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J Biol Chem, Vol. 274, Issue 46, 32699-32703, November 12, 1999
From the Department of Internal Medicine, University of Michigan
Medical Center, Ann Arbor, Michigan 48109-0936
Abnormal p53 cellular localization has been
considered to be one of the mechanisms that could inactivate p53
function. To understand the regulation of p53 cellular trafficking, we
have previously identified two p53 domains involved in its
localization. A basic domain,
Lys305-Arg306, is required for p53
nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic
sequestration domain (CSD) from residues 326-355, could block the
nuclear import of Lys305 or Arg306 mutated p53.
To characterize further the function of these two domains, we
demonstrate in this report that the previously described major nuclear
localization signal works together with
Lys305-Arg306 to form a bipartite and
functional nuclear localization sequence (NLS) for p53 nuclear import.
The CSD could block the binding of p53 to the NLS receptor, importin
The p53 tumor suppressor is a nuclear phosphoprotein whose
activities have been linked to cell cycle control, apoptotic cell death, DNA repair, stress responses, and genomic stability (1-3). p53
is thought to regulate cell growth and viability through
transcriptional dependent and independent pathways (3, 4). Since a
defect in p53 nuclear translocation would impair its biological
function (5-7), the cellular trafficking of p53 must be tightly
regulated for it to function normally. Indeed, in certain human cancers (breast cancers, colon cancers, and neuroblastoma), wild-type p53 is
localized predominantly in the cytoplasm, suggesting that the
inactivation of wild-type p53 in cancer cells could partly result from
a defect in the regulation of p53 subcellular localization (8-10).
Protein shuttling between the nucleus and the cytoplasm is
fundamentally controlled by both a nuclear localization sequence (NLS)1 and a nuclear export
sequence (NES). These topogenic sequences bind to specific receptors
that allow selective passage through the nuclear pore complex (reviewed
in Refs. 11-13). Classical NLSs are either a monopartite motif that
consists of a single, short stretch of several basic amino acids or a
bipartite motif that consists of two separated clusters of basic
residues (Table I). Both types of signals can be recognized by the
nuclear import factor, karyopherin It has been shown that the p53 protein is subject to both nuclear
import and export via a fast, energy-dependent pathway
(17). Similar to other nuclear proteins, the p53 protein contains both an NLS and an NES. Three putative NLSs residing in the carboxyl terminus of p53 were first identified based on sequence and mutagenesis analyses (18). The major one, NLSI (PQPKKKP), was proven to be
functionally important for p53 nuclear import and can direct a
cytoplasmic reporter protein, pyruvate kinase, completely into the
nucleus (19). A recent study has identified a Rev-like NES located in
the tetramerization domain that is involved in p53 nuclear export
mediated by the CRM1 (20). This intrinsic NES is sufficient to mediate
p53 nuclear export, although other studies have reported that Hdm2 can
also shuttle p53 from the nucleus to the cytoplasm (21, 22). It was
proposed that oligomerization can cause p53 nuclear retention via
masking of the NES (20).
We have identified two cis-acting domains important for p53
subcellular localization as follows: a basic domain
(Lys305-Arg306), in addition to the known NLSI,
that is required for p53 nuclear import, and a carboxyl-terminal
cytoplasmic sequestration domain (CSD, residues 326-355) that
interacts with this Lys-Arg domain to regulate p53 nuclear import (23,
24). The CSD contains an oligomerization domain and the NES. In the
present report, we demonstrate that NLSI alone is not sufficient to
direct p53 nuclear import due to a weak binding to importin Cell Culture--
The MCF-7 breast cancer cell line, which
expresses wild-type p53, and two p53-null cell lines, H1299 lung
adenocarcinoma and Saos-2 osteosarcoma, were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing 10%
(v/v) fetal bovine serum at 37 °C in a humidified 5%
CO2 atmosphere.
