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J. Biol. Chem., Vol. 275, Issue 30, 23139-23145, July 28, 2000

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Truncated Form of Importin  Identified in Breast Cancer Cell Inhibits Nuclear Import of p53^*

Il-Soo Kim, Dong-Hwan Kim, Su-Mi Han, Mi-Uk Chin, Hye-Jung Nam, Hyun-Pil Cho, Sang-Yong Choi, Byung-Joo Song, Eun-Ryoung Kim, Yong-Soo Bae, and Young-Ho Moon^§

From the Sung Ae Life Science Research Institute, Kyeonggi 423-030, South Korea and the  Department of Microbiology, Hannam University, Ojeong-dong 133, Daeduk-gu, Taejeon 306-791, South Korea

Received for publication, November 17, 1999, and in revised form, April 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Disruption of the function of tumor suppressor proteins occasionally can be dependent on their subcellular localization. In about 40% of the breast cancer tissues, p53 is found in the cytoplasm as opposed to the nucleus, where it resides in normal breast cells. This means that the regulation of subcellular location of p53 is an important mechanism in controlling its function. The transport factors required for the nuclear export of p53 and the mechanisms of their nuclear export have been extensively characterized. However, little is known about the mechanism of nuclear import of p53. p53 contains putative nuclear localization signals (NLSs) which would interact with a nuclear transport factor, importin . In this report we demonstrate that importin  binds to NLSI in p53 and mediates the nuclear import of p53. Reverse transcriptase-polymerase chain reaction and sequencing analyses showed that a truncated importin  deleted the region encoding the putative NLS-binding domain of p53, suggesting that it could not bind to NLSs of p53 proteins. Binding of importin  to p53 was confirmed by using yeast two-hybrid assay. When expressed in CHO-K1 cells, the truncated importin  predominantly localized to the cytoplasm. In truncated importin  expressing cells, p53 preferentially localized to cytoplasmic sites as well. A significant increase in the p21^waf1/cip1 mRNA level and induction of apoptosis were also observed in importin  overexpressing cells. These results strongly suggest that importin  functions as a component of the NLS receptor for p53 and mediates nuclear import of p53.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p53 is a tumor suppressor gene and various p53 gene mutations are found in over 50% of all human cancers (1). Although inactivation of tumor suppressor proteins is generally thought to originate in their genetic mutations, disruption of their function can occasionally be independent of such mutations. Moll et al. (2) have reported that about 37% out of 27 samples of breast cancer tissues showed cytoplasmic localization of wild-type p53, resulting in inhibition of normal p53 function (2). Nuclear exclusion of wild-type p53 has also been reported in neuroblastoma and colon carcinoma cells (3, 4). In another study, wild-type p53 was located in the cytoplasm of human cervical carcinoma cell lines with integrated human papillomavirus-18 or -16 (5). In colon carcinoma, cytoplasmic accumulation of p53 correlates with unfavorable prognosis (4). These data indicate that the regulation of p53 subcellular location is an important mechanism in controlling p53 function.

In eukaryotic cells, the nucleus is separated from the cytoplasm by the nuclear envelope. This spatial segregation requires a nuclear transport system to correctly import or export nuclear components at the proper time. The prototype of the nuclear transport signal is the classical nuclear localization signal (NLS),¹ and nuclear import of proteins bearing an NLS is dependent on two cellular factors termed importin  and importin  (6-11). The initial cytoplasmic event in NLS-dependent nuclear protein import is the binding of the import cargo to the importin / heterodimer. Importin  provides the NLS-binding site and then interacts via its importin -binding domain (IBB domain) with importin , which in turn interacts with the nuclear pore complex (10, 12-14). The transfer of the trimeric NLS-importin / complex through the NPC is energy-dependent and appears to require GTP hydrolysis by Ran (15).

Since p53 functions as a transcriptional activator (1), p53 must enter the cell nucleus where it can function as a transcription activator. p53 has three potential nuclear localization signals in the C terminus of the protein (1). The major one, PQPKKKP, is able to direct the cytoplasmic protein to the nucleus (16). Although a set of transport factors required for the nuclear export of p53 has been identified and extensively characterized (17), the precise mechanism of the nuclear import has not yet been elucidated in vivo.

Our work was initiated to study the mechanisms of importin -mediated nuclear import of p53. We have newly identified a truncated form of importin  in a breast cancer cell line. The truncated importin  was tested for its biological activities in line with the nuclear import of p53.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human breast cancer cell line ZR-75-1 was grown in RPMI medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) at 37 °C in a humidified 5% CO₂-containing atmosphere. HBL-100 (human mammary epithelial cell) and CHO-K1 (Chinese hamster ovary cell line) was grown in Dulbecco's modified Eagle's medium (Life Technologies) containing 10% fetal bovine serum.

