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Previous Article | Next Article 
Molecular and Cellular Biology, December 2006, p. 8857-8867, Vol. 26, No. 23
0270-7306/06/$08.00+0 doi:10.1128/MCB.01031-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Departments of Urology and Genetics, Stanford University School of Medicine, Stanford, California 94305-5118
Received 8 June 2006/ Returned for modification 19 June 2006/ Accepted 30 August 2006
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß-Catenin accumulates in the cytoplasm and nucleus in response to Wnt signaling (33). The nuclear shift of ß-catenin reflects both an increase in total proteins and an enhanced nuclear targeting of ß-catenin (2, 38). It has been shown that the central armadillo repeats of ß-catenin directly bind the nuclear pore complex, mediating its own nuclear import (20). The CRM1/exportin nuclear export pathway has been proposed in regulation of the nuclear export of ß-catenin. Amino acid sequences responsible for highly efficient nuclear export (NES) have been identified in an increasing number of proteins, such as the human immunodeficiency virus (HIV) type 1 Rev protein (8). The "classical" Rev-like NES is characterized by a leucine-rich sequence that binds CRM1/exportin to facilitate the nuclear export of the protein (10, 11). The tumor suppressor APC contains functional Rev-like NES motifs that are necessary for its leptomycin B (LMB)-sensitive nuclear exclusion (13, 27). It has been shown that ectopic and endogenous APC can associate with ß-catenin in the nucleus and induce a translocation of ß-catenin from the nucleus to the cytoplasm. While the precise role of APC in nuclear-cytoplasmic shuttling remains unclear, multiple lines of evidence suggest that it can affect the subcellular distribution and degradation of ß-catenin (13, 27).
Although previous studies have suggested that the nuclear accumulation of ß-catenin in colon cancer cells may correlate with a loss of nuclear export function of mutated APC (36), overwhelming evidence has suggested that it may also be regulated by an APC-independent mechanism (12, 18). Moreover, ß-catenin nuclear export can occur even when the CRM1 export pathway is blocked by LMB (6). Additionally, the overexpression of CRM1 in an APC-mutated colon cancer cell line, SW480, stimulated the export of endogenous nuclear ß-catenin (6). These findings suggest that other mechanisms may be involved in the regulation of the nuclear export of ß-catenin, including a yet-undefined CRM1-dependent but APC-independent pathway.
The FEZ1/LZTS1 (leucine zipper, putative tumor suppressor 1) gene was identified by positional cloning from 8p21-22, a region that is frequently lost in human tumors (15). Although previous studies have shown that FEZ/LZTS1 may regulate cell mitosis, little is known about its biological roles in cells. Using a BLAST search, a sequence similar to that of FEZ1/LZTS1, named leucine zipper tumor suppressor 2 (LZTS2)/LAPSER1, was identified within human 10q24.3, which is frequently deleted in various human cancers (1). Like FEZ1/LZTS1, LZTS2 contains several domains and motifs, including leucine zippers. Overexpression of LZTS2 showed an inhibitory effect on cell growth (1).