Plasmid Construction and Mutagenesis--
To analyze the
function of p53 nuclear localization signals (NLS), the DNA fragments
corresponding to p53 amino acid residues 316-322, 305-322, and
K305N-322 were amplified by PCR and subcloned into the KpnI
and NotI sites in the 3' end of a Myc-tagged chicken muscle
pyruvate kinase (PK) cDNA expression plasmid (25). To replace the
NLSI (residues 316-322; PQPKKKP) of p53 with the SV40 large T antigen
NLS (PKKKRKV), a two-step PCR mutagenesis protocol was performed as
described previously (23) using pC53-SN (26) as the template. The final
PCR fragments were subcloned into the BamHI and
EcoRI sites of the pK7-GFP plasmid (27). To analyze the
relationship between p53 oligomerization and nuclear import, the
FLAG-tagged DNA fragment corresponding to p53 residues 325-369 was
amplified by PCR with a 5' primer containing FLAG sequences, and the
PCR product was ligated to the BamHI and EcoRI
sites of the pcDNA3 plasmid. All other mutated p53 constructs were
made by two-step PCR and inserted into the plasmid pK7-GFP.
Cell Transfection and Immunofluorescence Microscopy--
Cells
were grown on glass coverslips in six-well plates and transfected using
LipofectAMINE (Life Technologies, Inc.) as described previously (23).
After transfection, cells were washed with PBS and fixed with 4%
paraformaldehyde for 10 min at room temperature. After three washes
with PBS, the fixed cell were then permeabilized with 0.2% Triton
X-100 in PBS for 2 min and incubated in PBS containing 0.5% bovine
serum albumin for 20 min. Where indicated, cells were incubated for
1 h with an anti-FLAG M2 monoclonal antibody (Sigma) or an
anti-c-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology) followed by 50 min incubation with fluorescein isothiocyanate or
lissamine rhodamine B sulfonyl chloride (LRSC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). After a final
wash with PBS, coverslips were mounted with antifade solution (100 mM NaHCO3 pH 9.0, 4 mg/ml
p-phenylenediamine, 50% glycerol) and examined by
fluorescence microscopy.
Analysis of Leptomycin B-treated Cells--
MCF-7 cells were
transiently transfected with plasmid p53K305N-GFP. Two days after
transfection, cells were treated with 100 nM leptomycin B
(LMB) for 4 h. The localization of cyclin B1, a control for
LMB-inhibited nuclear export (28), was then determined by indirect
immunostaining using a combination of monoclonal antibody to human
cyclin B1 (GNS1, Santa Cruz Biotechnology) and LRSC-conjugated goat
anti-mouse IgG. The cellular localizations of exogenous p53K305N-GFP and endogenous cyclin B1 in a sample were visualized and differentiated by fluorescence microscopy.
In Vitro Binding Assay of p53 and Importin Nuclear Export Is Not Associated with Cytoplasmic Sequestration of
Lys305-mutated p53--
Due to the fact that p53 cellular
localization is a dynamic equilibrium between rapid nuclear import and
ongoing export, it is possible that the cytoplasmic sequestration
effect of the Lys305 or Arg306 mutations is a
result of enhancement of p53 nuclear export, probably by increasing the
interaction with CRM1. To address this possibility, MCF-7 cells
transiently transfected with the p53K305N-GFP plasmid were treated with
leptomycin B (LMB). The transfected cells were immunostained for cyclin
B1 as the control of LMB inhibition of CRM1-mediated nuclear export
(28). The results showed that although cyclin B1 shifted from the
cytoplasm to the nucleus after LMB treatment,
Lys305-mutated p53 (p53K305) still accumulated in the
cytoplasm (Fig. 1). This indicates that
cytoplasmic sequestration of p53K305 is not a result of an increase in
nuclear export but a result of a defect in nuclear import.