RT-PCR and Southern Blot-- Total cell RNA was isolated from 2 × 10⁷ cells of ZR-75-1 cell line using Catrimox-14^TM surfactant solution (Iowa Biotechnology) as described by the manufacturer. The Takara RNA LA PCR kit (Takara) was used to RT-PCR with slight modification. In brief, 300 ng of total RNA was used for cDNA synthesis using avian myeloblastosis reverse transcriptase with oligo-d(T) adaptor primer and subsequent cDNA amplification using Takara LA Taq with upstream primer (5'-ATGTCCACCAACGAGAATGCTAATAC-3') and downstream primer (5'-CTAAAAGTTAAAGGTCCCAGGAGCCCC-3') for importin -specific primers. RT was performed at 42 °C for 30 min. This was followed by a 2-min denaturation of DNA at 94 °C and 30 cycles of PCR amplification using a 30-s 94 °C denaturation step, a 30-s 56 °C annealing step and a 90-s 72 °C primer extension step. PCR products were resolved on 1% agarose gel containing 0.5 µg/ml ethidium bromide. To ensure the PCR products were amplified from importin  transcripts, Southern blot analysis was performed. PCR products resolved on 1% agarose gel were transferred to Hybond N⁺ membrane (Amersham Pharmacia Biotech) and hybridized with a digoxigenin-11-dUTP-labeled importin  probe. For preparation of the digoxigenin-labeled importin  probe, pRIP vector (Bioneer) containing the full-length importin  was digested with PstI and fractionated in 1% agarose gel. About 0.35-kilobase PstI-digested fragment containing N-terminal fragment of importin  was isolated, labeled with digoxigenin-11-dUTP, and used as the probe. The detection of hybridized DNA was carried out using DNA labeling and detection kit (Roche Molecular Biochemicals).

Cloning and Sequencing Analysis-- The positive bands identified by Southern blot analysis were isolated from agarose gel using GENECLEAN kit (Bio-101), cloned to SmaI digested-pRIP cloning vector (Bioneer), and subjected to sequencing using universal primers. AccuPower^TM DNA sequencing kit and Silverstar^TM staining kit (Bioneer) were used for DNA sequencing.

NLS Mutagenesis-- Point mutation was constructed by a PCR-based technique (18). For the p53 double point mutation of K320A and K321A, the first PCR reaction amplified the upstream fragment using the 5' boundary primer (5'-GGAATTCATGGAGGAGCCGCAGTCAGAT-3') and one of the mutant primers (5'-TCCATCCAGTGGTGCCGCCTTTGGCTGGGG-3') readings from 3' to 5'. The second PCR amplified the downstream fragment using mutant primer (5'-CCCCAGCCAAAGGCGGCACCACTGGATGGA-3') readings from 5' to 3' and the 3' boundary primer (5'-CCGCTCGAGTCAGTCTGAGTCAGGCCCTTC-3'). These upstream and downstream PCR products, which overlap at the mutation region, were mixed together and used as templates in the last PCR reaction with 5' and 3' boundary primers. The end PCR product with the desired mutation was gel-purified and digested with EcoRI and XhoI before ligation to pAD-GAL4 phagemid vector (Stratagene). Accuracy of these constructs was confirmed by sequencing.

Yeast Two-hybrid Protein Interaction Assay-- To investigate interactions between importin  and p53, and between importin  and the NLSI mutant p53, yeast two-hybrid system was used. Yeast strain YRG-2 (Mat ura3-52 his3-200 ade2-101 lys2-801 trp1-90 leu2-3 112 gal4-542 gal80-538 LYS2::UAS_GAL1-TATA_GAL1-HIS3 URA3::UAS_{GAL4 17-mers(X3)}-TATA_CYC1-lacZ) was co-transformed with pBD-I, pBD-INLSb and pAD-p53, pBD-INLSb and pAD-I, and pBD-I and pAD-NLSmut p53, and assayed for -galactosidase activity as described below. For importin  expression (pBD-I), a full-length cDNA encoding amino acids 1-529 was fused to the DNA-binding domain of GAL4 in pBD-GAL4 Cam plasmid (Stratagene). pBD-INLSb was constructed by fusing the truncated importin  to the DNA-binding domain of GAL4 in pBD-GAL4 Cam. pAD-p53 was constructed by fusing p53 to the activation domain of GAL4 in pAD-GAL4. pAD-I was constructed by cloning a partial fragment of importin  (amino acids 331 to 876), covering the region to interact with importin , into the activation domain of GAL4 in pAD-GAL4. To investigate interaction of importin  with NLS mutant p53, the hybrid proteins were constructed as described above. Competent cells of yeast strain YRG-2 were prepared by the LiOAc/single stranded DNA/PEG method (19). Co-transformed cells were plated onto synthetic dropout medium lacking leucine and tryptophan and supplemented with 25 mM 3-aminotrizole (Sigma/Aldrich) to investigate interaction of the hybrid proteins. Interactions of two partners were confirmed by a 5-bromo-4-chloro-3-indolyl -D-galactoside filter-staining assay (20). Yeasts transformed with empty vector alone (pGAL4, Stratagene) or recombinant plasmid and its corresponding empty vector were used as negative controls, whereas yeast transformed with pBD-p53 (Stratagene) and pAD-SV40 (Stratagene) was used as a positive control.

