From the Department of Biotechnology, Graduate School
of Agriculture and Life Sciences, the University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, the
Department of Anatomy and Cell Biology,
Osaka University Medical School, Suita, Osaka 565, and
§ CREST Research Project, Japan Science and Technology
Corporation, Saitama 332-0012, Japan
Received for publication, January 10, 2001, and in revised form, January 30, 2001
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ABSTRACT |
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Mouse temperature-sensitive
p53Val-135 accumulates in the nucleus and acts as a
"wild-type" at 32 °C while it is sequestered in the cytoplasm at
37 °C. The cytoplasmic p53Val-135 relocalized into the
nucleus upon inhibition of the nuclear export at 37 °C, whereas a
mutation in a major bipartite nuclear localization signal (NLS) caused
constitutive cytoplasmic localization, indicating that it shuttled
between the cytoplasm and the nucleus by its own nuclear export signal
and NLS rather than tethered to cytoplasmic structures. Although the
full-length p53Val-135 did not bind the import receptor at
37 °C, a C-terminally truncated p53Val-135 lacking
residues 326-390 did bind it. Molecular chaperones such as Hsc70 were
associated with p53Val-135 at 37 °C but not at 32 °C.
When the nuclear export was blocked by leptomycin B, only a fraction
lacking Hsc70 was specifically accumulated in the nucleus.
Immunodepletion of Hsc70 from the reticulocyte lysate caused
p53Val-135 to bind the import receptor. This binding was
blocked by supplying the cell extract containing Hsc70 but not by the
addition of recombinant Hsc70 alone. We suggest that the association
with the Hsc70-containing complex prevents the NLS from the access of
the import receptor through the C-terminal region of
p53Val-135 at 37 °C, whereas its dissociation at
32 °C allows rapid nuclear import.
Cellular DNA damage caused the increased stability and
accumulation of the tumor suppressor protein p53 in the nucleus, which leads to cell cycle arrest or apoptosis by transcriptional activation of specific growth-inhibitory target genes (1-3). Cells lacking this pathway are more resistant to chemotherapeutic agents and exhibit
increased genomic instability, allowing them to gain a selective
advantage during tumor progression. It is therefore proposed that the
physiological function of p53 is a "guardian of the genome" (4). In
~50% of human tumors, the p53 gene is inactivated by a point
mutation that gives rise to a missense protein. In cells expressing
both the wild-type and the mutant alleles, mutant p53 may lead to a
dominant negative inactivation of the remaining wild-type protein
through oligomerization (5-7). In other cases, wild-type p53 is
functionally inactivated by sequestration in the cytoplasm or by
associating with other proteins such as MDM2 and viral oncoproteins
(8-10). Thus, appropriate subcellular localization is crucial for
regulating the p53 function.
Recently, p53 was shown to shuttle between the nucleus and cytoplasm
(11), and a functional leucine-rich nuclear export signal
(NES)1 was identified in the
p53 tetramerization domain (12). This finding led to an intriguing
model in which wild-type p53 subcellular localization is established
through tetramerization-regulated exposure of the NES to the export
machinery CRM1; p53 shuttles between the nucleus and the cytoplasm
through its intrinsic nuclear localization signal (NLS) and NES, but
tetramerization inhibits nuclear export by masking the NES, resulting
in the p53 nuclear retention. Several tumor types such as neuroblastoma
have wild-type p53 which is inactivated due to its cytoplasmic
sequestration (13). At least in neuroblastoma cells, the enhanced
nuclear export, rather than a tether retaining it in cytoplasmic
structures, is ascribed to the cytoplasmic localization (12). In some
cases, however, overproduction of MDM2, a p53-target gene product, may play an important role in the p53 cytoplasmic sequestration (14, 15).
Like wild-type p53, the subcellular localization of mutant p53 proteins
is subject to variation, ranging from exclusively cytoplasmic to
exclusively nuclear in tumors and transformed cells (16-19). It seems
likely that the subcellular localization of mutant p53 is not
determined by the mutation itself but may be dependent on the
intracellular environment (20, 21). Mutant p53 proteins, independent of
the mutation sites, exhibit the same epitopes recognized by the
mutant-specific antibodies and associate with heat-shock proteins to
prolong the half-lives, probably due to a conformation similar to that
among the mutant proteins but not to the wild-type one. It is therefore
unclear whether the mechanism by which mutant p53 is sequestered in the
cytoplasm is the same as that for the wild-type protein. It was
previously proposed that p53 with an aberrant conformation is
sequestered from the nucleus by Hsp70/Hsc70 binding (22-24). In some
cases, however, Hsc70 binding may not effectively sequester p53 to the
cytoplasm, since similar proportions of Hsc70-p53 complexes have been
found in both the cytoplasm and the nucleus (20).
The mouse temperature-sensitive mutant p53Val-135 serves as
a useful system to analyze the mechanism underlying the subcellular
localization of both mutant and wild-type p53 (25).
p53Val-135 is present in the nucleus with the
"wild-type" conformation at the permissive temperature (32 °C),
whereas it redistributes in the cytoplasm at the nonpermissive
temperature (37 °C) in the cells transformed with
p53Val-135 plus ras. Since
cycloheximide induced rapid nuclear import of the mutant form of
p53Val-135 at 37 °C, it has been postulated that
p53Val-135 is anchored to the cytoplasmic structures, which
is mediated by short lived protein(s) (26). This model is apparently
different from that for wild-type p53, explained by the hyperactive
nuclear export. In this study, using leptomycin B, a specific inhibitor of nuclear export (27-29), we show that p53Val-135 at
37 °C still shuttles between the nucleus and the cytoplasm as does
wild-type p53 but that the rate of nuclear import is markedly reduced.
Our data suggest that the formation of a multiprotein complex
containing Hsc70 causes NLS masking, resulting in the cytoplasmic
retention. This model is in contrast to the oligomerization-induced NES
masking of wild-type p53 for nuclear retention.