Here, we present multiple lines of evidence in vitro and in vivo to demonstrate that LZTS2 is a novel ß-catenin-interacting protein and represses ß-catenin-mediated transcription on T-cell factor/lymphoid enhancing factor (TCF/LEF) and androgen receptor-mediated transcription. Particularly, we identified a Rev-like leucine-rich, CRM1/exportin-regulated NES sequence at the C terminus of LZTS2, through which LZTS2 can regulate the cellular localization of ß-catenin. Our data show that LZTS2, acting as a ß-catenin-interacting protein, regulates the cellular distribution and transcriptional activity of ß-catenin through its nuclear export activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid construction. A yeast clone containing the truncated cDNA fragment of KIAA1813 (amino acids 118 to 669) was isolated in the screen. A BLAST search found that the sequence of KIAA1813 is identical with those of LZTS2, LAPSER, and several human expressed sequence tag cDNA clones. A full-length LZTS2 fragment was created by combining the cDNA fragment that encodes the amino-terminal region isolated from KIAA1813 cDNA and the yeast clone isolated from the yeast two-hybrid assay in the pcDNA3 vector with or without an amino-terminal FLAG epitope tag. Using this as a template, the C-terminal and internal deletions of LZTS2 clones were generated by PCR with specific primers containing the appropriate restriction enzyme sites. After cleavage, the fragments containing different portions of LZTS2 were cloned downstream of the GAL4 transactivation domain in the pACT2 vector (Clontech). Mutants of LZTS2 with single or double point mutations within the NES were generated by PCR-based mutagenesis. Plasmids encoding green fluorescent protein (GFP) fused to potential NES sequences from LZTS2 or from the human immunodeficiency virus type 1 Rev protein were generated by inserting double-stranded oligonucleotides into the pEGFP-C1 plasmid with appropriate restriction enzyme sites (Clontech). The oligonucleotide sequences included HIV-Rev (amino acids 75 to 83), 5'-TCGAGCTCTACCACCGCTTGAGAGACTTACTCTT-3'; LZTS2 (amino acids 134 to 143), 5'-TCGAGAGCTGGAGAAGAACATGGAGAAGATCCTGATC-3'; amino acids 389 to 397, 5'TCGAGAGCTGCTGCAGCTGCAGGTGTTCCAGCTG-3'; aa 471 to 478, 5'-AATTCAGCGCCACCCGCAGAGCCACCAGCTC-3; aa 571 to 580, 5'-TCGAGGCTTGCGGGCCCAGGTGGAGCGATTGCGGGTG-3'; and aa 631 to 642, 5'-TCGAGAGCTAGAGCAGGAGCTGCAGCAGCTCAGCCTGGAGCTG-3'. The pLentiviral LZTS2 expression constructs were generated in the pLenti-super vector (5).
Topflash (pGL3-OT) and Flopflash luciferease (pGL3-OF) reporters were obtained from Bert Vogelstein. pSV-ß-galactosidase is a commercial reporter plasmid (Promega, Madison, WI).
Cell cultures and transfections. The monkey kidney cell line, CV-1, human prostate cancer cell lines, PC3 and DU145, and human colon cancer cell line, SW480.7, were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (HyClone, Denver, CO). An androgen receptor-positive prostate cancer cell line, LNCaP, was maintained in T-medium (Invitrogene, Carlsbad, CA) with 5% fetal calf serum.
Transient transfections were carried out using a Lipofectamine transfection kit or Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transient transfection and reporter assays were performed as described previously (43). The relative light units from individual transfections were normalized by ß-Gal activity in the same samples. Individual transfection experiments were done in triplicate, and the results are reported as mean RLU/ß-Gal (± standard deviation).
In vitro binding assay. Glutathione S-transferase (GST)-ß-catenin fusion proteins were constructed in the pGEX-4T-1 vector (Amersham, Arlington Heights, IL). Expression and purification of GST fusion proteins were performed according to the manufacturer's instructions. Full-length human LZTS2 proteins were generated and 35S labeled by in vitro transcription and translation with a TNT-coupled reticulocyte lysate system (Promega). Equal amounts of GST fusion proteins coupled to glutathione-Sepharose beads were incubated with 35S-labeled proteins at 4°C for 2 h in the lysis buffer as described previously (43). GST fusion proteins were then eluted by incubating them with buffer containing 10 mM glutathione and 50 mM Tris-HCl (pH 8.0) for 10 min at room temperature. The bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography.
Antibody production. The polypeptide (amino acids 332-RDREAELQQLRDSLDEN-350) was chosen from the central region of LZTS2 and synthesized as a source of antigen for antibody production. A rabbit polyclonal, affinity-purified antibody against human LZTS2 was produced by Proteintech Group, Inc. (Chicago, IL). Antibody specificity for the LZTS2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay.
Preparation of whole-cell lysates, immunoprecipitation, and Western blotting. Cells transfected with ß-catenin and LZTS2 expression constructs were washed with phosphate-buffered saline (PBS) and resuspended in RIPA buffer (1% NP-40, 0.1% SDS, 50 mM NaF, 0.2 mM Na3VO4, 0.5 mM dithiothreitol, 150 mM NaCl, 2 mM EDTA, 10 mM sodium phosphate buffer, pH 7.2). Nuclear extracts were prepared according to the method of Dignam et al., with minor modifications (4).