Plasmid Construction and Expression of Fusion Proteins-- To express the normal and the truncated importin  in CHO-K1, we constructed expression vectors of green fluorescent protein (GFP)-fused proteins using pEGFP-N1 (CLONTECH). The open reading frames of the normal and truncated importin  were translationally fused to GFP in-frame. The coding region of importin  was amplified by using PCR method (primers: 5'-ATGTCCACCAACGAGAATGCTAATAC-3' as upstream primer and 5'-AAAAGTTAAAGGTCCCAG-3' as downstream primer) with Ex Taq DNA polymerase (Takara) and cloned to SmaI site of pEGFP-N1. The PCR fragment amplified by using PCR method (primers: 5'-ATGTCCACCAACGAGAATGCTAATAC-3' as upstream primer and 5'-CTAAAAGTTAAAGGTCCCAGGAGCCCC-3' as downstream primer) containing the truncated importin  was digested with HindIII and cloned into the HindIII site in pEGFP-N1. The constructed plasmids were isolated using the ammonium acetate method (21) and transfected to CHO-K1 cells as described below. Each 35-mm dish of cells grown to about 70% confluency was transfected with 2 µg of plasmid DNAs by using LipofectAMINE (Life Technologies, Inc.). The DNA-liposome complexes were left in the culture medium for 8 h. At that time the medium was drained, and the cells were refed with fresh medium. At 48 h after transfection, the cells were passaged 1:10 into selective medium containing 800 µg/ml G418 and the G418-resistant colonies from each transfection were pooled and expanded into stable cell lines for Northern blot analysis. To express the normal and NLSI-mutated p53 in CHO-K1, the normal and the NLSI-mutant p53 generated as described above were ligated to the C terminus of the cyan fluorescent protein (CFP) in pECFP-C1 vector (CLONTECH). The constructed plasmids were isolated with ammonium acetate method (21) and transfected to CHO-K1 as described above.

Fluorescence Microscopy-- The transfected cells grown on coverslips were rinsed in PBS solution, fixed for 30 min in 4% paraformaldehyde in PBS, and air dried. Expression of fusion proteins was monitored using fluorescence microscopy (Leica DMRBE). 4',6'-Diamidine-2'-phenylindole dihydrochloride (Roche Molecular Biochemicals) staining was done according to the manufacturer's instructions.

Immunohistochemistry-- Immunohistological staining was carried out using Histostain^TM-plus kit (Zymed Laboratories Inc.) as described by the manufacturer. In brief, the transfected cells grown on coverslips were washed in PBS and fixed for 30 min in 4% paraformaldehyde in PBS with 0.5% Triton X-100. The cells were blocked in PBS containing 10% normal goat serum. The cells were then incubated with anti-p53 antibodies (DO-1 for HBL-100 and ZR-75-1 cells, Pab 246 for CHO-K1; Santa Cruz Biotechnology) for 3 h, followed by three washes with PBS. The biotinylated secondary antibody was reacted with the primary antibody, followed by three rinses with PBS. Horseradish peroxidase-conjugated streptavidin was then bound to the biotinylated secondary antibody. Antigen-antibody-enzyme complex was visualized by using AEC chromogen/substrate solution for red signal. Cell staining was observed by light microscopy (Leica DMRBE).

Northern Blot-- Cellular RNA was isolated from G418-selected CHO-K1 cells with TRIzol reagent (Life Technologies, Inc.) according to manufacturer's instructions. For Northern analysis, 10 µg of total cellular RNA was heat denaturated, separated on 1% agarose-formaldehyde gels, transferred to Hybond-N⁺ nylon membrane and probed with ³²P-labeled p21^waf1/cip1. A probe specific for glyceraldehyde-3-phosphate dehydrogenase was used to confirm equal loading.

Western Blot-- Forty-eight hours after transfection, the cells were lysed with lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100) containing 0.2 mM phenylmethylsulfonyl fluoride and 1.0 µg/ml aprotinin and denatured by boiling in sample buffer for 5 min. After SDS-polyacrylamide gel electrophoresis, p53 protein was detected in Western blot experiments using anti-p53 antibody and ECL Western blot reagents (Amersham Pharmacia Biotech).