Lys305-Arg306 and NLSI Form a
Bipartite-like NLS--
Previously we showed that a spacer between the
Lys305-Arg306 motif and NLSI is necessary for
entrance of p53 into the cell nucleus (24). Thus, it is possible that
the Lys305-Arg306 motif and NLSI work together
as a bipartite NLS. To test this possibility, the NLSI alone or the
complete sequence from Lys305 to NLSI were fused with a
cytoplasmic protein, Myc-tagged pyruvate kinase, to analyze their
nuclear targeting efficiency. The NLSs were fused to a Myc-tagged
pyruvate kinase (Myc-PK, Ref. 25) at the carboxyl terminus, and all
constructs were transfected into MCF-7 cells. The results showed that
although the sequence composed of residues 305-322 was sufficient to
direct a complete nuclear import of Myc-PK, neither NLSI alone
(residues 316-322) nor Lys305-mutated sequences were able
to do so (Fig. 2). To determine further the effect of NLSI in its native context within the p53 protein, we
replaced NLSI in p53K305 with the SV40 large T antigen NLS (Tag NLS,
PKKKRKV), a well defined monopartite NLS. If NLSI functions the same
way as that of Tag NLS, this replacement should not change the
cytoplasmic sequestration of the protein. The result, however, showed
that p53K305 restored nuclear import when NLSI was substituted by Tag
NLS (Fig. 3), suggesting that NLSI is a
weak monopartite NLS compared with the Tag NLS. These data confirmed
that two basic domains, Lys305-Arg306 and
NLSI, are both required for nuclear import of p53. Taken together,
these data indicate that a bipartite-like NLS is required for p53
nuclear import.
Masking of p53 Nuclear Import Signals by a Cytoplasmic
Sequestration Domain--
It has been shown that deletion of any
region from residues 326 to 355, the so-called cytoplasmic
sequestration domain (CSD), results in entry of Lys305 or
Arg306 mutated p53 into the nucleus (23, 24). It is
possible that the CSD could inhibit the nuclear import of p53 by
masking the NLS and block the binding of importin
To delineate the role that the CSD plays in p53 nuclear import in the
cell, we analyzed the localization of transiently transfected GFP
fusions of WT p53 and CSD-deleted mutants in MCF-7, H1299, and Saos-2
cells. The subcellular localizations of the varied mutant and wild-type
p53 proteins, as determined by GFP fluorescence, were scored and
divided into three groups as nuclear (N), cytoplasmic (C), and both
nuclear and cytoplasmic (N + C) accumulation. The percentage of each
localization was determined from a total of ~500 fluorescent cells
observed in several fields of a slide. The results showed that although
WT p53 was distributed predominantly in both the nucleus and the
cytoplasm of an individual cell, the CSD-deleted mutants primarily
exhibited the nuclear localization, although the percentage of
distribution varied in different cell lines (Table II). The cellular
localization of a p53 mutant with the deletion outside of the CSD
region (residues 356-365) did not show a significant difference from
that of WT p53 (Table II). This result supports the observation that
the CSD could block the binding of importin p53 Can Enter the Nucleus as an Oligomerized Complex--
Due to
the fact that the CSD contains the p53 oligomerization domain (31, 32),
it is possible that the blocking effect of the CSD on nuclear import is
a result of oligomerization of p53 protein. To test this possibility,
MCF-7 cells were co-transfected with the GFP fusion of WT p53 or
p53K305 and a FLAG-tagged construct (residues 325-369) consisting of
the oligomerization domain. The localization of WT p53 or p53K305 and
the oligomerization domain peptide were differentiated by GFP
fluorescence and immunostaining of FLAG tags. The results showed that
the oligomerization domain itself was distributed to both the nucleus
and the cytoplasm (Fig. 5). When
co-transfected with WT p53-GFP, the oligomerization domain exclusively
accumulated in the nucleus as did WT p53-GFP (Fig. 5). Because this
oligomerization domain contains no NLS, it must rely on the p53 NLS to
enter the nucleus, suggesting that oligomerization of p53 does not
block the p53 nuclear import. Our data also showed that the
oligomerization domain accumulated in the cytoplasm together with
p53K305 (Fig. 5). Taken together, these data indicate that the blocking
effect of the CSD on p53 nuclear import is not associated with p53
oligomerization.
