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

The human Kank protein has a role in controlling the formation of the cytoskeleton by regulating actin polymerization. Besides the cytoplasmic localization as reported before, we observed the nuclear localization of Kank in OS-RC-2 cells. To uncover the mechanism behind this phenomenon, we focused on the nuclear localization signal (NLS) and the nuclear export signal (NES). We found one NLS (NLS1) and two NESs (NES1 and NES2) in the N-terminal region of Kank-L that were absent in Kank-S, and another NLS (NLS2) and NES (NES3) in the common region. These signals were active as mutations introduced into them abolished the nuclear import (for NLS1 and NLS2) or the nuclear export (for NES1 to NES3) of Kank. The localization of Kank in the cells before and after treatment with leptomycin B suggested that the transportation of Kank from the nucleus to the cytoplasm was mediated by a CRM1-dependent mechanism. TOPFLASH reporter assays revealed a positive relationship between the nuclear import of Kank and the activation of β-catenin-dependent transcription. Kank can bind to β-catenin and regulate the subcellular distribution of β-catenin. Based on the findings shown here, we propose that Kank has multiple functions in the cells and plays different roles in the cytoplasm and the nucleus.

Introduction specific nuclear localization of Kank protein, which is mediated by nuclear localization signals (NLSs) and nuclear export signals (NESs). We found one NLS motif (NLS1) predicted by the PSORT II software and three NES motifs (NES1 to NES3) in Kank-L predicted by the NetNES 1.1 software ( Fig. 1B and Fig. 1C). Although another NLS and two other NESs were also predicted by these software packages, the data are not shown here because they all turned out to be inactive. NLS2 was predicted manually by comparison with typical bipartite NLS motifs because it couldn't be recognized by PSORT II software.

Identification of NLSs in Kank-L
We first prepared GST-fused proteins to examine NLS1 located in the N-terminal region of Kank-L ( Fig. 2A). To make a mutation in the NLS1 motif, the amino acids KRRK were substituted with AAAA. The wild-type N-terminal GST-fused form of Kank-L (Nt-WT-GST) was distributed almost equally in the nucleus and cytoplasm in most cells, while the GSTfused construct containing mutant NLS1 (NLS1m-GST) was predominantly detected in the cytoplasm ( Fig. 2B and Fig. 2C). This suggested that NLS1 is a functional NLS and the fusion protein cannot be transported into the nucleus without this signal. Since the other NLS motif (NLS2) was located at the region close to the C-terminus (amino acids 979 to 992), we examined its function with a construct (NLS1m-NESnull-GST-NLS2), which was made by fusing NLS2 to the N-terminal fragment of Kank-L with function-less NLS1 and NESs (NLS1m-NESnull-GST) (Fig. 2D). As the motif clearly contributed to the nuclear localization of the Kank fragment ( Fig. 2E and Fig. 2F), NLS2 is likely to be another functional NLS.
Identification of NESs in Kank-L We next prepared N-terminal GST-fused versions of Kank-L with mutations in either the NES1 or NES2 motif (Fig. 3A). Note that NES1 and NES2 were located close to NLS1 and these constructs contained NLS1. In the mutant constructs, the region of amino acids LDFLKYVDDI was substituted with ADFAKYADDA (NES1m-GST) or the region LETSLPFLTI was substituted with AETSAPFATA (NES2m-GST). As shown in Fig. 3B (a summary in Fig. 3C), both the NES1 and NES2 mutants were predominantly found in the nucleus although the wild-type construct was distributed both in the nucleus and in the cytoplasm. These results suggested that NES1 and NES2 were both functional NESs for the transportation of the fusion protein from the nucleus to the cytoplasm.