Flow Cytometry-- At 24 h post-transfection, adherent and floating cells were collected, fixed in 2% paraformaldehyde supplemented with 0.1% Nonidet P-40 and stained with propodium iodide (Sigma). The cell cycle distribution of each sample was determined by measuring the DNA content through propidium iodide staining. Samples were analyzed in a cell sorter (FACSCalibur) using the CellQuest software (Becton Dickson). The apoptotic fraction was determined by quantitating the number of cells possessing a sub-G₁ DNA content (22).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Truncated Transcripts of Importin  Are Observed in Breast Cancer Cell-- Different from other normal cell lines like HBL-100, a breast cancer cell ZR-75-1 showed substantial amounts of p53 locating in the cytoplasm (Fig. 1A). In ZR-75-1 cells, we have newly identified a truncated form of importin  in the experiments of RT-PCR with importin -specific primers (Fig. 1B). The mRNA level for the truncated importin  was much higher than that for normal importin  in the cells when tested by quantitative RT-PCR (data not shown). The truncated cDNA fragments shown in Fig. 1B were extracted from the gel, cloned into a vector, and sequenced. Sequencing analysis of the truncated transcript of importin  showed that the truncated mutant of importin  was 387 base pairs in size and contained internal deletion in the cDNA of importin  (Fig. 1C). The truncated mutant, which we termed INLSb, deleted nucleotides from 251 to 1458 and contained an open reading frame encoding 89 amino acids, resulting in premature protein of importin  (Fig. 1C). The truncated transcript INLSb contained the IBB domain whereas a part of the importin  transcript segment encoding the putative NLS-binding domain was deleted, suggesting that this deletion mutant would not bind to NLSs of p53 proteins (Fig. 1D).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   p53 localization in the breast cancer cell ZR-75-1 and detection and sequence analysis of truncated importin . A, immunohistochemistry for p53 localization. ZR-75-1 and HBL-100 cells grown on glass coverslips were fixed and stained with anti-p53 antibody (DO-1, Santa Cruz Biochem) as described under "Experimental Procedures." Stained cells were photographed at X400. B, RT-PCR with RNA prepared from ZR-75-1 breast cancer cell line and importin -specific primers. The amplified DNA fragments were size fractionated on 1% agarose containing ethidium bromide. B, the cDNA fragment of truncated importin  in a breast cancer cell line (ZR-75-1) was cloned to pRIP vector (Bioneer) and sequenced. An arrow indicates the putative splicing junction in the truncated transcript. C, nucleotide and putative amino acid sequences of truncated transcript of importin . An arrow indicates the putative splicing junction in the sequence of the truncated transcript. Asterisk indicates premature stop codon. The putative mature stop codon is underlined. D, the schematic diagram of wild-type and the truncated importin . Each solid bar represents functional domains in importin ; an Arm repeat (23), acidic domain (24), IBB domain (10, 12), and NLS-binding domain. The dashed line represents the deleted portion of the truncated transcript of importin . Numbers indicate the numbers of amino acids in the truncated mutant.

Importin  Interacts with p53-- Importin  has been reported as a mediator for a classical nuclear transport (8). The nuclear localization signals in the C terminus of the p53 protein are directly involved in its subcellular localization (16). To be transported from the cytoplasm to the nucleus, the p53 protein must bind to a transport mediator through interaction between the NLS-binding domain of importin  and its NLSs. To investigate whether importin  interacts with p53, yeast two-hybrid assays were performed. Interaction between importin  and p53 was detected when yeast strain YRG-2 was co-transformed with pBD-I and pAD-p53 and monitored for leucine and tryptophan prototrophy, and -galactosidase activity was measured by filter lift assay (Fig. 2). On the other hand, the truncated importin  lacking the NLS-binding domain did not interact with p53, as expected (Fig. 2). When the truncated importin -expressing plasmid (pBD-INLSb) was co-transfected with importin -expressing plasmid (pAD-I) in yeast, the -galactosidase gene was normally turned on (Fig. 2). None of the negative controls (pBD-I plus pAD, pBD-INLSb plus pAD, and pBD-p53 plus pAD) showed any detectable blue color in filter lift assay. These results strongly suggest that the truncated region of the importin  is essential for the p53 binding.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Mutant importin  does not interact with wild-type p53 in yeast two-hybrid assay. Recombinant yeast cells, transformed with recombinant plasmids shown in the legend, were plated onto selective medium lacking leucine and tryptophan, and then tested for -galactosidase activity in a filter lift assay with 5-bromo-4-chloro-3-indolyl -D-galactoside. Combination of p53 and SV40 was used as positive control.