Three novel findings about p53 subcellular localization are
demonstrated in this study. First, the nuclear localization signal of
p53 is bipartite. Second, a CSD from residues 326 to 355 can reduce the
efficiency of p53 nuclear import by blocking the binding of NLS to
importin These observations extend our previous studies that identified a basic
domain, Lys305-Arg306, required for p53
nuclear import. We show here that this domain works together with NLSI
(PQPKKKP) to bind to importin Deletion of the CSD enhances the binding of p53 to importin
A Bipartite Nuclear Localization Signal Is Required for p53
Nuclear Import Regulated by a Carboxyl-terminal Domain*
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, and reduce the efficiency of p53 nuclear import in MCF-7, H1299,
and Saos-2 cells. The blocking effect of the CSD is not due to the
enhancement of nuclear export or oligomerization of the p53. These
results indicate that the CSD can regulate p53 nuclear import by
controlling access of the NLS to importin binding.
INTRODUCTION
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DISCUSSION
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or importin , to initiate
nuclear entry of the protein. On the other hand, the NES is
characterized by a leucine-rich sequence. An NES receptor, exportin 1 or CRM1, has been identified to be responsible for protein nuclear
export (14-16).
. The
Lys-Arg and NLSI together form a bipartite NLS that allows importin to bind more tightly to p53, therefore mediating the nuclear import of
p53. Furthermore, we show that the CSD modulates the binding of
importin to the NLS. In contrast to nuclear export, oligomerization does not inhibit p53 nuclear import.
EXPERIMENTAL PROCEDURES
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--
The
wild-type or mutated p53 DNA fragments were ligated into the
BamHI and EcoRI sites of vector pGEX-2T (Amersham
Pharmacia Biotech) and transformed into the Escherichia coli
strain DH5. The cell culture and batch purification of glutathione
S-transferase (GST) fusion proteins were performed according
to manufacturer's instructions. The E. coli strain BLR
containing GST fusions of a functional SV40 large T antigen nuclear
localization signal (Tag NLS) or an inverse version of Tag NLS (Tag
NLSinv) were cultured, and the proteins were purified as described
(29). The plasmid pRSET-hSRP1 containing a cDNA of human
importin (30) was used to produce a
[35S]methionine-labeled protein using the Promega TNT T7
Quick Coupled Translation System. Fifteen micrograms of GST-p53 or p53
mutants and 7 µg of GST-Tag NLS or GST-TagNLSinv were incubated with
40 µl of glutathione-agarose beads (Santa Cruz Biotechnology) in 0.5 ml of binding buffer (20 mM HEPES, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 2 mM dithiothreitol, 0.1% Tween 20) for 2 h at 4 °C.
The beads were collected and washed three times with binding buffer.
After washing, one-twentieth of the beads were removed to analyze the
amount of immobilized GST fusion proteins by immunoblotting with the
anti-p53 pAb122 hybridoma supernatant (ATCC TIB116) or the anti-GST
mouse IgG1 monoclonal antibody (Santa Cruz Biotechnology). The rest of the beads were incubated with 90 µl of in
vitro translated importin reaction mixture for 4 h at
4 °C. The beads were then washed six times with binding buffer,
boiled in 30 µl of sample buffer, and the immobilized proteins were
resolved on a SDS-10% polyacrylamide gel. The 35S-labeled
importin bound to the GST fusion proteins was detected by
fluorography using Amplify Reagent (Amersham Pharmacia Biotech). The
relative intensity of importin was measured by NIH Image software.
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DISCUSSION
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View larger version (84K):
[in a new window]
Fig. 1.
Effect of LMB on the subcellular localization
of Lys305-mutated p53. MCF-7 cells grown on coverslips
were transfected with p53K305-GFP and treated with or without 100 nM LMB for 4 h. The cellular localization of cyclin B1
in transfected cells was determined by immunostaining with a monoclonal
anti-cyclin B1 antibody and an LRSC-conjugated goat anti-mouse IgG. The
localizations of Lys305-mutated p53 and cyclin B1 were
differentiated by GFP and LRSC fluorescence using fluorescence
microscopy.
View larger version (123K):
[in a new window]
Fig. 2.