Since NES3 is localized far from NLS1, we used a different strategy to analyze its function. We prepared a GST-fused protein without NES1 but with NLS1 and a mutant NES2 (Fig.  3D). This fusion construct was predominantly distributed to the nucleus because it lacked a functional NES ( Fig. 3E; a summary in Fig. 3F). However, after insertion of the region containing the NES3 motif (amino acids LTLLKTNLNL) into this construct, the fusion protein was predominantly localized to the cytoplasm (Fig. 3E). This suggested that NES3 was functional for the transportation of the fusion protein from the nucleus to the cytoplasm.
Confirmation of the NLSs and NESs in the full-length Kank-L As both the NLSs and NESs were examined using GST-fused proteins, we examined whether these NLSs and NESs can function in the full-length Kank-L (Fig. 4). For this purpose, we prepared full-length constructs of Kank-L with mutations in NLSs (NLSm) or NESs (NESm) (Fig. 4A). Since mutations in a single NLS or NES did not result in a clear change in the localization (data not shown), we prepared mutant NLS and NES constructs in which all NLSs or NESs were mutated (NLSm and NESm, respectively). NLSm showed a much greater cytoplasmic localization of Kank-L than the wild type of Kank-L (Kank-L-WT), whereas NESm showed a clear nuclear localization (Fig. 4B and Fig. 4C), confirming that these signals function in the full-length Kank-L. To exclude the possibility that the cytoplasmic localization of NLSm resulted from rapid exonuclear transportation by NESs as seen with Kank-L-WT, the cells were treated with leptomycin B (LMB), which blocks the nuclear export (described below). After the treatment with LMB, the subcellular localization of NLSm was still almost the same as untreated cells (Fig. 4C). This suggests that Kank-L protein possesses only two functional NLSs, as reported here.
Effect of leptomycin B on the subcellular localization of Kank protein It is known that a protein containing an NES can be transported from the nucleus to the cytoplasm mediated by CRM1 (Fornerod et al., 1997;Stade et al., 1997;Gama-Carvalho and Carmo-Fonseca, 2001;Harel and Forbes, 2004), and this process can be inhibited specifically by an antibiotic, leptomycin B (LMB) (Wolff et al., 1997;Kudo et al., 1998;Kudo et al., 1999;Fasken et al., 2000). To examine whether the export of Kank depends on CRM1, we examined the effects of LMB. While Kank protein in human OS-RC-2 cells showed a cytoplasmic localization without LMB treatment, Kank was detected in the nucleus in most cells after treatment with LMB Journal of Cell Science 119 (19) for 8 hours (Fig. 5A). Since the anti-Kank antibody cannot discriminate Kank-L from Kank-S, we transfected vectors expressing FLAG-tagged Kank-L and Kank-S into mouse NIH3T3 cells, respectively. While both Kank-L and Kank-S showed a cytoplasmic localization without LMB treatment, the localization of Kank-L and Kank-S gradually shifted to the nucleus after LMB treatment ( Fig. 5B-D). When the shift of Kank-L was compared with that of Kank-S, it seemed that Kank-L was accumulated in the nucleus more quickly than Kank-S (Fig. 5C,D). Effect of nucleo-cytoplasmic shuttling of Kank on ␤catenin-dependent transcription and nuclear localization In an attempt to explore the function of Kank protein, we screened for the proteins binding to Kank. We found that endogenous Kank can bind to endogenous ␤-catenin, which was revealed by immunoprecipitation (Fig. 6). We then examined the effect of nucleo-cytoplasmic shuttling of Kank on ␤-catenin-dependent transcription (Fig. 7A). After being transported into the nucleus, ␤-catenin associates with the transcription factor LEF/TCF, which results in the transcription of target genes (Logan and Nusse, 2004). We wondered whether Kank protein and its mutants have any effect on this process and examined the transactivation of ␤catenin-LEF using the TOPFLASH/ FOPFLASH reporter genes which were co-expressed with the plasmids used in Fig.  4 (Kank-L-WT, NESm and NLSm), or plasmids either expressing Kank-S or Kank-L with mutated NLS1 (NLS1m). In the FOPFLASH samples where there was a mutation at the LEF/TCF binding site, there was no difference among the samples (data not shown), suggesting that Kank and its mutants had no effect on this process without binding between the ␤-catenin-LEF complex and LEF/TCF binding sites on the reporter gene. In the TOPFLASH samples, conversely, the wild-type Kank-L (Kank-L-WT) up-regulated the expression and an NES-null mutant (NESm) had an effect stronger than the wild type, while an NLS-null mutant (NLSm) had a weaker effect than the wild type, NLS1m and Kank-S. Kank-S showed an intermediate effect, which was similar to that of NLS1m (Fig. 7A). This suggests that the nuclear accumulation of Kank protein could enhance ␤-catenin-dependent transcription and Kank-L is more efficient than Kank-S because of NLS1 in its extra N-terminal region.