The NLS-mutant p53 Does Not Interact with Importin  and Locate to the Cytoplasm-- In p53, three potential nuclear localization signals reside in the C terminus of the protein. NLSI (PQPKKKP), especially, is the key player for the nuclear import of p53 (16). As expected, importin  would bind to p53 at the NLSI locus. To investigate whether this NLSI site mediates interaction between importin  and p53, we have carried out yeast two-hybrid assay with p53-expressing constructs containing the double mutation at NLSI site (KK320, 321AA) (Fig. 3A). The yeast cells were co-transformed with pBD-I and pAD-NLSwt p53 or pAD-NLSmut p53, and the colonies showing leucine and tryptophan prototrophy were subjected to filter lift assay. Importin  did not interact with the NLS mutant while it did interact with wild-type NLS showing blue color in the 5-bromo-4-chloro-3-indolyl -D-galactoside assay (Fig. 3B). NLS mutant showed blue color when co-transformed with pBD-SV40 (Fig. 3B), suggesting that the NLS mutant p53 was normally expressed in the transformed yeast. The expression of the NLS mutant p53 in yeast was also confirmed by Western blot analysis (data not shown). Because importin  did not interact with the p53 containing the double mutations (K320A/K321A) at NLSI site, the NLS mutant p53 might be sequestered in the cytoplasm. To test this hypothesis, we constructed an NLS mutant p53/CFP fusion protein containing KK320, 321AA in the putative p53 NLS (Fig. 3A). When CHO-K1 cells were transfected with p53-expressing plasmid, the NLS mutant p53 were located only to the cytoplasm (Fig. 3C, lower panel), while NLS wild-type p53 located to the nucleus (Fig. 3C, upper panel). These results suggest that the NLS mutant p53 does not bind to importin  and thus results in sequestration of p53 in the cytoplasm, and supports that the nuclear import of p53 is mediated by importin .

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Interaction of importin  with the NLS mutant p53 and the subcellular localization of the p53 mutant. A, the schematic diagram of the expression plasmids for wild-type and NLS mutant p53. The amino acids of the major nuclear localization signal of wild-type p53 and the NLS mutant p53 are given in a single-letter symbol. Substitution of these amino acids was conducted as described under "Experimental Procedures." The wild-type and NLS mutant p53 were ligated to the C terminus of the CFP in pECFP-C1 vector (CLONTECH). B, interactions between the NLS mutant p53 and importin  were tested by yeast two-hybrid and filter lift assay as described previously. Each spot shows the signal of the recombinant yeast co-transformed with pBD-I + pAD-p53 and pBD-I + pAD-NLSmut p53. C, the subcellular localization of the NLS1 mutant p53. CHO-K1 cells were cultured on glass coverslips and transfected with the CFP-fused expression plasmids, pNLSwt p53/CFP, and pNLSmut p53/CFP. After 24 h, the cells were fixed in paraformaldehyde, and examined with a fluorescent microscope. The NLSmut p53/CFP fusion protein was only observed in the cytoplasm of CHO-K1 cell (d) while the NLSwt p53/CFP fusion protein was preferentially observed in the nucleus (a). 4',6'-Diamidine-2'-phenylindole dihydrochloride (DAPI) staining is shown in b and e to indicate nuclei of the cells, and c and f are the corresponding phase-contrast images.

The Truncated Mutant of Importin  Is Preferentially Located to the Cytoplasm-- Importin  is only known as a cargo receptor and importin  thus must first bind to importin -binding domain of importin  for its nuclear translocation (8). It is also known that only the IBB domain could transport the fusion protein to the nucleus (10, 25). Notably, although the INLSb mutant deleted the region encoding the NLS-binding domain, it contained the IBB domain. To investigate whether the truncated importin  is functional in nuclear localization, expression vectors for fusion proteins in which the open reading frames of wild-type importin  and the truncated-type importin  were fused with that of GFP and were constructed and termed pI/GFP and pINLSb/GFP, respectively. Using this method, the localization in cells of the fused proteins could be easily monitored by fluorescence microscopy. When expressed in CHO-K1 cell, the INLSb/GFP fusion protein was preferentially observed in the cytoplasm and accumulated at the cytoplasmic periphery of the nuclear envelope (Fig. 4, g and h) while control GFP protein was in both the nucleus and the cytoplasm (Fig. 4, a and b). In contrast, the I/GFP fusion protein was preferentially observed in the nucleus (Fig. 4, d and e). The same result was obtained when the fusion proteins were expressed in HBL-100 and 293 cells (data not shown)). These data strongly suggest that truncated importin  is not functional for nuclear localization even though it contains intact IBB domain.

View larger version (135K): [in this window] [in a new window]   Fig. 4.   The subcellular localization of the truncated importin . CHO-K1 cells were cultured on glass coverslips and transfected with the GFP-fused expression plasmids, pI/GFP and pINLSb/GFP for the wild-type and mutant-importin  cDNA, respectively. After 24 h, the cells were fixed in paraformaldehyde, and examined with a fluorescent microscope. GFP-fused mutant p53 was preferentially observed in the cytoplasm and accumulated at the cytoplasmic periphery of the nuclear envelope (g and h) while control GFP protein was in both nucleus and cytoplasm (a and b). In contrast, GFP-fused importin  was preferentially observed in the nucleus (d and e). 4',6'-Diamidine-2'-phenylindole dihydrochloride staining is shown in b, e, and h to indicate nuclei of the cells. Panels c, f, and i indicate the phase-contrast images.