Subcellular localizations of p53 nuclear
localization signals and PK fusion derivatives. The p53 residues
316-322 (NLSI), 305-322, or K305N-322 were linked to the
KpnI sites in the carboxyl terminus of a Myc-tagged PK
cDNA. The different fusion constructs were transiently transfected
into MCF-7 cells, and the cellular localization of fusion proteins was
determined by immunostaining with a monoclonal anti-c-Myc antibody and
a fluorescein isothiocyanate-conjugated goat anti-mouse IgG.
View larger version (34K):
[in a new window]
Fig. 3.
Effect of NLSI replacement by SV40 large T
antigen (Tag NLS) on the subcellular localization of
Lys305-mutated p53. The GFP fusion proteins of
wild-type (a) and mutated p53 proteins including
K319A/K320A/K321A (b), K305N (c), and replacement
of NLSI by Tag NLS (d) were expressed in MCF-7 cells, and
the subcellular localization of each fusion protein was determined by
GFP fluorescence. Two basic domains required for p53 nuclear import are
underlined. The italic letters indicate the
mutated amino acid residues.
. To test this
hypothesis, we first performed a binding assay using in
vitro translated importin and recombinant GST fusion proteins
of either wild-type (WT) or p53 mutations of Lys305, the
CSD, or both. The GST fusions with SV40 Tag NLS (GST-NLS) and an
inverse version of Tag NLS (GST-NLSinv) served as positive and negative
controls, respectively, for the importin binding. As shown in Fig.
4, p53K305, which accumulates in the
cytoplasm of MCF-7 cells, had 3-fold less binding to importin compared with that of WT p53. Deletion of part of the CSD (residues
346-350), however, enhanced the binding of p53K305 to importin by
2-fold. This suggests that the in vivo data showing that
CSD-deleted p53K305 restored nuclear import ability (23) can be
explained by increased binding of the CSD mutant to importin . In WT
p53, deletion of the CSD also increased binding to importin by
~3-fold (Fig. 4). There is a weak but detectable binding of importin
to the p53 with the NLS (residues 305-322) deletion. This may
result from binding of importin to the NLSII and NLSIII in the
carboxyl terminus (18) of p53 or a nonspecific binding to the mutant. Nonetheless, these data indicate that the CSD can inhibit the binding
of importin to p53.
View larger version (49K):
[in a new window]
Fig. 4.
Binding of importin to wild-type and mutated p53 proteins. Immobilized GST-p53
(15 µg per 40 µl agarose beads) or GST-NLS and GST-NLSinv (7 µg
per 40 µl agarose beads) were incubated with 90 µl of in
vitro translated importin labeled with
[35S]methionine for 4 h at 4 °C. The proteins
were separated by SDS-10% PAGE gel, and bound importin was
analyzed by fluorography. The bottom panel is the immunoblot
showing the relative amount of immobilized GST fusion proteins
subjected to the binding assay. The difference of importin binding
between wild-type (WT) and the mutant p53 proteins was
quantitated by measuring the intensity of each band with NIH Image
software. The intensity is relative to the binding of WT p53 to
importin . The results are representative of two independent
experiments.
to the NLS and hence
reduce the efficiency of p53 nuclear import.
View larger version (66K):
[in a new window]
Fig. 5.
Co-localization of the p53 oligomerization
domain with wild-type and Lys305-mutated p53. A FLAG
fusion protein containing the p53 oligomerization domain (residues
325-369) was expressed alone or co-expressed with p53-GFP or
p53K305N-GFP in MCF-7 cells. The cellular localization of
FLAG-p53-(325-369) was determined by immunostaining with a monoclonal
anti-FLAG antibody and an LRSC-conjugated goat anti-mouse IgG.
