Truncated Form of Importin α 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, and the ¶Department of Microbiology,

Hannam University, Ojeong-dong 133, Daeduk-gu, Taejeon 306-791, South Korea

‡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: hjyh@hitel.net

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running Title

Truncated Importin α and Nuclear Import of p53

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SUMMARY

Disruption of the function of tumor suppressor proteins occasionally can be dependent on their subcellular

localization. About forty percent 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 α. In this report we demonstrate that importin α binds to NLSI in p53 and

mediates the nuclear import of p53. RT-PCR and sequencing analyses showed that a truncated importin α 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 α to p53 was confirmed by using yeast two-hybrid assay. When expressed in CHO-

K1 cells, the truncated importin α predominantly localized to the cytoplasm. In truncated importin α expressing

cells, p53 preferentially localized to cytoplasmic sites as well. A significant increase in the p21WAF1/CIP1 mRNA

level and induction of apoptosis were also observed in importin α overexpressing cells. These results strongly

suggest that importin α functions as a component of the NLS receptor for p53 and mediates nuclear import of p53.

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INTRODUCTION

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 the 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), and nuclear import of

proteins bearing an NLS is dependent on two cellular factors termed importin α and importin β (6-11). The initial

cytoplasmic event in NLS-dependent nuclear protein import is the binding of the import cargo to the importin α/β

heterodimer. Importin α provides the NLS-binding site and then interacts via its importin β binding domain (IBB

domain) with importin β, which in turn interacts with the nuclear pore complex (NPC) (10, 12-14). The transfer of

the trimeric NLS/importin α/β 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 α↑mediated nuclear import of p53. We have newly

identified a truncated form of importin α in a breast cancer cell line. The truncated importin α was tested for its

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biological activities in line with the nuclear import of p53.

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EXPERIMENTAL PROCEDURES

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 (FBS; Life Technologies) at 37 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% FBS.

RT-PCR and Southern Blot Total cell RNA was isolated from 2 X 107 cells of ZR-75-1 cell line using Catrimox-

14 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 AMV 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 α specific primers. RT was performed at 42 for 30

min. This was followed by a 2-min denaturation of DNA at 94 and 30 cycles of PCR amplification using a 30-s 94

denaturation step, a 30-s 56 annealing step and a 90-s 72 primer extension step. PCR products were resolved on 1%

agarose gel containing 0.5 •/– ethidium bromide. To ensure the PCR products were amplified from importin α

transcripts, Southern blot analysis was performed. PCR products resolved on 1% agarose gel were transferred to

Hybond-N+ membrane (Amersham) and hybridized with a DIG-11-dUTP-labeled importin α probe. For

preparation of the DIG-labeled importin α probe, pRIP vector (Bioneer) containing the full-length importin α was

digested with PstI and fractionated in 1% agarose gel. About 0.35 kb PstI-digested fragment containing N-terminal

fragment of importin α was isolated, labeled with DIG-11-dUTP and used as the probe. The detection of

hybridized DNA was carried out using DNA labeling and detection kit (Boehringer Mannheim).

Cloning and Sequencing analysis The positive bands identified by Southern blot analysis were isolated from agarose

gel using GENECLEAN kit (Bio101), cloned to SmaI digested-pRIP cloning vector (Bioneer) and subjected to

sequencing using universal primers. AccuPower DNA sequencing kit and Silverstar staining kit (Bioneer) were

used for DNA sequencing.

NLS mutagenesisPoint mutation was constructed by a PCR-based technique (18). For the p53 double point

mutation of Lys320Ala and Lys321Ala, the first PCR reaction amplified the upstream fragment using the 5’ 6

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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 another 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 α and p53, and between

importin α and the NLSI mutant p53, yeast two-hybrid system was used. Yeast strain YRG-2 (Matα ura3-52 his3-

200 ade2-101 lys2-801 trp1-90 leu2-3 112 gal4-542 gal80-538 LYS2::UASGAL1-TATAGAL1-HIS3