Cells, Antibodies, and Reagents--
Clone 6, a rat fibroblast
cell line transformed with p53Val-135 and activated
ras (25), was kindly provided by S. Khochbin. 2KO, a mouse
embryonic fibroblast cell line established from a p53 Plasmid Construction and
Mutagenesis--
pBS-p53Val-135 and
pEGFP-p53Val-135 were constructed by inserting the mouse
p53Val-135-coding sequence (kindly provided by C. Delphin)
between the EcoRI and HindIII sites of
pBluescript II SK- (Stratagene) and pEGFP-C1 (CLONTECH Laboratories), respectively. To construct
the NES mutant of p53Val-135
(pEGFP-p53Val-135-L345A/L347A), we carried out the
PCR-based mutagenesis using pBS-p53Val-135 as the template
and 5'-GAGCTGAATGAGGCCGCAGAGGCAAAGGATGCCCATGCT-3' (sense) and
5'-AGCATGGGCATCCTTTGCCTCTGCGGCCTCATTCAGCTC-3' (antisense) as primers.
For construction of the NLS mutant of p53Val-135
(pEGFP-p53Val-135-K302N), primers
5'-CCCCCAGGGAGCGCAAATAGAGCGCTGCCCACC-3' (sense) and
5'-GGTGGGCAGCGCTCTATTTGCGCTCCCTGGGGG-3' (antisense) were used. The
EcoRI-HindIII fragment obtained from the PCR
product was ligated into pEGFP-C1. All constructs generated in this way
were sequenced. To conjugate the p53 NLS to GFP-p53Val-135,
we synthesized double-stranded oligonucleotide DNA (sense,
5'-GATCTATGCCCCCAGGGAGCGCAAAGAGAGCGCTGCCCACCTGCACAAGCGCCTCTCCCCCGCAAAAGAAAAAACCACTTGATGGACG-3', and antisense,
5'-AGCTCGTCCATCAAGTGGTTTTTTCTTTTGCGGGGGAGAGGCGCTTGTGCAGGTGGGCAGCGCTCTCTTTGCGCTCCCTGGGGGCATAGA-3'), and inserted it into the BglII-XhoI site of
pEGFP-p53Val-135 or pEGFP-p53Val-135-K302N.
Immunofluorescent Staining--
Clone 6 cells grown on glass
coverslips to 70% confluence were incubated with or without drugs for
12 h or incubated at 32 °C for 12 h. They were then rinsed
with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room
temperature. The fixed cells were washed with PBS and permeabilized
with 0.1% Triton X-100 in PBS for 10 min. For minimizing nonspecific
binding, the samples were preincubated with 2% fetal bovine serum in
buffer containing 150 mM NaCl, 10 mM Tris-HCl
(pH 7.5), 0.1% (v/v) Tween 20, and then treated with the primary
antibody pAb421 followed by the incubation with the secondary antibody
(fluorescein isothiocyanate goat anti-mouse IgG, Amersham Pharmacia
Biotech). The coverslips were washed with PBS, rinsed with water,
mounted with 1 µg/ml DAPI in Vectashield (Vector Laboratories), and
observed under a Zeiss Axiophoto 2 fluorescent microscope (Carl Zeiss).
Cell Transfection--
Clone 6 and 2KO cells grown on glass
coverslips in 6-well plates (35-mm diameter) to about 80%
confluence were transfected with various plasmid constructs using
LipofectAMINE (Life Technologies, Inc.) according to manufacturer's
instructions. For each transfection, 3 µg of plasmid DNA and 7 µl
of LipofectAMINE were mixed in 1 ml of Opti-MEM (Life Technologies,
Inc.), and overlaid onto the cells. After 5 h of incubation at
37 °C, the medium was changed to Dulbecco's modified Eagle's
medium supplemented with 12% fetal bovine serum. At 24 h after
the transfection, cells were treated with LMB or CHX for 12 h,
washed with PBS, and then fixed with 4% paraformaldehyde in PBS
for 15 min at room temperature. Cells were then washed with PBS and
permeabilized with 0.1% Triton X-100 for 10 min, washed with PBS,
rinsed with water, and mounted with 1 µg/ml DAPI in Vectashield for
microscopic observation.
Immunoprecipitation, Immunoblotting, and
Immunodepletion--
Cells collected were sonicated for 5 s two
times in ice-cold immunoprecipitation (IP) buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2,
0.2% (v/v) Tween 20, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium molybdate, which stabilizes the unstable chaperone complex (32).
The lysates were centrifuged at 15,000 × g for 20 min at 4 °C, and the protein concentrations of the supernatants were determined by the Bradford method (Bio-Rad). Immunoprecipitation was
performed with 1 mg of total protein. After the supernatants had been
precleared with protein A/G-agarose beads (Santa Cruz Biotechnology)
for 10 min, followed by centrifugation at 5,000 × g,
the cleared supernatants were successively incubated with primary
antibodies for 1 h with gentle agitation and with the protein
A/G-agarose beads for 1 h. The agarose pellets were then washed
three times with lysis buffer, and the bound proteins were extracted
with 5× Laemmli sample loading buffer by heating to 95 °C for 5 min. Proteins eluted were separated by sodium dodecyl sulfate, 10%
polyacrylamide gel electrophoresis and transferred to an Immobilon-P
membrane (Millipore Co, Bedford, MA) by electroblotting. After the
membranes had been incubated with primary and secondary antibodies, the
immune complexes were detected with an ECL Western blotting kit
(Amersham Pharmacia Biotech). For immunodepletion of Hsc70, the
anti-Hsc70 polyclonal antibody (20 µl) was added into 15 µl of
rabbit reticulocyte lysate (Promega) following the in vitro
transcription/translation reaction and incubated at 37 or 32 °C for
3 h. The immune complex containing Hsc70 was depleted at 4 °C
by adsorption to the protein A/G-agarose beads from the lysate. The
successful depletion was monitored by immunoblotting.
In Vitro Transcription/Translation and Binding
Assay--
[35S]Methionine-labeled
p53Val-135, importin LMB Causes Nuclear Accumulation of Temperature-sensitive
p53--
To assess the proposed model in which a tether of mutant p53
to cytoplasmic structures via short lived protein(s) prevents its
release and subsequent movement into the nucleus, we analyzed the
effect of leptomycin B (LMB), an inhibitor of the nuclear export signal
(NES)-dependent protein nuclear export, on the subcellular localization of p53 in rat embryonic fibroblast clone 6 cells (25)
expressing an activated Ras and temperature-sensitive
p53Val-135 at a high level (Fig.