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. Third, p53 can enter the nucleus as an oligomerized complex.
and direct p53 to the nucleus. The
NLSI was originally considered as a functional NLS based upon the
observation that NLSI is sufficient to direct the nuclear import of PK
when it was linked at the amino terminus of PK and transfected into
COS-7 cells (19). The NLSI motif may function as an NLS (19), but due
to the CSD in the p53 protein, it is an extremely weak NLS resulting
from a weak binding to importin . Therefore, the activity of the p53
NLS is dependent on the protein context within which it is located. The
replacement of NLSI with the Tag NLS could restore the nuclear import
of Lys305-mutated p53, further demonstrating that NLSI
itself is a rather weak NLS. The Lys305-Arg306
domain separated from the NLSI by 9 amino acids is necessary for
binding to importin and p53 nuclear import. This redefines the NLS
of p53 as a bipartite NLS
(305KRALPNNTSSSPQPKKKP322). This bipartite NLS,
however, is different from that of nucleoplasmin (Table
I) whose upstream Lys residue can be
substituted by another basic residue, Arg, without affecting its
nuclear transport (33). Our previous study showed that mutation of
Lys305 to Arg or Arg306 to Lys could not
restore the ability of p53 nuclear import (24).
Comparison of nuclear localization signals in human p53 and other
nuclear proteins
and
increases the efficiency of p53 nuclear import, indicating that the CSD
can regulate p53 cellular trafficking by controlling its entrance into
the nucleus. The CSD also contains an oligomerization domain (31, 32)
and a functional nuclear export signal (NES, see Ref. 20). Several
lines of evidence suggest that oligomerization and nuclear export of
p53 are not involved in the control of p53 nuclear import by the CSD.
First, inhibition of protein nuclear export mediated by CRM1 could not
rescue the nuclear import of Lys305-mutated p53 which,
however, happened when the CSD was deleted (Fig. 1 and Ref. 23).
Second, a deletion in the CSD region (residues 326-335), but not
within the NES (residues 340-351), increased the efficiency of p53
nuclear import (Table II), indicating
that the blocking of p53 nuclear import by the CSD is not solely the counter effect of the NES residing in the CSD. Third, association of a
peptide (residues 325-369) consisting of the p53 oligomerization domain with the p53 protein has no effect on p53 nuclear import (Fig.
5), indicating that the blocking of p53 nuclear import by the CSD does
not result from oligomerization of p53 through this domain.
Subcellular localization of mutated p53 in MCF-7, HI299, and Saos-2
cells
The in vitro binding assay indicated that the CSD can
structurally mask the access of importin to the NLS of p53. It is likely that this masking is regulated in the cell via a change in
protein conformation, thus the CSD may serve as a guarding sequence for
the activity of p53 by controlling the availability of the NLS to
importin .
Although it is still unclear how the cellular trafficking of p53 is
regulated by the cell, studies have indicated that protein synthesis is
required for the nucleo-cytoplasmic transport of p53 protein (34, 35).
We have observed in this study that the effect of the CSD is varied in
different cell lines. Taken together, these results imply the existence
of a mechanism involved in the regulation of CSD function, and this
regulation is cell type-dependent. It will be interesting
to see if there is any upstream signal or molecule involved in the
regulation of CSD function.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Gideon Dreyfuss and Haruhiko
Siomi for the Myc-tagged pcDNA3-PK plasmid; Dr. Arnold J. Levine
and Jiayuh Lin for the pC53-SN plasmid and H1299 cells; Dr. Ian G. Macara for the pK7-GFP plasmid; Dr. Michael F. Rexach for GST-NLS and
GST-NLSinv constructs; and Dr. Karsten Weis for the pRSET-hSRP1
plasmid. We also thank Dr. Minoru Yoshida and Nobuaki Kudo for the
supply of leptomycin B and Dr. Adam Goldsmith for thoughtful reading of
the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by NCI Grant CA67140 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Internal
Medicine, University of Michigan Medical Center, 4310 CCGC, 1500 East
Medical Center Dr., Ann Arbor, MI 48109-0936 Tel.: 734-764-8195; Fax:
734-763-4226; E-mail: mclarke@umich.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: NLS, nuclear localization sequence; NLSI, nuclear localization signal; NES, nuclear export sequence; CSD, cytoplasmic sequestration domain; PCR, polymerase chain reaction; PK, pyruvate kinase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; LRSC, lissamine rhodamine B sulfonyl chloride; LMB, leptomycin B; GST, glutathione S-transferase; WT, wild type.
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