Whole-cell lysates or nuclear extracts were first precleared with protein A-Sepharose beads for 1 h and then incubated with mouse or rabbit normal immunoglobulin G (IgG) or specific antibodies conjugated with preequilibrated protein A-Sepharose beads at 4°C for 2 h. The beads were collected by centrifugation and washed. Proteins were eluted by boiling in SDS sample buffer, resolved on 10% polyacrylamide gels, and transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in Tris-buffered saline with Tween for 1 h and then probed with anti-FLAG M2 (Sigma, St. Louis, MO) and anti-ß-catenin (Signaling Transduction lab, Lexington, KY), followed by incubation with species-specific horseradish peroxidase-conjugated antibodies.
Immunofluorescence. CV-1, PC3, or SW480.7 cells were plated onto chamber slides 16 h before transfection. pcDNA3-FLAG-LZTS2, pcDNA3-FLAG-LZTS2 (L638/640A), and pcDNA3-FLAG-APC were transfected with or without pCMG2-HA-ß-catenin into cells with the Lipofectamine reagent (Invitrogen). Transfected cells were incubated for 24 h and fixed for 10 min (CV-1) or 30 min (PC3 and SW480) with 4% paraformaldehyde. Cells were then permeablized and blocked for nonspecific sites with 0.04% Triton X-100-5% goat serum-PBS buffer for 30 min and incubated with appropriate primary antibodies overnight at 4°C. Cells were washed three times, followed by incubation with appropriate secondary antibodies. Samples were also counterstained with 1 ng/ml 4',6'-diamidino-2-phenylindole (DAPI). The pEGFP-C1-NES constructs were transfected into CV-1 or PC3 cells and were examined with an inverted epiflourescence microscope (Zeiss, Thornwood, NY) after 16 h.
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RESULTS |
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Alignment of LZTS2 with FEZ1/LZTS1 and LZTS3, which was identified recently (39), showed significant sequence similarity among these proteins, particularly in the C-terminal potential leucine zipper regions (Fig. 1A). Interestingly, the nuclear export sequence identified in LZTS2 is not conserved in both the LZTS1 and the LZTS3 proteins but is highly conserved in the orthologs of LZTS2 across species (Fig. 1B). These observations suggest a potential role of LZTS2 in the regulation of nuclear and cytoplasmic trafficking, which may distinguish it from LZTS1 and LZTS3.
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LZTS2 interacts with ß-catenin in vitro and in vivo. The physical interaction between LZTS2 and ß-catenin was further assessed by GST pull-down experiments. A series of GST fusion proteins containing full-length ß-catenin and truncated mutants was generated and immobilized onto a glutathione-Sepharose matrix. The binding of [35S]methionine-labeled LZTS2 to GST-ß-catenin fusion proteins was analyzed by SDS-PAGE and visualized by autoradiography. As shown in Fig. 2D, the GST fusion protein containing full-length armadillo repeats, GST-ß-cat(134-671), showed a more pronounced interaction with LZTS2 than full-length ß-catenin or the other truncated mutants. The data are consistent with results of our yeast two-hybrid experiments and suggest a structural significance of the armadillo repeats for the interaction with LZTS2.
To confirm that full-length LZTS2 and ß-catenin proteins are physically associated in intact cells, we carried out coimmunoprecipitation assays. ß-Catenin and FLAG-tagged LZTS2 were coexpressed in CV-1 cells. Whole-cell lysates containing equal amounts of the proteins were immunoprecipitated with normal mouse IgG or a ß-catenin or FLAG monoclonal antibody. As shown in Fig. 2E, the FLAG-LZTS2 protein was detected in the immunoprecipitates with the ß-catenin antibody but not with IgG. Additionally, in the reverse coimmunoprecipitation assay, ß-catenin was found specifically in the immunoprecipitates with the FLAG antibody (Fig. 2F). To detect the interaction between endogenous proteins, we produced a specific antibody against the LZTS2 protein and used it in the coimmunoprecipitation experiments. As shown in Fig. 2G, endogenous ß-catenin was specifically detected in the immunoprecipitates pulled down by the LZTS2 antibody. Taken together, these data demonstrate a biologically relevant interaction between LZTS2 and ß-catenin.