1). If p53 in these cells is tethered in the cytoplasm at 37 °C, it should remain there even when the nuclear export is blocked by LMB. The subcellular localization of
p53Val-135 in clone 6 cells, as determined by
immunofluorescent staining, is divided into three groups as follows:
predominantly nuclear (N), cytoplasmic (C), and both nuclear and
cytoplasmic (N = C) localizations. More than 80% of cells showed
p53 cytoplasmic localization (C and N = C) at 37 °C. In
contrast, in ~90% of cells p53Val-135 was relocalized
into the nucleus from the cytoplasm when treated with 10 nM
LMB for 12 h at 37 °C. Since LMB binds directly to CRM1 and
inhibits the formation of complexes containing CRM1, RanGTP, and
NES-containing proteins, the CRM1-mediated nuclear export may be
involved in the cytoplasmic localization of p53Val-135 at
37 °C. To rule out the possibility that LMB induced a general stress
response thereby leading to the nuclear translocation of p53
independently of CRM1, we tested whether the expression of the
LMB-resistant CRM1 mutant causes the recovery of the cytoplasmic localization of p53Val-135 in the presence of LMB. We
previously showed that LMB covalently bound a cysteine residue in the
central conserved region of CRM1 by a Michael-type addition and that
the mutation at this residue caused the protein to be insensitive to
LMB (35). When the clone 6 cells were transfected with the human
CRM1-K1 mutant having Ser instead of Cys-528, the cells containing
nuclear p53 in the presence of LMB decreased to about 40% (Fig. 1,
LMB+CRM1-K1). Since the transfection efficiency was about
50% in this particular experiment, the cells showing p53 cytoplasmic
localization are reasonably expressing CRM1-K1. In agreement with this,
only a marginal increase in the cells showing p53 cytoplasmic
localization was observed for the wild-type CRM1 construct (Fig. 1,
LMB+CRM1). We assume that the CRM1-dependent
nuclear export of p53Val-135 is responsible for the
cytoplasmic localization of p53Val-135 at 37 °C.
Transcriptional activity of p53Val-135 was not recovered
even when p53Val-135 was accumulated in the nucleus by LMB
treatment (data not shown).
Temperature-sensitive p53 Shuttles between the Nucleus and the
Cytoplasm by Its Own NLS and NES--
The clone 6 cells express MDM2
that can bind and functionally inactivate p53 (data not shown). MDM2
has been shown to promote the nuclear export of p53 in a
CRM1-dependent manner by its NES (36, 37) or stimulating
the p53 NES activity (38, 39). We therefore tested whether the
cytoplasmic localization of p53Val-135 is mediated by MDM2.
To this end, we expressed the GFP-p53 fusion in
p53
An NES, which is sufficient to mediate the export of wild-type human
p53, has recently been identified in the tetramerization domain of p53
encompassed by the proposed cytoplasmic sequestration domain (12). We
therefore constructed a mutant GFP-p53Val-135 fusion
consisting of leucine to alanine conversions at residues 345 and 347, which corresponded to the leucine residues at 348 and 350 essential for
the NES of wild-type human p53 (Fig. 2B), and we analyzed
the subcellular localization of the putative NES mutant. The 2KO cells
expressing the GFP-p53Val-135 L345A/L347A had
exclusively nuclear p53 at not only 32 °C but also 37 °C (Fig.
2C). This finding demonstrates that p53Val-135
uses its own NES for the nuclear export.
Three potential NLSs have been proposed to reside in the C terminus of
p53 (40). A mutation in the major one, NLS1
(313PPQKKKP319) in mouse p53, caused a defect
in the nuclear localization. In addition to NLS1, however, Lys-305 in
human p53 has recently been shown to be essential for the nuclear
import of p53 (41, 42), suggesting that the two basic amino acid
clusters, 305-306 (KR) and 319-321 (KKK), constitute a bipartite-type
NLS (Fig. 2B). To see if the NLS is necessary for the
nuclear localization of p53Val-135, we constructed a
GFP-p53Val-135 fusion containing a K302N mutation
corresponding to the human K305N, and we expressed it in 2KO cells. As
shown in Fig. 2C, the K302N mutant localized in the
cytoplasm even at 32 °C. Cycloheximide or LMB treatment could not
induce the nuclear accumulation of the K302N mutant. The constitutive
cytoplasmic localization of this mutant indicates that the NLS directly
mediates the nuclear import of p53Val-135.
The Rate of Nuclear Import of Temperature-sensitive p53 Is Reduced
at 37 °C--
The subcellular localization of shuttling proteins
reflects the equilibrium of the nuclear import and export rates. Since LMB does not affect the nuclear import of proteins (27, 29), specific
inhibition of nuclear export by LMB allows us to determine the kinetics
of the nuclear import of shuttling proteins. The percentage of cells
with p53 nuclear localization (nuclear > cytoplasmic), as calculated
from total 400-500 fluorescent cells, was ~15% in the control clone
6 culture at 37 °C, and it was almost unchanged during the further
cultivation at 37 °C for 12 h (Fig.
3A). Cells having nuclear p53
rapidly increased upon the temperature shift to 32 °C and the
cycloheximide treatment. About only a 1- or 2-h treatment was
sufficient for the half-maximum nuclear import. When the clone 6 cells
were treated with LMB at 37 °C, the cells showing p53 nuclear
localization gradually increased to reach a plateau 12 h after the
LMB addition. The time for the half-maximum induction was 7 h,
indicating that the import rate is greatly reduced at 37 °C. In
contrast, the import rate of p53 during the temperature shift and
cycloheximide treatment was almost unaffected by LMB. These results
suggest that the increase in the import rate is responsible for the
rapid nuclear accumulation of p53Val-135 upon the
temperature shift and cycloheximide treatment.
Association of p53Val-135 with the Import Receptor
Complex Is Reduced at 37 °C--
NLS-dependent nuclear
translocation of proteins is mediated by importins Addition of the p53 NLS to the N Terminus Suppresses the
Temperature-sensitive Nuclear Import--
If the NLS is masked by
interacting with other proteins or p53 itself, then the addition of the
NLS motif to the unmasked region would rescue the temperature-sensitive
nuclear import. To test this possibility, we conjugated the 26-amino
acid sequence (residues 297-322) containing both Lys-302 and NLS1
(Fig. 2B) to the N terminus of p53Val-135, and
we examined the subcellular localization of the fusion proteins (Fig.