p53 Accumulates at the Cytoplasm in the Cells Overexpressing the Truncated Importin -- Different from other cell lines, ZR-75-1 cells were nicely stained by anti-p53 antibody at the cytoplasm as well as the nucleus in the experiments of immunohistochemical staining (Fig. 1A). Our accumulative results, showing that importin  binds to p53 protein, the truncated importin  loses its binding capacity to p53, and the truncated importin  is localized predominantly to the cytoplasm of CHO-K1 cells expressing the truncated importin -GFP fusion protein, led us to assume that the p53 enhancement in the cytoplasm of ZR-75-1 is likely to be associated with the expression of truncated importin . We have examined the p53 localization in the mutant I-expressing CHO-K1 cells. The expression of the truncated importin  and the location of p53 proteins were monitored by GFP fluorescence and immunohistochemical staining with a monoclonal anti-p53 antibody, respectively. As shown in Fig. 5, p53 was accumulated at the cytoplasm in truncated importin -expressing CHO-K1 cells (Fig. 5B) while preferentially localized to the nuclei in normal importin -expressing CHO-K1 cells (Fig. 5A). These results, together with our previous results, strongly suggest that the p53 accumulation at the cytoplasm rather than efficient localization to the nucleus in the ZR-75-1 cells is, at least in part, due to the existence of dysfunctional importin  in the breast cancer cells.

View larger version (60K): [in this window] [in a new window]   Fig. 5.   Overexpression of truncated importin  changes subcellular localization of wild-type p53. CHO-K1 cells grown on glass coverslip were transfected with pI/GFP (A) and pINLSb/GFP plasmids (B), respectively. After 48 h, the cells were fixed in neutral paraformaldehyde. The coverslips were incubated with anti-p53 antibody (Pab 246, Santa Cruz Biotechnology) and stained with a HistostainTM plus kit for detection, and then examined with a light microscope (Leica DMRBE). In CHO-K1 cells expressing the truncated importin , p53 predominantly localized to the cytoplasm (B), while it localized to the nucleus in wild-type importin  expressing cells (A).

Overexpression of Importin  Induces p53-mediated Biological Activity-- Biologically active p53 in nucleus modulates the transcription of its downstream target genes, such as bax, p21, GADD45, etc, resulting in growth arrest or apoptosis (1). To see whether the importin -mediated nuclear-imported p53 shown in Fig. 5 is functional or not, we examined the expression level of the p21^waf1/cip1 gene, which is considered to be the most important p53-responsive gene (26), by Northern blot analysis in importin -overexpressing CHO-K1 cells. A significant increase in waf1/cip1 mRNA level was observed in CHO-K1 cells transfected with pI/GFP plasmid, but not in GFP-transfected control cells nor in pINLSb/GFP-transfected CHO-K1 cells (Fig. 6A). Total protein level of p53 was slightly increased in the cells transfected with pI/GFP plasmid (Fig. 6B).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Overexpression of importin  induces p21waf1/cip1 expression and apoptosis. A, Nothern blot hybridization of p21waf1/cip1 mRNA expressed in the CHO-K1 cells transfected with wild-type and truncated importin . Total RNA was purified from the transfected cells, separated on denaturing agarose gel electrophoresis, and then Northern blot hybridized with 32P-labeled human p21waf1/cip1 probe. The upper panel shows Nothern signals for p21waf1/cip1 message and the lower panel shows subsequent hybridization with a glyceraldehyde-3-phosphate dehydrogenase probe for normalization of each RNA sample. B, quantitation of p53 in Western blot analysis. Immunoblot signals display the amounts of p53 expressed in CHO-K1 cells without transfection (lane 1) or transfected with pGFP (lane 2), pI/GFP (lane 3), and pINLSb/GFP (lane 4). Actin was used for normalization of each sample. C, DNA content profiles. CHO-K1 cells were transfected with the indicated plasmids and then subjected to flow cytometric DNA content analysis. Cells were fixed and stained with propodium iodide. Cells with sub-G1 DNA content were taken to be apoptotic. Percentages of the cells with sub-G1 DNA content are indicated below the corresponding plasmids.