LZTS2 represses ß-catenin-mediated transcription. Transient transfection assays were performed to investigate the possible effect of LZTS2 on ß-catenin-mediated transcription on TCF/LEF (25). First, we examined the repressive effect of LZTS2 on ß-catenin-mediated enhancement of TCF-1 regulated transcription. Expression constructs of TCF-1 and ß-catenin were transfected with pGL3-OT and pGL3-OF in three prostate cancer cell lines, LNCaP, PC3, and DU145. Coexpression of TCF-1 and ß-catenin showed a transcription induction of the pGL3-OT promoter in all three cell lines (Fig. 3A, B, and C). Cotransfection of LZTS2 showed a dosage-dependent repression on OT promoters in all cell types examined. However, there was no significant change in the samples transfected with the OF promoter/reporter. These data provide the first line of evidence that demonstrates that LZTS2 represses the activity of ß-catenin on TCF-mediated transcription.
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LZTS2 localizes mainly in the cytoplasm, and its subcellular distribution is affected by leptomycin B. Next, we examined the subcellular localization of the LZTS2 protein. Transfection with expression plasmids encoding FLAG-tagged LZTS2 in CV-1 cells showed that most of the exogenous LZTS2 proteins were exclusively located in the cytoplasm, surrounding the nuclear envelope (Fig. 4A). To confirm this observation, we generated a specific antibody to LZTS2 and used it to assess the cellular distribution of the endogenous protein. As shown in Fig. 4B, endogenous proteins showed a staining similar pattern to that of ectopically expressed LZTS2 proteins, confirming the validity of the overexpression models. To assess the mechanism of the exclusive cytoplasmic localization of LZTS2, we examined the effect of LMB, a specific inhibitor for CRM1/exportin-alpha-dependent nuclear export, on the subcellular distribution of LZTS2 (28). In the presence of 60 ng/ml LMB, either exogenous or endogenous LZTS2 show a clear nuclear retention in CV-1 and PC3 cells, respectively (Fig. 4C and D). These data suggest that the exclusive cytoplasmic localization of LZTS2 is the result of the specific export of LZTS2 proteins from the nuclei, which appears to be regulated by the CRM-1/exportin-alpha pathway.
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The consensus sequence of NES is defined as a set of critically spaced hydrophobic residues, usually leucines, such as L(XXX/XX)L(XX/XXX)LXL (10, 28). The sequence between amino acids 631 and 640 in LZTS2 shows a perfect match to the consensus NES sequence (Fig. 4E). To confirm that this sequence is responsible for the nuclear export of LZTS2, we replaced leucine with alanine at either aa 638 or 640 or both positions by a PCR-based, site-directed mutagenesis technique (Fig. 4G). These mutants and wild-type LZTS2 were ectopically expressed in CV-1 cells. Staining of transfected cells with anti-FLAG antibodies showed that FLAG-LZTS2 NES mutated proteins are mainly retained in the nuclei either in the presence or absence of LMB (Fig. 4H). In contrast, as we have observed above, wild-type LZTS2 was excluded from the nuclei (Fig. 4A and C). These data confirm that the sequence between 631 and 640 is a functional NES site and is required for the nuclear export of LZTS2 through the CRM1/exportin-alpha pathway.
