoplasm, or located equally in both, were classified and scored respectively. (D) Schematic structures of GST constructs of FLAG-tagged N-terminal Kank with mutant NLS1 and NESs (NLS1m-NESnull-GST), or mutant NLS1 and NESs, and intact NLS2 (NLS1m-NESnull-GST-NLS2). (E) Representative images of NIH3T3 cells transiently transfected with the fusion constructs shown in (D). The localization of FLAG-tagged GST-fused protein was monitored by immunostaining with an FITC-conjugated antibody and the nucleus was stained with DAPI. (F) Quantification of the cells according to the localization of GST-fused proteins. Bar, 20 µm.

View larger version (54K): [in a new window]   Fig. 3. Identification of NESs. (A) Schematic structures of FLAG-tagged GST (FLAG-GST) or GST constructs of FLAG-tagged N-terminal Kank with wild-type (Nt-WT-GST), or mutant NES1 (NES1m-GST) or mutant NES2 (NES2m-GST). (B) Representative images of NIH3T3 cells transiently transfected with the fusion constructs shown in (A). The localization of FLAG-tagged GST fused protein was monitored by immunostaining with an FITC-conjugated antibody and the nucleus was stained with DAPI. LM, light microscopy. (C) Quantification of the cells according to the localization of GST-fused proteins. (D) Schematic structures of GST constructs of FLAG-tagged partial Kank without NESs (NESnull-GST) or with NES3 (NESnull-GST-NES3). In NESnull-GST, the first NES was deleted and the second was mutated. (E) Representative images of NIH3T3 cells transiently transfected with the fusion constructs shown in (D). The localization of FLAG-tagged GST fused protein was monitored by immunostaining with an FITC-conjugated antibody and the nucleus was stained with DAPI. (F) Quantification of the cells according to the localization of GST-fused proteins. Bar, 20 µm.

View larger version (39K): [in a new window]   Fig. 4. Localization of full length Kank-L and its mutants. (A) Schematic structures of FLAG-tagged full-length Kank-L constructs with wild-type NLS and NES motifs (Kank-L-WT), or with mutant NLS (NLSm) or NES (NESm) motifs. (B) Representative images of NIH3T3 cells transiently transfected with the fusion constructs shown in (A). The cells transfected with NLSm were treated with or without leptomycin B. The localization of FLAG-tagged GST fused protein was monitored by immunostaining with an FITC-conjugated antibody and the nucleus was stained with DAPI. LM, light microscopy. (C) Quantification of the cells according to the localization of GST-fused proteins. Bar, 20 µm.

View larger version (60K): [in a new window]   Fig. 5. The effect of leptomycin B on the localization of Kank protein. (A) Representative images of OS-RC-2 cells showing endogenous Kank protein detected with anti-Kank antibody before or after treatment with leptomycin B (LMB). LMB-, the cells without LMB treatment; LMB+, the cells treated with LMB for 8 hours. (B) Representative images of NIH3T3 cells transiently transfected with the constructs of Kank-L or Kank-S and the nucleus was stained with DAPI. (C) Quantification of the cells according to the localization of FLAG-tagged Kank-L protein after treatment with LMB for the periods indicated. (D) Quantification of the cells according to the localization of FLAG-tagged Kank-S protein after treatment with LMB for the periods indicated. The results in (C) and (D) were scored from triplicate transfections. Bar, 20 µm.

View larger version (34K): [in a new window]   Fig. 6. In vivo binding assay between Kank and ß-catenin. Immunoprecipitation (IP) of the lysate from VMRC-RCW cells with an anti-Kank antibody, an anti-ß-catenin antibody or with control IgG was examined by western blotting using antibodies against Kank or ß-catenin.

View larger version (36K): [in a new window]   Fig. 7. The effect of Kank protein on ß-catenin-dependent transcription and nuclear localization. (A) The effect of Kank protein on ß-catenin-dependent transcription examined by TOPFLASH reporter gene assay. SW480 cells were transfected with the reporter gene plasmid (TOPFLASH) together with vectors expressing wild-type and mutant Kank proteins. The pCMV Tag-2B cloning vector (Vector) was used to show the background level of ß-catenin-dependent transcription. The constructs for Kank-L-WT, NESm and NLSm are the same as shown in Fig. 4. The constructs for Kank-L with a function-less NLS1 (NLS1m) and Kank-S were also included. Luciferase activity was normalized against Renilla luciferase activity. The results were obtained from triplicate transfections. (B) The efficiency of Kank RNAi. The level of endogenous Kank protein was examined by western blotting (top) and quantified (bottom). (C) The requirement of endogenous Kank protein for ß-catenin-dependent transcription examined by TOPFLASH reporter gene assay. The reporter gene plasmid (TOPFLASH) with or without ß-catenin-S33Y or esiRNA against Kank were transfected into HEK293T cells. Luciferase activity was normalized against Renilla luciferase activity. The results were obtained from triplicate transfections. (D) Representative images of NIH3T3 cells transiently transfected with the constructs expressing ß-catenin-S33Y alone or ß-catenin-S33Y with FLAG-tagged wild-type Kank-L or FLAG-tagged Kank-L with mutant NLS (NLSm) or NES (NESm) motifs. ß-catenin was visualized with an FITC-conjugated antibody and Kank was visualized with a rhodamine-conjugated antibody. The nucleus was stained with DAPI. (E) Quantification of the Kank-positive cells according to the localization of ß-catenin protein. The asterisks indicate P<0.05 by t test. (F) Binding between FLAG-tagged Kank and ß-catenin. All constructs used were the same as those used in (D). IP, immunoprecipitation. Bar, 20 µm.