4). The p53 NLS was able to direct the
nuclear import of GFP alone or GFP-p53Val-135 at both 32 and 37 °C. We also tested whether the constitutive cytoplasmic
sequestration of the K302N mutant can be rescued by the NLS supply at
its N terminus, to rule out the possible activation of endogenous NLS
by a conformational change upon the fusion. The GFP-p53 NLS-K302N also
showed nuclear localization even at 37 °C. These results clearly
indicate that the NLS does work irrespective of temperatures in
culture, when localized at the N terminus of p53Val-135.
The NLS is probably masked at 37 °C only when it is localized in the
C-terminal region.
Part of the C-terminal Domain Is Involved in the NLS
Masking--
The C-terminal region of p53 containing the NES has been
described previously to be responsible for the cytoplasmic
sequestration of wild-type p53 (41, 46). To determine whether the
C-terminal region of p53Val-135 plays a role in the NLS
masking, we synthesized three C-terminally truncated mutants of
p53Val-135 with the rabbit reticulocyte lysate, and we
examined their ability to bind importin Inhibition of Nuclear Export Results in the Nuclear Accumulation of
the p53 Complex Lacking Hsc70--
Molecular chaperones, Hsc70 and
Hsp90 family proteins, associate with mutant p53 in the cytoplasm (22,
23, 47-49). The multiprotein complex dissociates from
p53Val-135 upon its nuclear translocation (25, 26, 48). To
obtain a clue to understanding the mechanism by which the NLS is masked at 37 °C, we compared the association of p53Val-135 with
these chaperones between cytoplasmic and nuclear
p53Val-135. We first determined whether Hsp90 could be
co-precipitated with p53Val-135 in the presence of sodium
molybdate that stabilized the chaperone complex (32). As described by
others (48, 49), Hsp90 and its co-chaperone p23 became almost
undetectable in the precipitates containing the wild-type conformation
of p53Val-135 when cultured at 32 °C (Fig.
6A). On the other hand, when
the nuclear export was blocked by LMB or the nuclear import was
stimulated by cycloheximide, p53Val-135 accumulated in the
nucleus was still associated with both Hsp90 and p23. We next asked if
the Hsc70 binding to p53Val-135 was affected by
drug-induced nuclear translocation (Fig. 6B). p53Val-135 with the wild-type conformation at 32 °C did
not interact with Hsc70. In contrast to the case of Hsp90, the amount
of p53Val-135 co-precipitated with Hsc70 was greatly
reduced in the cells treated with LMB for 12 h, at which time in
more than 80% of cells p53Val-135 were accumulated in the
nucleus (Fig. 3A). As shown in Fig. 6C, the time
course of the dissociation of p53Val-135 from the Hsc70
complex during LMB treatment coincided well with the increase in the
p53 nuclear accumulation (see Fig. 3A). In the
cycloheximide-treated cells, however, the p53Val-135 was
slightly decreased but still present in the precipitate (Fig. 6B). These results demonstrate that the composition of
molecular chaperones in the nuclear p53 complex is different between
the two drug treatments. Namely, the inhibition of the nuclear export by LMB causes selective nuclear import of the complexes containing Hsp90 but lacking Hsc70, whereas cycloheximide treatment can induce nuclear translocation of the complexes containing Hsp90, Hsc70, or
both. To test the possibility that LMB directly promotes dissociation of the p53Val-135-Hsc70 complex, we examined the effect of
LMB on in vitro interaction between p53 and Hsc70 in the
rabbit reticulocyte lysate. As shown in Fig. 6D, the
association of the in vitro translated proteins was
unaffected by LMB.
Depletion of Hsc70 from the Reticulocyte Lysate Restores the p53
Association with the Import Receptor--
To investigate further the
role of Hsc70 in the regulation of p53Val-135 subcellular
localization, we examined the effect of Hsc70 removal from the
reticulocyte lysate by immunoadsorption on the in vitro binding of p53Val-135 to importin Temperature-sensitive Nuclear Import of p53--
In this study, we
show that cytoplasmic p53Val-135 at the nonpermissive
temperature still shuttles between the nucleus and the cytoplasm by
using its own NLS and NES. The specific inhibition of the nuclear export by LMB revealed that the decreased nuclear import is responsible for the cytoplasmic localization of p53Val-135 at 37 °C.
Binding to the import receptor was not detected at nonpermissive
temperature in the rabbit reticulocyte lysate, suggesting that the NLS
function in p53Val-135 is temperature-sensitive. Although
three potential monopartite NLS sequences (NLS1-NLS3) have been
identified in the p53 C-terminal region, it was recently shown that
NLS1, which alone directed the nuclear import of proteins, constituted
a strong bipartite-type NLS with an upstream KR sequence, inducing
sufficient nuclear transport of p53 (42). The constitutive cytoplasmic
localization of the NLS mutant of p53Val-135 indicates that
the bipartite-type NLS containing NLS1 is required for the nuclear
import of p53Val-135 and that NLS2 and NLS3 may have a
marginal activity to induce the nuclear import of
p53Val-135 (Fig. 2C). Since the NLS sequence
itself has no mutation in p53Val-135, it is unlikely that
the structure of the NLS itself is impaired at 37 °C. We showed that
the defect in the NLS function at 37 °C is completely rescued by the
same NLS when attached to the N terminus of p53Val-135
(Fig. 4). Therefore, the C-terminal region containing the NLS may be
locally masked at 37 °C. This mechanism of cytoplasmic retention is
in contrast to that for the nuclear retention by the
tetramerization-induced NES masking (Fig.
8).
A Model for the Cytoplasmic Sequestration of
p53Val-135--
We showed that immunodepletion of
Hsc70-containing complex from the reticulocyte lysate rescued the
p53Val-135 binding to importin
It is still unclear how the p53 NLS is masked. It was previously
proposed that p53 with an aberrant conformation is sequestered from the
nucleus by Hsp70/Hsc70 binding (22-24). Suppression of the high
transforming capacity of specific p53 mutants by overexpression of
Hsc70 suggests a role of Hsp70/Hsc70 in regulating transformation (52).