Since the expression of Kank shown in Fig. 7A was directed by ectopic vectors, we wonder whether the endogenous expression of Kank regulates ␤-catenin-dependent transcription. We performed RNA interference using the esiRNA technique to suppress the expression of endogenous Kank in HEK293T cells. The efficiency of RNAi was examined by western blot (Fig. 7B, upper panel), in which a Xenopus elongation factor was used as a negative control. As shown in Fig. 7B (lower panel), esiRNA reduced the level of endogenous Kank protein to a fourth compared with the negative control. After transfecting a plasmid expressing ␤-catenin-S33Y, a mutant ␤-catenin resistant to degradation, luciferase activity was increased more than 2.5-fold in TOPFLASH samples (Fig.  7C). However, in the Kank knockdown sample, the activity was decreased to about two-thirds compared with the sample without the knockdown (Fig. 7C). This suggests that Kank affected ␤-catenin-dependent transcription in mammalian cells.
To understand how Kank regulates ␤-catenin-dependent transcription, we examined whether Kank affects the subcellular distribution of ␤-catenin (Fig. 7D,E). While ␤catenin-S33Y was distributed equally in the nucleus and the cytoplasm, the percentage of nuclear ␤-catenin was increased by the co-expression of wild-type Kank-L (Fig.  7D,E). In the cells expressing ␤-catenin with the NLS-null mutant (NLSm), the percentage of cytoplasmic ␤-catenin increased compared with wild-type Kank-L. By contrast, the expression of the NES-null mutant (NESm) resulted in an increased percentage of nuclear ␤-catenin. These results suggested that Kank can promote the nuclear localization of ␤-catenin and potentiate ␤-catenin-dependent transcription.
To examine whether the mutations in the NLS and NESs of Kank affect the binding between Kank and ␤-catenin, we co-transfected the vectors expressing wild-type or mutant Kank and ␤-catenin-S33Y into HEK293T cells. Immunoprecipitation of wild-type and mutant Kank proteins with the anti-FLAG antibody all resulted in co-precipitation of ␤-catenin (Fig. 7F), suggesting that the mutations in the NLS and NESs of Kank did not affect the binding between Kank and ␤-catenin. Binding between Kank and ␤-catenin was detected using wild-type ␤-catenin (data not shown).

Discussion
Nucleo-cytoplasmic transportation of proteins plays an important role in the regulation of many cellular processes, including signal transduction, cell cycle progression and cell proliferation (Gama-Carvalho and Carmo-Fonseca, 2001). In general, these shuttling proteins contain both a nuclear localization signal (NLS) and a nuclear export signal (NES), which are required for the import to the nucleus and the export to the cytoplasm, respectively (Gorlich and Kutay, 1999;Moroianu, 1999;Harel and Forbes, 2004). A well-documented example is p53 protein, which was identified as a nucleocytoplasmic shuttling protein. According to a current model, the activity of p53 is regulated both by restraining the import to the nucleus and by enhancing the exit to the cytoplasm under the normal condition (Boyd et al., 2000;Geyer et al., 2000;Liang and Clarke, 2001).