URA3::UASGAL4 17mers(X3)-TATACYC1-lacZ) was co-transformed with pBD-Iα, pBD-Iα∆NLSb and pAD-

p53, pBD-Iα∆NLSb and pAD-Iβ, and pBD-Iα and pAD-NLSmut p53, and assayed for β-galactosidase activity as

described below. For importin α expression (pBD-Iα), a full-length of cDNA encoding amino acid 1-529 was

fused to the DNA binding domain of GAL4 in pBD-GAL4 Cam plasmid (Stratagene). pBD-Iα∆NLSb was

constructed by fusing the truncated importin α 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-Iβ was constructed by

cloning a partial fragment of importin β (amino acid 331 to 876), covering the region to interact with importin α,

into the activation domain of GAL4 in pAD-GAL4. To investigate interaction of importin α 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 straned DNA/PEG method (19). Cotransformed 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

β-D-galactoside (X-gal) 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.7

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Plasmid Construction and Expression of Fusion Proteins To express the normal and the truncated importin α 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 imoprtin α were translationally fused to GFP in

frame. The coding region of importin α was amplified by using PCR method (primers: 5’-

ATGTCCACCAACGAGAATGCTAATAC-3’ as upstream primer and 5’-AAAAGTTAAAGGTCCCAG–3’ as

down stream 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 down stream primer) containing the truncated

importin α was digested with HindIII and cloned into HindIII site in pEGFP-N1. The constructed plasmids were

isolated with ammonium acetate method (21) and transfected to CHO-K1 cell as described below. Each 35 mm dish

of cells grown to about 70% confluency was transfected with 2 • of plasmid DNAs by using lipofectAMINE (Gibco

BRL). 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 •/– of 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 cover slips 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 (DAPI; Boehringer Mannheim)

staining was done according to the manufacturer’s instructions.

Immunohistochemistry Immuno-histological staining was carried out using Histostain-plus kit (Zymed

laboratory) as described by the manufacturer. In brief, the transfected cells grown on cover slips 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

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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 streptavidine 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 (Gibco BRL)

according to manufacturer’s instructions. For Northern analysis, 10 • of total cellular RNA was heat denaturated,

separated on 1% agarose-formaldehyde gels, transferred to Hybond-N+ nylon membrane and probed with 32P-

labeled p21WAF1/CIP1. A probe specific for GAPDH 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 blots experiments using anti-p53 antibody and ECL Western blot reagents (Amersham).

Flow cytometry At twenty-four hours post-transfection, adherent and floating cells were collected, fixed in 2%

paraformaldehyde supplemented with 0.1% NP-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).

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RESULTS

Truncated Transcripts of Importin α 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 α in the experiments of RT-PCR with

importin α↑specific primers (Fig. 1B). The mRNA level for the truncated importin α was much more higher than

that for normal importin α 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 α showed that the truncated mutant of importin α was 387 bp in size and

contained internal deletion in the cDNA of importin α (Fig. 1C). The truncated mutant, which we termed Iα∆NLSb,

deleted nucleotides from 251 to 1458 and contained an open reading frame encoding 89 amino acids, resulting in

premature protein of importin α (Fig. 1C). The truncated transcript Iα∆NLSb contained the IBB domain whereas a

part of the importin α 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).

Importin α Interacts with p53 Importin α has been reported as a mediator for a classical nuclear transport (8). The

nuclear localization signals in the C terminus of 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 α and its NLSs. To investigate whether the importin α

interacts with p53, yeast two-hybrid assays were performed. Interaction between importin α and p53 was detected

when yeast strain YRG-2 was cotransformed with pBD-Iα and pAD-p53 and monitored for leucine and tryptophan

prototrophy, and β-galactosidase activity was measured by filter lift assay (Fig. 2). On the other hand, the truncated

importin α lacking NLS binding domain did not interact with p53, as expected (Fig. 2). When the truncated importin

α-expressing plasmid (pBD-Iα∆NLSb) was co-transfected with importin β-expressing plasmid (pAD-Iβ) in

yeast, β-galactosidase gene was normally turned on (Fig. 2). None of the negative controls (pBD-Iα plus pAD,

pBD-Iα∆NLSb 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 α is essential for the p53-binding.