LZTS2-dependent nuclear export of ß-catenin requires an intact NES. A physical interaction between LZTS2 and ß-catenin has been demonstrated in this study. One possible mechanism for the regulation of ß-catenin by LZTS2 is through the modulation of subcellular localization of ß-catenin. To test this hypothesis, we cotransfected LZTS2 and ß-catenin into CV-1 cells. As reported earlier (17), ectopically expressed ß-catenin mainly localized in the nuclei (Fig. 5A). Interestingly, coexpression of LZTS2 with ß-catenin showed that both proteins were excluded from nuclei and colocalized in the cytoplasm in the absence of LMB (Fig. 5B, top panels). However, most of the LZTS2 and ß-catenin proteins colocalize and accumulate in nuclei in the presence of LMB, whereas some cells showed a nuclear and cytoplasmic distribution of the proteins (Fig. 5B, bottom panels). To further evaluate the effect of LZTS2 on the subcellular localization of ß-catenin in a physiologically relevant cellular context, we examined the colocalization of endogenous LZTS2 and ß-catenin in PC3 cells. The cells treated with 60 ng/ml LMB for 24 h showed an accumulation of both LZTS2 and ß-catenin in the nuclei (Fig. 5D, bottom panels). In contrast, most ß-catenin and LZTS2 colocalized in the cytoplasm of untreated cells (Fig. 5D, top panels).
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Using the shRNA construct of LZTS2, we also assessed the effect of endogenous LZTS2 in regulation of cellular distribution of ß-catenin. As shown in Fig. 5G, knockdown of endogenous LZTS2 proteins results in an accumulation of nuclear ß-catenin proteins in SW480 cells. However, no specific effect was observed in cells transfected with a control shRNA vector. The above results are correlated with our previous observation from the transient transfection experiments (Fig. 3E), suggesting that the repressive effect of LZTS2 on ß-catenin-regulated transcription is mainly mediated through regulation of ß-catenin nuclear export.
LZTS2 affects endogenous ß-catenin in SW480 cells. As described above, SW480 cells possess mutated APC (29), resulting in a marked accumulation of ß-catenin in the nucleus of cells (26). Introducing wild-type APC into the cells increases the degradation of ß-catenin in SW480 cells. In order to assess a direct role of LZTS2 in the regulation of ß-catenin, we expressed both the wild type and the NES mutant of LZTS2 in SW480 cells. Full-length LZTS2, as well as APC, mainly localize in the cytoplasm of SW480 cells (Fig. 6A and B, left panels). As reported earlier (7), the nuclear level of ß-catenin is significantly decreased in the cells transfected with wild-type APC (Fig. 6A). Intriguingly, overexpression of wild-type LZTS2 also reduces the level of nuclear ß-catenin (Fig. 6B), but the LZTS2 NES mutant shows no significant effect on endogenous ß-catenin (Fig. 6C). The intensity of ß-catenin staining was measured and scored in the above cells (Fig. 6D). It appears that expression of the ectopic APC or LZTS2 wild-type protein reduces the nuclear level of ß-catenin. In addition, the level of cytoplasmic ß-catenin was also slightly reduced in the above cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we provide several lines of evidence to explore a link between LZTS2 and the ß-catenin-mediated cell signaling pathway. We first identified that LZTS2 interacts with ß-catenin using a yeast two-hybrid approach. The interaction was further confirmed by both in vitro GST pulldown and coimmunoprecipitation experiments. The full-length armadillo repeats of ß-catenin (amino acids 134 to 671) were mapped as being responsible for the interaction. Binding of LZTS2 to the central armadillo domain is consistent with previous findings, which have shown that the armadillo repeats provide a long positively charged groove for binding (14). In this study, we also showed that the C terminus of LZTS2 (aa 447 to 669) contains a unique leucine zipper region and is responsible for binding to ß-catenin. Interestingly, a sequence similar to a ß-catenin binding motif was found between amino acids 608 and 613 (EqVIrY) within a largely acidic region of LZTS2 (3). It should be noted that this region is very similar to the one of FEZ1/LZTS1, suggesting a potential link between FEZ1/LZTS1 and ß-catenin.