To determine the presence of NLSs and NESs in shuttling proteins, a series of deletion mutants are needed for examining their subcellular localization. Recently, the prediction of NLS and NES motifs by bioinformatics became available (Nair et al., 2003;la Cour et al., 2004), which enable us to save a time for predicting these motifs and for preparing point mutants. Since the human Kank protein is very large (more than 1000 amino acids for both Kank-L and Kank-S isoforms), it is rather difficult to identify NLSs or NESs in the full-length protein directly. Our strategy adopted here is: first to predict NLS and NES motifs in Kank protein by bioinformatic methods, then to prepare small fragments containing both NLSs and NESs, which are fused to GST protein for quick screening, and finally, to examine point mutations in the full-length Kank protein for

Journal of Cell Science
confirmation. Using this strategy, we demonstrated that Kank is a nucleo-cytoplasmic shuttling protein. The nuclear import or export of Kank-L is likely mediated respectively by two NLS motifs or by three NES motifs through the CRM1/exportin1 pathway. Among a total of five potential NES motifs in Kank-L (data not shown), only three were verified by the experiments reported here. Similarly, among three NLS motifs predicted, two NLSs were experimentally confirmed, one in the N-terminal region of Kank-L (KRRK from positions 65 to 68; NLS1) and the other, a bipartite NLS, in the common region of Kank-L and Kank-S (KKKDGNKDSNGAKK from positions 979 to 992; NLS2).
Kank was detected in the cytoplasm by immunofluorescence microscopy (Sarkar et al., 2002;Rodley et al., 2003;Roy et al., 2005). After the treatment with LMB for a long time (more than 8 hours, Fig. 5B-D), nuclear accumulation of Kank protein was observed. However, the cytoplasmic localization of Kank protein was still detected at a higher level compared with some ␤-catenin binding proteins (Henderson, 2000;Wiechens et al., 2004), which indicates that Kank might bind to some proteins in the cytoplasm and keep a part of Kank protein in the cytoplasm. Therefore, our findings demonstrated that the cytoplasmic distribution of Kank was achieved, if not all, by the NES-mediated nuclear export. Since Kank has a role in controlling the formation of the cytoskeleton through the polymerization of actin (Sarkar et al., 2002;Rodley et al., 2003), it is possible that Kank protein has multiple functions in the cell and play different roles in different subcellular localizations. The Kank protein molecules localized in the cytoplasm might be involved in the polymerization of actin, while the molecules transported into the nucleus might be involved in some other molecular events as discussed below.
When we searched for the binding proteins of Kank, we found that ␤-catenin could bind to Kank in mammalian cells (Fig. 6). However, this binding is likely to be indirect from the results of in vitro pull-down assay (data not shown). Then, we wondered what was the effect of Kank on the function of ␤catenin. Here, we have demonstrated that the nuclear import of Kank promoted the nuclear localization of ␤-catenin, which resulted in the activation of ␤-catenin-mediated transcription (Fig. 7A-E). Although we detected binding between endogenous Kank and endogenous ␤-catenin (Fig. 6), the subcellular distributions of Kank and ␤-catenin do not overlap completely (Fig. 7D). Our explanation for this phenomenon is that the nuclear export of Kank protein is greater than its nuclear import because of preferential localization of Kank in the cytoplasm, and thus, Kank protein might be transported to the cytoplasm soon after its import into the nucleus. By contrast, ␤-catenin is kept in the nucleus to exert its function. However, we cannot exclude the possibility that Kank affects ␤-catenin-dependent transcription through other signaling pathways because a mutant completely depleting of the functional nuclear import signal (NLSm) still showed a low but distinct level of enhancement in ␤-catenin-dependent transcription (Fig. 7A).
In conclusion, we show that Kank is a nucleo-cytoplasmic shuttling protein and the nuclear import of Kank protein accompanies with the nuclear import of ␤-catenin, which upregulates ␤-catenin-dependent transcription. Our data indicate that one should not consider Kank protein as a static molecule that merely localizes in the cytoplasm. On the contrary, Kank shuttles in and out of the nucleus so as to exert multiple functions in the cell and play different roles in the cytoplasm and the nucleus.