In some cases, however, Hsc70 binding may not effectively sequester p53
to the cytoplasm, since similar proportions of Hsc70-p53 complexes have
been found in both the cytoplasm and the nucleus (20). Our experiments
with recombinant Hsc70 ruled out the possibility that Hsc70 itself
interacts directly with the NLS (Fig. 7B). It is therefore
possible that a protein that masks the p53 NLS depending on the
p53Val-135 C-terminal domain is associated with the
Hsc70-containing complex (Fig. 8, protein X). It was
recently reported that the NLS in wild-type p53 was masked in some
tumor cells probably through its cytoplasmic sequestration domain of
p53, a region from residues 326 to 355 (42). We also showed that a
C-terminally truncated p53Val-135 (residues 1-326) could
bind importin A Yet Unsolved Question, Cycloheximide-induced Nuclear
Import--
Since cycloheximide induces nuclear translocation of
p53Val-135, it has long been postulated that a protein(s)
that rapidly turns over mediates the anchoring of
p53Val-135 to the cytoplasmic structure (26). However, the
present study demonstrated that cycloheximide induced recovery of the
NLS binding to the NLS receptor in cells (Fig. 3B) but not
in the reticulocyte lysate (Fig. 3C). In agreement with
this, the nuclear import rate in the cells treated with cycloheximide
was much greater than that in the control cells cultured with LMB at
37 °C (Fig. 3A). It is therefore unlikely that
cycloheximide stabilizes p53Val-135, leading to the
tetramerization-mediated inhibition of the nuclear export. These
results suggest that de novo protein synthesis is required
for the NLS masking. As described in the early study (26), the nuclear
p53Val-135 complex in the cells treated with cycloheximide
at 37 °C contained Hsc70 (Fig. 6B), supporting the idea
that Hsc70 itself is not the masking protein. Since the cell extract
from the untreated cells, but not cycloheximide-treated cells, blocked
the NLS binding to p53Val-135 in the Hsc70-depleted lysate
(Fig. 7C), it seems likely that a short lived protein(s),
which was lost in the nuclear p53Val-135 complex in the
presence of cycloheximide, is directly responsible for the NLS masking
or indirectly associated with specific chaperone activity to form the
NLS-masked conformation (Fig. 8). A protein of unknown function,
Spot-1, has been shown to interact with NLS1, but the significance of
this binding remains to be elucidated (55). Recently, Mot-2, a member
of the Hsp70 family (also known as mtHSP70 and Grp75), was shown to
interact directly with p53 and induce p53 cytoplasmic sequestration,
thereby inactivating the normal p53 function (56). The half-lives of
these proteins are unknown. The in vitro importin binding
assay in the Hsc70-depleted lysate used in this study provides a useful
system to identify the protein involved in the NLS masking.
p53 NLS Masking and Cancer--
Besides p53 gene
mutations in many human cancers, a substantial fraction of cancers
contain p53 that is functionally inactivated by other mechanisms.
Aberrant subcellular localization is one such mechanism found in breast
carcinoma (9), colorectal adenocarcinoma (57), undifferentiated
neuroblastoma (13), hepatocellular carcinoma (58), and retinoblastoma
(59). It is also associated with metastasis and poor prognosis (60).
p53 cytoplasmic sequestration could result from its anchorage to a
cytoplasmic tether or by an imbalance in nucleocytoplasmic shuttling,
i.e. increased nuclear export or decreased nuclear import.
Discrimination of these mechanisms had been difficult, until LMB became
available as a specific nuclear export inhibitor. Although
MDM2-dependent nuclear export may be involved in some
cases, we showed that the cytoplasmic sequestration of
p53Val-135 was independent of MDM2. It was recently shown
that in neuroblastoma cells the energy-dependent shuttling
of p53 is altered so that hyperactive nuclear export results in net
cytoplasmic accumulation (12). This finding implies that an upstream
signaling event which results in nuclear export is constitutively
active in these cells or that signals required for nuclear retention
are missing. It is currently unclear whether the cytoplasmic
sequestration of p53 in other cancers is also a result of hyperactive
nuclear export. The present study implies that the NLS masking
mechanism should exist in some cases that are believed to be
cytoplasmic anchoring. It is apparently important to explore the
possibility of the p53 cytoplasmic sequestration by NLS masking for the
better understanding of the mechanism by which p53 is inactivated in human cancers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mdm2
/
mouse (30), was kindly provided by G. Lozano. These cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated fetal bovine serum (HyClone Laboratories). Cells were
grown at 37 °C in a 5% CO2 atmosphere, and the
temperature was shifted to 32 °C when necessary. An anti-p53
monoclonal antibody (pAb421) and an anti-p53 polyclonal antibody
(FL393) were purchased from Oncogene Research and Santa Cruz
Biotechnology, respectively. Monoclonal antibodies against Hsp90 and
importin
were purchased from Transduction Laboratories. An
anti-Hsc70 polyclonal antibody was from Santa Cruz Biotechnology, and
an anti-p23 monoclonal antibody JJ3 was a gift from D. Toft (Mayo
Clinic). Recombinant Hsc70 was purchased from Medical and Biological
Laboratories. Leptomycin B (LMB) was prepared as described previously
(31). All other chemical reagents were purchased from Sigma unless
otherwise stated.