The NLS-mutant p53 Does Not Interact with Importin α and Locate to the Cytoplasm In p53, three potential10

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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 α would bind to p53 at the NLSI locus. To investigate

whether this NLSI site mediates interaction between importin α 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 cotransformed with pBD-Iα and pAD-NLSwt p53 or pAD-NLSmut p53, and the colonies

showing leucine and tryptophan prototrophy were subjected to filter lift assay. Importin α did not interact with the

NLS mutant while it did interact with wild-type NLS showing blue color in the X-gal assay (Fig. 3B). NLS mutant

showed blue color when cotransformed 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 α 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 α and thus results in

sequestration of p53 in the cytoplasm, and supports that the nuclear import of p53 is mediated by importin α.

The Truncated Mutant of Importin α Is Preferentially Located to the Cytoplasm Importin α is only known as a cargo

receptor and importin β thus must first bind to importin β binding domain of importin α for its nuclear translocation

(8). It also known that only IBB domain could transport the fusion protein to the nucleus (10, 25). Notably, although

the Iα∆NLSb mutant deleted the region encoding the NLS-binding domain, it contained the IBB domain. To

investigate whether the truncated importin α is functional in nuclear localization, expression vectors for fusion

proteins in which the open reading frames of the wild-type importin α and the truncated-type importin α were

fused with that of GFP and were constructed and termed pIα/GFP and pIα∆NLSb/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 Iα∆NLSb/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

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both the nucleus and the cytoplasm (Fig. 4, a and b). In contrast, Iα/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 α is not functional for nuclear

localization even though it contains intact IBB domain.

p53 Accumulates at the Cytoplasm in the Cells Overexpressing the Truncated Importin α 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 α binds

to p53 protein, the truncated importin α loses its binding capacity to p53, and the truncated importin α is localized

predominantly to the cytoplasm of CHO-K1 cells expressing the truncated importin α↑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 α. We have examined the p53 localization in the mutant Iα-expressing CHO cells. The

expression of the truncated importin α 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 α↑expressing CHO cells (Fig. 5B) while preferentially localized

to the nuclei in normal importin α↑ expressing CHO 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 α in the breast cancer cells.

Overexpression of Importin α 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 α-mediated nuclear-imported p53 shown in Fig. 5 is functional

or not, we examined the expression level of the p21WAF1/CIP1 gene, which is considered to be the most important

p53-responsive gene (26), by Northern blot analysis in importin α↑overexpressing CHO cells. A significant

increase in waf1/cip1 mRNA level was observed in CHO cells transfected with pIα/GFP plasmid, but not in GFP-

transfected control cells nor in pIα∆NLSb/GFP-transfected CHO cells (Fig. 6A). Total protein level of p53 was

slightly increased in the cells transfected with pIα/GFP plasmid (Fig. 6B).

In addition to the up-regulation of p21WAF1/CIP1, functional p53 in nucleus has been shown to transactivate12

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several other genes that could be involved in cell cycle arrest and apoptosis (1, 27). If the hypothesis that the

importin α facilitate the nuclear import of p53 is true, overexpression of importin α 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, pIα/GFP, pIα∆NLSb/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 2N (Sub-G1 in Fig. 6C) are considered apoptotic (28). As shown in Fig.

6C, wild-type p53- and importin α-transfected cells induced apoptosis by 17% and 24%, respectively, whereas

GFP- transfected cells and truncated importin α -transfected cells induced only by 4% and 6%, respectively. These

experimental results strongly suggest that importin α mediates the nuclear import of p53, followed by

transactivation of p53-responsive genes in nucleus and induction of consecutive p53-mediated biological activities.