In this study, we also showed a functional consequence of the interaction between LZTS2 and ß-catenin. Expression of LZTS2 can repress ß-catenin-mediated transcription on the TCF/LEF-regulated promoter. However, no significant repression was observed in transient-transfection assays when full-length and truncated LZTS2 were targeted through fusion to the GAL4 DNA binding domain (data not shown). These data indicate that LZTS2 may not contain an intrinsic transcriptional repressive domain, and the repression of ß-catenin by LZTS2 may be mediated through other mechanisms. In the course of a search for the mechanisms by which LZTS2 regulates ß-catenin-mediated transcription, we demonstrated that both exogenous and endogenous LZTS2 localize exclusively in the cytoplasm, with a perinuclear staining pattern (Fig. 4A to D). Using an immunofluorescence approach, we further observed that the nuclear exporting of LZTS2 is blocked by LMB, suggesting that the CRM-1-dependent pathway is involved in the regulation of the subcellular distribution of LZTS2. Several NES-like sequences appear in the LZTS2 protein. Expression of the GFP fusion proteins with these NES sequences in frame identified the C-terminal sequence (amino acids 631 to 642) as capable of regulating the nuclear export of the GFP fusion protein. Mutation of this NES site by substitution of either one or two leucines with alanine diminishes the nuclear export of LZTS2. Overall, these data suggest an important role of LZTS2 in nuclear and cytoplasmic trafficking.
Previous studies have suggested that the intracellular trafficking of ß-catenin is regulated by the CRM/exportin nuclear export pathway (6, 13). Since LZTS2 interacts with ß-catenin and its subcellular distribution is regulated by the CRM-dependent nuclear export signal through the intrinsic NES, we presented several lines of evidence to explore the possibility that LZTS2 regulates ß-catenin nuclear export. First, the expression of wild-type LZTS2 increased the cytoplasmic level of endogenous or ectopically expressed ß-catenin, and treating cells with leptomycin B blocked the effect of LZTS2, resulting in the nuclear retention of both the ß-catenin and LZTS2 proteins. Moreover, the LZTS2 NES mutants showed no effect on ß-catenin under similar experimental conditions. Furthermore, the expression of wild-type LZTS2 but not the NES mutant significantly reduced the level of endogenous nuclear ß-catenin in SW480 cells. These data suggest that LZTS2 regulates the nuclear trafficking of ß-catenin and affects the cellular level of ß-catenin through an APC-independent degradation pathway in SW480 cells.
The tumor suppressor APC has been shown to affect the nuclear-cytoplasm shuttling of ß-catenin (36). Several functional NES sequences have been identified within the APC protein. However, the significant exodus of ß-catenin from the nucleus appears in APC-mutated tumor cells (6), which suggests that an additional CRM1-dependent export pathway may exist and be involved in the regulation of ß-catenin. Our data that show the role of LZST2 in the regulation of ß-catenin nuclear export agree with the previous observation and provide evidence demonstrating a novel regulatory pathway for ß-catenin. Since cellular levels of ß-catenin are tightly regulated in normal cells, mutations affecting the degradation of ß-catenin can result in increasing the cellular level of ß-catenin, which has been considered a key event in tumorigenesis (33). APC-dependent proteasomal degradation pathways for ß-catenin are mediated through either the APC/Axin complex (32) or a p53-inducible pathway involving Siah-1 (22, 23). In addition, other APC-independent pathways have also been suggested to regulate the cellular level of ß-catenin (37, 42). In this study, we observed that overexpression of LZTS2 reduces the level of ß-catenin in SW480 cells. Given the fact that LZTS2 has been shown previously to inhibit the proliferation of human tumor cells (1), it would be interesting to further investigate whether the inhibitory role of LZTS2 in cell growth is mediated through the regulation of ß-catenin.
The sequence analysis showed that LZTS2 is very similar to a putative tumor suppressor, FEZ1/LZTS1. The FEZ1/LZTS1 gene was mapped to chromosome 8p22, a region that is frequently deleted in human tumors (15). Previous studies have shown that the expression of FEZ1/LZTS1 was altered in multiple human malignancies (30, 40, 41). Currently, the precise role of FEZ1/LZTS1 as a tumor suppressor still remains unclear. Based on the sequence similarity between LZTS2 and FEZ1/LZTS1, defining the functions of these two proteins in tumorigenesis will be extremely important.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grants CA70297, CA87767, and DK61002.
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FOOTNOTES |
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Published ahead of print on 25 September 2006.
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