, and Hsc70 were synthesized by
using a TNT T7 Quick-coupled Transcription/Translation System (Promega)
according to the manufacturer's instructions. Templates for
transcription and translation were prepared by PCR by using
pBS-p53Val-135 as the template and
5'-TAATACGACTCACTATAGGGAGACCACCATGACTGCCATGGAGGAGTCA-3' (sense)
and 5'-GGGCTCGAGCAGATCTCCTCAGCTAGCGTCTGAGTCAGGCCCCACTTTC-3' (antisense)
as the primers for full-length p53Val-135, pTAC97 (33) as
the template and
5'-TAATACGACTCACTATAGGGAGACCACCATGGAGCTGATCACCATTCTC-3' (sense) and
5'-GGGCTCGAGCAGATCTCCTCAGCTAGCAGCTTGGTTCTTCAGTTTCCTC-3' (antisense) as
the primers for importin
, and pETHsc70 (34) as the template and
5'-TAATACGACTCACTATAGGGAGACCACCATGTCCAAGGGACCTGCAGTTGGT-3' (sense) and
5'-GGGCTCGAGCAGATCTCCTCAGCTAGCATCAACCTCTTCAATGGTGGGCCC-3' (antisense)
as the primers for Hsc70. To synthesize the three C-terminal deletion
mutants of p53Val-135, we used antisense primers
5'-GGGCTCGAGCAGATCTCCTCAGCTAGCGGTGAAATACTCTCCATC-3' for
p53Val-135 (1),
5'-GGGCTCGAGCAGATCTCCTCAGCTAGCTAACTCTAAGGCCTCATT-3' for p53Val-135 (1), and
5'-GGGCTCGAGCAGATCTCCTCAGCTAGCTCCAGACTCCTCTGTAGC-3' for
p53Val-135 (1-357). All the sense primers
contained the T7 promoter and the Kozak sequence. A portion of each
lysate containing synthesized [35S]methionine-labeled
p53Val-135 was mixed with that containing
[35S]methionine-labeled importin
or Hsc70 and
subjected to the following in vitro binding assay. After the
labeled proteins had been incubated at 32 or 37 °C for 3 h with
or without 30 nM LMB, the anti-p53 polyclonal antibody
(FL393) was added to the mixture. The bound proteins were precipitated
by the protein A/G-agarose beads as described above and analyzed by
autoradiography. For competition assays, various amounts of recombinant
Hsc70 or cell extracts were added to the mixture 3 h before p53
immunoprecipitation. For preparation of the cell extracts, clone 6 cells (3 × 106) were treated with or without 1 µg/ml cycloheximide for 12 h, and then cells collected were
lysed by sonication in IP buffer. The supernatants after centrifugation
at 15,000 × g for 20 min at 4 °C were collected as
the cell extracts. One mg of total protein was added to the mixture
3 h before p53 immunoprecipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nuclear accumulation of
p53Val-135 by LMB treatment. Clone 6 cells cultured at
37 °C were treated with 10 nM LMB for 12 h, and
p53Val-135 was detected by indirect immunofluorescent
staining with an anti-p53 antibody pAb421. Effect of transient
expression of CRM1 or CRM1-K1 on the LMB-induced nuclear localization
was examined by transfection of clone 6 cells with 10 µg per dish of
the CRM1 or CRM1-K1 mutant construct before the treatment with LMB. The
nuclei of the fixed cells were stained with 1 µg/ml DAPI, and their
localization of p53Val-135 was analyzed by
immunofluorescent microscopy. At least 300 cells were counted, and the
results of three independent experiments are shown as percentages with
the standard errors. N, predominantly nuclear;
N = C, nuclear and cytoplasmic;
C, predominantly cytoplasmic localization.
/
/mdm2
/
mouse embryonic fibroblast (2KO) cells (30) (Fig.
2A). As in the clone 6 cells,
p53Val-135 showed the temperature-sensitive nuclear
localization in 2KO cells; it localized in the cytoplasm at 37 °C
but became present in the nucleus when cultured at 32 °C.
Cycloheximide treatment rapidly induced nuclear translocation of
p53Val-135. These patterns of subcellular localization were
identical to those in the clone 6 cells (26). LMB treatment induced its
nuclear accumulation also in this cell line, and LMB-induced nuclear
transport of p53Val-135 was almost completely suppressed by
co-transfection of the CRM1-K1 construct. These results clearly
indicate that p53Val-135 is exported in an
MDM2-independent, CRM1-mediated manner, although they do not rule out
the possibility that other NES-bearing protein partners participate in
the nuclear export of p53Val-135.
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Fig. 2.
Nucleocytoplasmic shuttling of
p53Val-135. A, effect of LMB and
cycloheximide (CHX) on the localization of
GFP-p53Val-135 which is transiently expressed in 2KO cells
(mdm2 /
p53
/
). Effect of co-expression of
CRM1 or CRM1-K1 on the LMB-induced nuclear localization was examined by
transfection of 2KO cells with the CRM1 or CRM1-K1 mutant construct
before the treatment with LMB for 12 h. The fixed cells were
mounted with 1 µg/ml DAPI, and their localization was analyzed by
immunofluorescent microscopy. B, the C-terminal sequences
containing the NLS and NES in mouse and human p53. Possible
bipartite-type NLS sequences consisting of two clusters of basic amino
acids are underlined. The leucine-rich NES sequences (12)
are shown by boxes. C, subcellular localization
of GFP-p53Val-135 containing mutations in the NES and NLS.
An NES mutant (L345A/L347A) and an NLS mutant (K302N) were transiently
expressed in 2KO cells, and the effects of CHX and LMB were analyzed by
immunofluorescent microscopy.
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Fig. 3.
Temperature-sensitive association of
p53Val-135 with importin .
A, kinetics of nuclear import of p53Val-135. The
nuclear import rate of p53Val-135 in clone 6 cells was
determined by inhibiting the nuclear export by LMB. Clone 6 cells were
incubated with CHX or at 32 °C in the presence and absence of LMB.
Cells were fixed at the indicated times and stained with the anti-p53
antibody, and the proportion of cells having nuclear p53 was determined
by immunofluorescent microscopy. At least 300 cells were counted, and
the results of three independent experiments are shown as percentages
with the standard errors. B, in vivo
p53Val-135 binding to importin
. p53 in the clone 6 cells incubated in the absence and presence of CHX at 37 or 32 °C
was immunoprecipitated using the anti-p53 antibody, and the precipitate
was analyzed by immunoblotting (IB) using an anti-importin
antibody. C, in vitro p53Val-135
binding to importin
. 35S-p53Val-135 and
35S-importin
synthesized in the rabbit reticulocyte
lysate were mixed and incubated in the absence and presence of CHX at
37 or 32 °C. The amount of importin
associated with
p53Val-135 was analyzed by immunoprecipitation using the
anti-p53 antibody followed by autoradiography.
and
(also
known as karyopherins
and
or PTACs), which together constitute
the NLS receptor, in a manner dependent on the GTPase Ran/TC4 and
p10/NTF2 (nuclear transport factor 2) (43, 44). At least three major
forms of importin
(NPI-1, Rch-1, and Qip1) have been shown to bind
conventional NLSs rich in the basic amino acids and interact
specifically with importin
1. We next tested whether the reduced
nuclear import of p53Val-135 at 37 °C is due to the
reduced association with the NLS receptor. Since it is unknown which
form of importin
carries the p53 NLS, we monitored the p53 binding
to importin
. p53 was immunoprecipitated from the lysates of clone 6 cells cultured at 32 or 37 °C, and importin
in the precipitates
was detected by immunoblotting with an anti-importin
antibody (Fig.