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DISCUSSION

We found a truncated form of importin α in the breast cancer cell ZR-75-1 in which substantial amounts of

p53 were localized in the cytoplasm (Fig. 1A). The truncated importin α 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 α interacts with p53 (Fig. 2), while the NLS1 mutant p53 does not bind to importin α (Fig. 3B), suggesting

that the NLS1 of the p53 is an important binding site to importin α. 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 α

preferentially localized to the cytoplasm (Fig. 4). In truncated importin α↑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 α↑overexpressing cells (Fig. 6),

suggesting that p53 is efficiently transported to the nucleus through the importin α-mediated nuclear transport

system. Based on our present results, we address that the cytoplasmic sequesteration of p53 in the breast cancer cell

ZR-75-1 is likely to be associated with the presence of truncated importin α 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 α exists in a cell line ZR-75-1 (Fig. 1B). Generally, ZR-75-1 cells show

predominant mRNA level for truncated importin α as compared to that for normal importin α 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 α 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 α 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

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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 α is unlikely to act as

a direct tumor suppressor, alternation of its phenotype may have an 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 α, which may provide many important insights into the tumorigenesis of the

breast cancer.

Sequence analysis showed that a truncated importin α contains the IBB domain but lacks NLS-binding

domain. It has been reported that the importin β binds to the IBB domain of importin α and transports it into the

nucleus (10). In our present study, however, the truncated importin α containing 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 α was confirmed still to have a binding capacity to importin β (Fig. 2) as reported previously

in the experiment with other deletion mutants of importin α (23). Whereas, normal importin α was detected

predominantly in the nucleus in the same condition. These results suggest that IBB domain may not be enough for

the importin β↑mediated translocation of importin α 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 α, resulting in a defect for nuclear localization of p53. In our experiments, however, NLS1 mutant p53

completely lost its binding capacity to importin α (Fig. 3), while K305A mutant p53 did not, and the nuclear

localization of 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 α and each NLS of p53, our experimental

results suggest that the NLS1 of p53 would be much more essential than other NLSs for importin α-binding and

nuclear import of p53. Precise interaction mechanisms remain to be discovered.

It has been proposed that export of importin α into cytoplasm is mediated by the Crm1 nuclear export protein

which would interact with a leucine-rich nuclear export signal (NES) located between residues 207 and 217 in Arm

repeat 3 of importin α (17, 32). On the other hand, it has also been proposed that export of importin α is dependent

on the distinct nuclear export factor cellular apoptosis susceptibility (CAS) which is another member of the importin

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β family and interacts with an unmapped NES located near the C-terminus of importin α (23). Although the precise

mechanism of the importin α export into the cytoplasm is still unclear, it is generally accepted that the importin α in

the nucleus should be transported to the cytoplasm where it could be recharged with cargoes and re-transported by

importin β. When expressed by transfection with pIα/GFP plasmid, however, the fusion protein Iα/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 α at C-terminus may inhibit the binding of nuclear export factor to importin α by

masking or changing the conformation of the essential CAS/Crm1-binding domain in importin α.

Even though the total amount of p53 was not markedly enhanced when cells were transfected with importin α-

expression plasmid (Fig. 6B), p53 was efficiently localized to the nucleus (Fig. 5), suggesting that the enhancement

of importin α 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 non-specific or

other secondary effects that may lead to p21 up-regulation and apoptosis, for importin α is not an exclusive import

factor for p53. Also, our illustration does not exclude the possibilities of plethoric effects of importin α-

overexprssion, 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 the breast cancer cells (34). Upon receiving the appropriate environmental stimulus,

BRCA1 is dissociated from BRAP2 and bound to importin α for moving into the nucleus. Besides, importin α 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

α with the cytoskeleton would be important for anchoring the NLS receptor in the cytoplasm, revealing that

importin α 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

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mechanisms for the truncated importin α mutant-mediated inhibition of p53 nuclear import in breast cancer: i)

overexpression of mutant importin α may cause a feedback inhibition of functional importin α expression, resulting

in the inhibition of p53 nuclear import, or ii) even though the wild type and mutant importin α have a similar

binding capacity to importin β, the relative amounts of importin β available for this bindings may not be enough to

cover the mutant and wild-type importin α, thus functional importin α has only a little chance to bind importin β,

resulting in inefficient translocation of p53 into the nucleus.

During the period of this work, we have identified similar truncated importin α mutants lacking NLS-binding

domain from several breast cancer cells and tumor tissues (unpublished result). 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 α 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 α in the breast cancer and other tumors.

Acknowledgements We thank Drs. J. O. Rimas, Bryan Show and W. -H. Lee for their valuable advice and

comments during the preparation of this manuscript. We also acknowledge Dr. D. Richardson for his warm-hearted

editorial help. This work was supported in part by grant from the Korea Research Foundation (1998-019-F00034).