In addition to the up-regulation of p21^waf1/cip1, functional p53 in nucleus has been shown to transactivate several other genes that could be involved in cell cycle arrest and apoptosis (1, 27). If the hypothesis that the importin  facilitate the nuclear import of p53 is true, overexpression of importin  may cause cell growth arrest and apoptosis by enhancement of nuclear import of p53 and transactivation of p53-responsive apoptotic factors. CHO-K1 cells were transfected with the expression plasmids, pGFP, pI/GFP, pINLSb/GFP, and pp53. Forty-eight hours post-transfection, cells were harvested, stained with propodium iodide, and subjected to flow-cytometric analysis. Cells with DNA content less than 2 N (sub-G₁ in Fig. 6C) are considered apoptotic (28). As shown in Fig. 6C, wild-type p53- and importin -transfected cells induced apoptosis by 17 and 24%, respectively, whereas GFP- transfected cells and truncated importin -transfected cells induced only by 4 and 6%, respectively. These experimental results strongly suggest that importin  mediates the nuclear import of p53, followed by transactivation of p53-responsive genes in nucleus and induction of consecutive p53-mediated biological activities.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We found a truncated form of importin  in the breast cancer cell ZR-75-1 in which substantial amounts of p53 were localized in the cytoplasm (Fig. 1A). The truncated importin  has a deletion in the middle of the RNA transcript (Fig. 1C). The deleted region covers the putative p53 NLS-binding domain (Fig. 1D). As expected, this deletion mutant could not bind to the wild-type p53 protein (Fig. 2). Yeast two-hybrid assay showed that normal importin  interacts with p53 (Fig. 2), while the NLS1 mutant p53 does not bind to importin  (Fig. 3B), suggesting that the NLS1 of the p53 is an important binding site to importin . The NLS mutant p53 was also sequestered in the cytoplasm (Fig. 3C). When expressed in CHO-K1 cells, the GFP-fused truncated mutant of importin  preferentially localized to the cytoplasm (Fig. 4). In truncated importin -expressing cells, p53 predominantly localized to the cytoplasm (Fig. 5B). Particularly noteworthy is that a significant increase in p21^waf1/cip1 mRNA level and enhancement of apoptosis were observed in the wild-type importin -overexpressing cells (Fig. 6), suggesting that p53 is efficiently transported to the nucleus through the importin -mediated nuclear transport system. Based on our present results, we address that the cytoplasmic sequestration of p53 in the breast cancer cell ZR-75-1 is likely to be associated with the presence of truncated importin  lacking in the NLS-binding domain, and is thus inefficient to transport p53 to the nucleus.

We found that the breast cancer cell ZR-75-1 shows distinct immunohistological patterns for p53 localization. Large amounts of p53 were detected in the cytoplasm of the cells (Fig. 1A). RT-PCR analysis demonstrated that the truncated transcript of importin  exists in a cell line ZR-75-1 (Fig. 1B). Generally, ZR-75-1 cells show predominant mRNA level for truncated importin  as compared with that for normal importin  as shown in Fig. 1B and in repeated quantitative RT-PCRs (data not shown). At times, however, the expression amount of mutant mRNA was not so much enhanced as usual. The fluctuation of mutant mRNA level is thought to be associated with the cell growth rate and culture conditions.

Although a genomic sequence of importin  has not yet been identified, sequence analysis of the RNA transcripts at the junction of the deleted sequence suggested that these truncated transcripts might be generated by the aberrant RNA splicing. The truncated transcript of importin  containing AAG at the end of 5' exon sequence and G/A at the 3' exon sequence (Fig. 1) match the canonical consensus sequences of the upstream and downstream splice donor and acceptor sites. It has been shown that the truncated transcripts of tumor suppressor genes, such as WT1, BRCA1, and TSG101, are frequently found in breast cancer (29-31). Although importin  is unlikely to act as a direct tumor suppressor, alternation of its phenotype may have influence on the functions of other important tumor suppressors that should be transported to the nucleus to be functionally active. With this regard, we have characterized the truncated importin , which may provide many important insights into the tumorigenesis of the breast cancer.

Sequence analysis showed that a truncated importin  contains the IBB domain but lacks NLS-binding domain. It has been reported that importin  binds to the IBB domain of importin  and transports it into the nucleus (10). In our present study, however, the truncated importin  containing the IBB domain was preferentially detected at the cytoplasm rather than in the nucleus of both CHO-K1 (Fig. 4) and HBL-100 cells (data not shown). The truncated importin  was confirmed still to have a binding capacity to importin  (Fig. 2) as reported previously in the experiment with other deletion mutants of importin  (23). Whereas, normal importin  was detected predominantly in the nucleus in the same condition. These results suggest that the IBB domain may not be enough for the importin -mediated translocation of importin  into the nucleus.

Lang and Clarke (33) have reported that both NLSI and two other NLSI basic domains are concomitantly required for p53 nuclear import. This means that the NLS1 alone in p53 may not be enough to be bound to the importin , resulting in a defect for nuclear localization of p53. In our experiments, however, NLS1 mutant p53 completely lost its binding capacity to importin  (Fig. 3), while K305A mutant p53 did not, and the nuclear localization of the K305A mutant was as efficient as that of wild-type p53 (data not shown). Although we did not perform the quantitative analysis of the binding affinity between importin  and each NLS of p53, our experimental results suggest that the NLS1 of p53 would be much more essential than other NLSs for importin -binding and nuclear import of p53. Precise interaction mechanisms remain to be discovered.