3B). The amount of importin
bound to p53 decreased to an
almost undetectable level in the cells cultured at 37 °C. When the
cells were treated with cycloheximide at 37 °C, however, it was
recovered to the level similar to that in the cells cultured at
32 °C. To detect the in vitro association of
p53Val-135 with importin
, we used
35S-p53Val-135 and 35S-importin
synthesized in the rabbit reticulocyte lysate, in which the
temperature-dependent conformational status is reproducible in terms of the immunoreactivity and T antigen binding (45). Since the
lysate contains endogenous importins but not a detectable amount of p53
(data not shown), it seemed possible to detect importin
bound to
the p53Val-135 NLS via the adapter importin
present in
the reticulocyte lysate. After the lysates containing
35S-p53Val-135 and 35S-importin
had been mixed and incubated at 37 or 32 °C, p53Val-135
was immunoprecipitated, and the bound importin
protein was detected
by autoradiography (Fig. 3C). Importantly, importin
bound to p53Val-135 was undetectable in the lysate kept at
37 °C, whereas it was clearly detected when the lysate was incubated
at 32 °C. These results suggest that the NLS is masked at 37 °C,
but the reversion of the conformation at 32 °C causes exposure of
the NLS to the import receptor, leading to rapid import and subsequent
nuclear accumulation. Since the addition of cycloheximide into the
lysate could not restore the binding at 37 °C, the cycloheximide
effect may be seen only in the living cells.
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Fig. 4.
Suppression of temperature-sensitive nuclear
import by N-terminal addition of the p53 NLS. The 26-amino acid
sequence containing the p53 NLS (residues 297-322) was added to the N
terminus of GFP, GFP-p53Val-135, and
GFP-p53Val-135-K302N, and their subcellular localization
was analyzed by transient expression in 2KO cells. AD,
activation domain; PR, proline-rich domain; DBD,
DNA-binding domain; Oligo, oligomerization (tetramerization)
domain.
in vitro
at 37 °C (Fig. 5). Like the
full-length protein, p53-(1-357) did not bind importin
, and
p53-(1-347) containing the NES bound very weakly. On the other hand,
p53-(1-326) lacking the whole cytoplasmic sequestration domain almost
completely recovered the NLS binding. It seems unlikely that the
recovery of the NLS binding is due to the conversion of the mutant
conformation to the wild-type one, since the amount of Hsc70 associated
with these truncated mutants was essentially unchanged (data not
shown). These results suggest that the cytoplasmic sequestration domain is involved in not only the nuclear export but also the NLS
masking.
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Fig. 5.
Recovery of NLS binding by C-terminal
deletion of p53Val-135. C-terminally truncated
p53Val-135 proteins, p53-(1-326) (lane 1),
p53-(1-347) (lane 2), and p53-(1-357) (lane 3),
as well as the full-length protein (lane 4) were synthesized
in the rabbit reticulocyte lysate, and their in vitro
binding to 35S-importin was detected by
immunoprecipitation (IP) and autoradiography.
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Fig. 6.
Dissociation of Hsc70 from p53 during LMB
treatment. A, effect of LMB and CHX on the association
of p53Val-135 with Hsp90 and its co-chaperone p23. Clone 6 cells were treated with LMB or CHX for 12 h, and the amounts of
Hsp90 and p23 associated with p53Val-135 in cells were
determined by immunoprecipitation (IP) using the anti-p53
antibody followed by immunoblotting using an anti-Hsp90 and an anti-p23
antibody. B, effect of LMB and CHX on the association of
p53Val-135 with Hsc70. Clone 6 cells were treated with LMB
or CHX for 12 h, and the amounts of Hsc70 associated with
p53Val-135 in cells were determined by immunoprecipitation
using an anti-Hsc70 antibody followed by immunoblotting using the
anti-p53 antibody. C, time course of Hsc70 dissociation.
Clone 6 cells were treated with LMB for the indicated times, and the
amount of p53 in the immune complex with the anti-Hsc70 antibody was
determined by immunoblotting using the anti-p53 antibody. D,
effect of LMB on in vitro binding of p53Val-135
to Hsc70. 35S-p53Val-135 and Hsc70 were
synthesized in the rabbit reticulocyte lysate and incubated for 3 h in the absence or presence of LMB, and the amount of Hsc70 associated
with p53Val-135 was determined by immunoprecipitation using
the anti-p53 antibody followed by autoradiography.
. Since in
vitro translation in the lysate was strongly inhibited by the
depletion of Hsc70, we synthesized labeled p53 and importin
in the
presence of Hsc70, and then Hsc70 was removed by an anti-Hsc70 antibody
at 4 °C, at which temperature p53 was completely dissociated from
the Hsc70 complex (data not shown). The successful depletion was
monitored by immunoblot analysis using the antibodies to Hsc70 and
Hsp90 (Fig. 7A). In the
presence of Hsc70, in vitro binding of
p53Val-135 to importin
was detected at 32 °C but not
at 37 °C, as shown in Fig. 7A and Fig. 3C.
When Hsc70 was removed, however, the association of
p53Val-135 with importin
became detectable even at
37 °C. This binding was specific, because the K302N mutant defective
in the NLS function did not bind importin
under the same
conditions. We next examined if the purified recombinant Hsc70 protein
could inhibit the association with importin
, which had been
restored by the Hsc70 depletion. As shown in Fig. 7B, Hsc70
even at a high concentration did not interfere with the protein-protein
interaction, suggesting that the Hsc70-containing complex is required
but Hsc70 alone is insufficient for masking the NLS at 37 °C.
Interestingly, a soluble cell extract prepared from the clone 6 cells
could block this interaction, whereas an extract from the cells treated
with cycloheximide failed to inhibit it (Fig. 7C).