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1 The abbreviations used are: NLS(s), nuclear localization signal(s); IBB, importin β binding; NPC, nuclear pore

complex; FBS, fetal bovine serum; PBS, phosphate-buffered saline; X-gal, 5-bromo-4-chloro-3-indolyl β-D-

galactoside; GFP, green fluorescent protein; CFP, cyan fluorescence protein; DAPI, 4’,6’-diamidine-2’-

phenylindole dihydrochloride; NES, nuclear export signal; CAS, cellular apoptosis susceptibility.

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FIGURE LEGEND

FIG. 1. p53 localization in the breast cancer cell ZR-75-1 and detection and sequence analysis of truncated importin

α. A, Immunohistochemistry for p53 localization. ZR-75-1 and HBL-100 cells grown on cover glass were fixed

and stained with anti-p53 antibody (DO-1, Santa Cruz Biochem.) as described in EXPERIMENTAL

PROCEDURES. Stained cells were photographed at X400. B, RT-PCR with RNA prepared from ZR-75-1 breast

cancer cell line and importin α-specific primers. The amplified DNA fragments were size-fractionated on 1%

agarose containing ethidium bromide. B, The cDNA fragment of truncated importin α 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 α. 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 α. Each solid bar represents functional domains in importin α; an Arm repeat (23), acidic

domain (24), importin β binding domain (IBB; 10, 12) and NLSs binding domain. The dashed line represents the

deleted portion of the truncated transcript of importin α. Arabic numerals indicate the numbers of amino acids in the

truncated mutant.

FIG. 2. Mutant Importin α 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 β-galactosidase activity in a filter lift assay with X-gal. Combination of p53 and

SV40 was used as positive control.

FIG. 3. Interaction of importin α 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 in 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

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between the NLS mutant p53 and importin α 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-Iα+ pAD-p53 and

pBD-Iα + pAD-NLSmut p53. C, The subcellular localization of the NLS1 mutant p53. CHO-K1 cells were

cultured on glass cover slips 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). DAPI staining is shown in b and e

to indicate nuclei of the cells, and c and f are the corresponding phase contrast images.

FIG. 4. The subcellular localization of the truncated importin α. CHO-K1 cells were cultured on glass cover slips

and transfected with the GFP-fused expression plasmids, pIα/GFP and pIαNLSb/GFP for the wild-type and

mutant- importin α 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 α was preferentially observed in the nucleus (d and e). DAPI

staining is shown in b, e and h to indicate nuclei of the cells. Panels c, f and i indicate the phase contrast images.

FIG. 5. Overexpression of Importin α changes subcellular localization of wild-type p53. CHO-K1 cells grown on

glass cover slip were transfected with pIα/GFP (A) and pIα∆NLSb/GFP plasmids (B), respectively. After 48 h, the

cells were fixed in neutral paraformaldehyde. The cover slips were incubated with anti-p53 antibody (Pab 246,

Santa Cruz Biotechnology) and stained with a Histostain-plus kit for detection, and then examined with a light

microscope (Leica DMRBE). In CHO-K1 cells expressing the truncated importin α, p53 predominantly localized to

the cytoplasm (B), while it localized to the nucleus in wild-type importin α expressing cells (A).

FIG. 6. Overexpression of importin α 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 truncated22

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importin α. Total RNA was purified from the transfected cells, separated on a denaturing agarose gel electrophoresis

and then Northern blot hybridized with 32P-labeled human p21WAF1/CIP1 probe. The upper panel shows Nothern

signals for p21 WAF1/CIP1 message and the lower panel shows subsequent hybridization with a GAPDH 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),

pIα/GFP (lane 3), and pIαNLSb/GFP (lane 4). Actin was used for normalization of each sample. C, DNA contents

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.

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

Import of p53 Identified in Breast Cancer Cell Inhibits NuclearαTruncated Form of Importin

published online April 28, 2000J. Biol. Chem. 

  10.1074/jbc.M909256199Access the most updated version of this article at doi:

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