Plasmid construction
For the construction of FLAG-tagged Kank N-terminus-GST plasmids, a BamHI-EcoRI cDNA fragment encoding the N-terminal 158 amino acids in Kank-L was inserted into the BamHI/XhoI site of the vector pCMV-Tag 2B (Stratagene) together with a PCR-amplified EcoRI-XhoI fragment from the GST gene. Deletion and mutant constructs of FLAG-tagged Kank-L N-terminus-GST were generated by PCR amplification using the wild-type construct as a template and appropriate primer pairs. To generate NLS1m-NESnull-GST-NLS2, a pair of oligonucleotides encoding NLS2 (KKKDGNKDSNGAKK), 5Ј-AATTCAAGAAGAAAGATG-GTAACAAAGATTCAAATGGCGCAAAAAAGG-3Ј (forward) and 5Ј-AATTC-CTTTTTTGCGCCATTTGAATCTTTGTTACCATCTTTCTTCTTG-3Ј (reverse), were annealed and inserted into the EcoRI site, which was located between the Kank N-terminal fragment and GST. To generate NESnull-GST-NES3, a pair of primers encoding NES3 and its neighboring amino acids (DLTLLKTNLNLK), 5Ј-AATTC-GACCTCACACTCCTCAAGACAAACTTGAATCTCAAAG-3Ј (forward) and 5Ј-AATTCTTTGAGATTCAAGTTTGTCTTGAGGAGTGTGAGGTCG-3Ј (reverse), were annealed and inserted into the EcoRI site, which was located between the Kank N-terminal fragment and GST. The mutant constructs of full-length Kank-L were generated by PCR amplification using the FLAG-tagged wild-type Kank-L Fig. 7. The effect of Kank protein on ␤-catenin-dependent transcription and nuclear localization. (A) The effect of Kank protein on ␤-catenin-dependent transcription examined by TOPFLASH reporter gene assay. SW480 cells were transfected with the reporter gene plasmid (TOPFLASH) together with vectors expressing wildtype and mutant Kank proteins. The pCMV Tag-2B cloning vector (Vector) was used to show the background level of ␤-catenindependent 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 ␤-catenin-dependent transcription examined by TOPFLASH reporter gene assay. The reporter gene plasmid (TOPFLASH) with or without ␤-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 ␤-catenin-S33Y alone or ␤-catenin-S33Y with FLAG-tagged wild-type Kank-L or FLAG-tagged Kank-L with mutant NLS (NLSm) or NES (NESm) motifs. ␤-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 ␤-catenin protein. The asterisks indicate P<0.05 by t test. (F) Binding between FLAG-tagged Kank and ␤-catenin. All constructs used were the same as those used in (D). IP, immunoprecipitation. Bar, 20 m. construct as a template and appropriate primers. All of the constructs were verified by DNA sequencing.

Immunofluorescence staining and microscopy
Plasmid DNA (1.5 g) was transiently transfected into NIH3T3 cells grown on fibronectin-coated glass coverslips with PolyFect (QIAGEN). Twenty-four hours after transfection, the cells were fixed with 3.7% formaldehyde in PBS for 15 minutes. The cells were then treated with 0.5% Triton X-100 for 5 minutes and with fresh 0.1% sodium borohydride in PBS for another 5 minutes (sodium borohydride treatment was omitted in the case of examination of ␤-catenin localization). Next, after the cells had been treated with 5% goat serum for 30 minutes, they were incubated with anti-FLAG M2 (Sigma) or anti-␤-catenin (BD Biosciences) antibodies for 1 hour, and visualized using FITC-or Rhodamine-labeled antibodies (Molecular Probe) under a fluorescence microscope (Axioskop, Zeiss). DAPI staining was performed for 10 minutes after incubation with a secondary antibody when necessary. In all experiments presented here, more than one hundred FITCpositive cells were counted and classified according to their subcellular localization.