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Fig. 7.
Effect of Hsc70 immunodepletion on in
vitro p53Val-135 binding to importin
in rabbit reticulocyte lysate. A,
restoration of the p53Val-135 binding to importin
in
the absence of Hsc70. 35S-Labeled p53Val-135,
p53Val-135-K302N, and importin
were synthesized in the
rabbit reticulocyte lysate (input). Hsc70 was immunodepleted
from these lysates at 4 °C using the anti-Hsc70 antibody
(Hsc70-depleted). The lysates containing
35S-p53Val-135 and 35S-importin
were mixed and incubated at 37 or 32 °C for 3 h, and the amount
of importin
associated with p53Val-135 was determined
by immunoprecipitation (IP) and autoradiography. The amounts
of Hsc70 and Hsp90 in the mixtures were determined by immunoblotting
(IB). B, effect of recombinant Hsc70 on in
vitro binding to importin
. Various amounts of the recombinant
Hsc70 protein were added into the mixture (100 µl), and the effect
on the interaction between p53Val-135 and importin
in
the Hsc70-depleted lysates was analyzed by autoradiography.
C, effect of cell extract on in vitro binding to
importin
. The cell extract prepared from the clone 6 cells treated
with or without CHX was added into the mixtures, and the effect on the
interaction between p53Val-135 and importin
in the
Hsc70-depleted lysates was analyzed by autoradiography. The amounts of
Hsc70 in the mixtures were also determined by immunoblotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
A model for subcellular localization of
p53Val-135 regulated by temperature-sensitive nuclear
import. At 37 °C, most p53Val-135 is associated
with a multiprotein complex containing Hsc70, thereby being sequestered
in the cytoplasm, because its major NLS is masked through a possible
conformational change or by a yet unknown masking protein
(X) associated with the complex. A fraction of
p53Val-135 that was dissociated from the Hsc70-containing
complex can shuttle between the cytoplasm and the nucleus by its own
NLS and NES. At 32 °C, p53Val-135 dissociates from both
Hsc70 and Hsp90 and can relocalize into the nucleus. Tetramerization in
the nucleus may prevent the nuclear export of p53Val-135 by
the NES masking, resulting in the nuclear retention as described
(12).
(Fig. 7). This finding
strongly suggests that the formation of the p53Val-135
multiprotein complex with Hsc70-containing chaperones is important for
the NLS masking. Thus, we present a model for the temperature-sensitive nuclear import in that the NLS is masked by the Hsc70-containing complex (Fig. 8). Dissociation of p53Val-135 from the
chaperone complex at 32 °C may result in the rapid nuclear import.
If the p53 NLS is completely masked at 37 °C, it should remain in
the cytoplasm even when the nuclear export is blocked by LMB. During
LMB treatment, however, the protein complex containing Hsc70, but not
Hsp90, was gradually dissociated from p53Val-135 in clone 6 cells, which coincided well with the slow nuclear translocation of
p53Val-135 in the presence of LMB (see Figs. 3A
and 6C). Because in the reticulocyte lysate LMB did not
induce separation of p53Val-135 from the Hsc70-containing
complex (Fig. 6D), it is unlikely that LMB treatment
directly causes unmasking of the NLS by dissociating p53Val-135 from the complex. In this model, there are at
least two distinct physical complexes of p53Val-135 at
37 °C, one containing both Hsc70 and Hsp90 and the other containing Hsp90 but lacking Hsc70, the former is incapable of entering the nucleus due to the NLS masking, but the latter is able to shuttle. The
subcellular localization of Hsc70 is predominantly cytoplasmic in clone
6 cells, and it was unaffected by LMB
treatment.2 Since
reticulocyte lysate does not contain nuclei, the increased dissociation
of p53Val-135 from the Hsc70 complex in the LMB-treated
living cells may be due to compartmentalization of
p53Val-135 by the inhibition of the nuclear export after
the nuclear import, which prevents re-association with cytoplasmic
Hsc70. The dissociation of p53Val-135 from the
Hsc70-containing complex may be the rate-limiting step for the
p53Val-135 nuclear import at 37 °C. It is possible that
the NLS-masked, Hsc70-associated complex of p53 is reversibly tethered
to a cytoplasmic structure. Thus, the current model is not inconsistent
with the observation that the mutant p53 is sometimes tethered to
intermediate filaments (50) and mitochondria (51).
at 37 °C without affecting the Hsc70 binding,
suggesting that the cytoplasmic sequestration domain regulates the
nuclear import by controlling access of the NLS to the import receptor
in the presence of the Hsc70-containing complex (Fig. 5). Therefore, it
is also possible that p53Val-135 is intramolecularly masked
in the Hsc70-containing complex. Since it is established that
Hsp70/Hsc70 is a component required for a minimal chaperone system
containing five proteins (Hsp90, Hsp70, Hop, Hsp40, and p23)
reconstituted from reticulocyte lysate (53, 54), the
p53Val-135 polypeptide may be folded by the chaperone
complex leading to the NLS masking with the cytoplasmic sequestration
domain (Fig. 8).
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ACKNOWLEDGEMENTS |
---|
We thank Saadi Khochbin for providing the clone 6 cell line, Guillermina Lozano for the 2KO cell line, David Toft for the anti-p23 antibody, and Christian Delphin for the p53Val-135 plasmid. We also thank David P. Lane for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by the CREST Research Project, Japan Science and Technology Corporation, a special grant for Advanced Research on Cancer from the Ministry of Education, Culture and Science of Japan, the Senri Life Science Foundation, and the Takeda Science Foundation, Japan.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 and reprint requests should be addressed: Dept. of Biotechnology, Graduate School of Agriculture and Life Sciences, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-5124; Fax: 81-3-5841-5337; E-mail: ayoshida@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100200200
2 S. Akakura, M. Yoshida, Y. Yoneda, and S. Horinouchi, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NES, nuclear export signal; CRM1, chromosome region maintenance 1; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; Hsc70, heat-shock protein 70 cognate protein; LMB, leptomycin B; NLS, nuclear localization signal; PBS, phosphate-buffered saline; pAb, polyclonal antibody; PCR, polymerase chain reaction; CHX, cycloheximide.
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