It has been proposed that export of importin  into cytoplasm is mediated by the Crm1 nuclear export protein which would interact with a leucine-rich nuclear export signal located between residues 207 and 217 in Arm repeat 3 of importin  (17, 32). On the other hand, it has also been proposed that export of importin  is dependent on the distinct nuclear export factor, CAS, which is another member of the importin  family and interacts with an unmapped nuclear export signal located near the C terminus of importin  (23). Although the precise mechanism of the importin  export into the cytoplasm is still unclear, it is generally accepted that importin  in the nucleus should be transported to the cytoplasm where it could be recharged with cargoes and re-transported by importin . When expressed by transfection with pI/GFP plasmid, however, the fusion protein I/GFP was preferentially localized to the nuclei of CHO-K1 (Fig. 4) and HBL-100 cells (data not shown). We assume that the GFP fusion to the importin  at the C terminus may inhibit the binding of nuclear export factor to importin  by masking or changing the conformation of the essential CAS/Crm1-binding domain in importin .

Even though the total amount of p53 was not markedly enhanced when cells were transfected with importin -expression plasmid (Fig. 6B), p53 was efficiently localized to the nucleus (Fig. 5), suggesting that the enhancement of importin  expression may facilitate the nuclear localization of p53, resulting in the induction of p53-mediated biological activities, such as growth arrest or apoptosis (Fig. 6C). Nevertheless, we cannot exclude nonspecific or other secondary effects that may lead to p21 up-regulation and apoptosis, for importin  is not an exclusive import factor for p53. Also, our illustration does not exclude the possibilities of plethoric effects of importin -overexpression, which may cause apoptosis through other unrevealed mechanisms rather than p53-mediated apoptotic procedures.

Other tumor suppressor nuclear proteins such as BRCA1 and, most recently, WT1 have also been observed to be mislocated in the cytoplasm of breast cancer cells (28). Recently, BRAP2, a novel cytoplasmic protein that interacts with two functional NLS motifs of BRCA1, has been reported to retain a newly synthesized BRCA1 protein in the cytoplasm of breast cancer cells (34). Upon receiving the appropriate environmental stimulus, BRCA1 is dissociated from BRAP2 and bound to importin  for moving into the nucleus. Besides, importin  has the intriguing features of the armadillo family proteins that can be attached to the cytoskeleton through the interaction of nuclear regulatory proteins anchored to the cytoskeleton (35). NLS-dependent association of importin  with the cytoskeleton would be important for anchoring the NLS receptor in the cytoplasm, revealing that importin  could serve as both an anchor and a carrier protein of nuclear regulatory proteins. These data suggest that a certain defect or phenotypic changes of transport factors may cause mislocation of tumor suppressor proteins, leading to inactivation of their tumor suppressor functions. With this regard, we are assuming two possible mechanisms for the truncated importin  mutant-mediated inhibition of p53 nuclear import in breast cancer: (i) overexpression of mutant importin  may cause a feedback inhibition of functional importin  expression, resulting in the inhibition of p53 nuclear import, or (ii) even though the wild-type and mutant importin  have a similar binding capacity to importin , the relative amounts of importin  available for this binding may not be enough to cover the mutant and wild-type importin , thus functional importin  has only a little chance to bind importin , resulting in inefficient translocation of p53 into the nucleus.

During the period of this work, we have identified similar truncated importin  mutants lacking NLS-binding domain from several breast cancer cells and tumor tissues.² Although there are still many questions remaining unanswered for its detail mechanisms, considering the report that mere reduction in p53 levels is sufficient to promote tumorigenesis (36), the truncated importin  is likely to be involved in the tumorigenesis or tumor progression of breast cancer and other tumors by causing inefficient nuclear import of functional p53 and/or other tumor suppressor nuclear proteins. Further intensive studies are required to reveal the precise biological role of truncate importin  in the breast cancer and other tumors.

	ACKNOWLEDGEMENTS

We thank Drs. J. O. Rimas, Bryan Show, and W.-H. Lee for valuable advice and comments during the preparation of this manuscript. We also acknowledge Dr. D. Richardson for his warmhearted editorial help.

	FOOTNOTES

^* This work was supported in part by Korea Research Foundation Grant 1998-019-F00034.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: 389 Chulsan, Kwangmyeong, Kyeonggi 423-030, Korea. Tel.: 82-2-6807-164/165; Fax: 82-2-6807-162; E-mail: hjyhdjsj@sungae.co.kr.

Published, JBC Papers in Press, April 28, 2000, DOI 10.1074/jbc.M909256199

² D.-H. Kim, S.-M. Han, Y.-S. Bae, I.-S. Kim, and Y-H. Moon, unpublished results.

	ABBREVIATIONS

The abbreviations used are: NLS(s), nuclear localization signal(s); IBB, importin  binding; PBS, phosphate-buffered saline; GFP, green fluorescent protein; CFP, cyan fluorescence protein; RT-PCR, reverse transcriptase-polymerase chain reaction; CHO, Chinese hamster ovary.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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