From the Department of Molecular Biology, University
of Occupational and Environmental Health, 1-1 Iseigaoka
Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan and the
§ Orthopaedic Surgery, Biomedical Regulation,
Integrative Biomedical Sciences, Graduate School of Medical Sciences,
Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
Received for publication, September 6, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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A nonhistone chromosomal protein, high mobility
group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds
preferentially to cisplatin-modified DNA. HMG1 also functions as a
coactivator of p53, a tumor suppressor protein. We investigated
physical interactions between HMG1 and p53 and the influence of p53 on
the ability of HMG1 to recognize damaged DNA. Using immunochemical
coprecipitation, we observed binding of HMG1 and p53. Interaction
between HMG1 and p53 required the HMG A box of HMG1 and amino acids
363-376 of p53. Cisplatin-modified DNA binding by HMG1 was
significantly enhanced by p53. An HMG1-specific antibody that
recognized the A box of this protein also stimulated cisplatin-modified
DNA binding. These data suggest that an interaction with either p53 or
antibody may induce conformational change in the HMG1 A box that
optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA
damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and
p53-mediated DNA repair.
The cytotoxic effect of cisplatin is believed to result from
formation of covalent adducts with DNA (1). Cisplatin treatment induces
a tumor suppressor protein, p53, to suppress cell proliferation through
p21 induction (2). Interestingly, p53 possesses 3' to 5' exonuclease
activity (3). The recent observation that another protein,
HMG1,1 can enhance
p53-mediated transactivation suggests links between HMG1 and p53 in
other physiologic settings (4). The p53 protein has been reported to
accumulate in cisplatin-resistant cells (5). In addition to
transactivation, p53 is involved in recognition and repair of DNA
damage (5). HMG1 has three domains. The N-terminal domain (A box) and
the central domain (B box) are positively charged and bind to DNA,
whereas the acidic C-terminal domain interacts with histones (6). The
amino acid sequences of the A and B boxes of HMG1 are homologous to a
segment of about 70 amino acid residues called the HMG box. HMG1 does
not bind to DNA in a sequence-specific manner; rather, it functions as
an architectural protein for structuring chromatin. Notably, HMG1 binds
preferentially to cisplatin-modified DNA (7). Yeast mutants lacking the
HMG domain protein Ixr1 have been found to be significantly more
resistant to cisplatin than wild type cells (8). On the other hand,
distribution of HMG1 protein is changed after cisplatin treatment (9),
and HMG1 protein genes usually are up-regulated in cisplatin-resistant human cancer cells.2 Thus,
uncertainty prevails as to whether cellular levels of HMG proteins
correlate with cellular resistance to cisplatin. HMG proteins enhance
sequence-specific DNA binding of a variety of transcription factors,
but whether these factors can modulate HMG1 binding to damaged DNA is
not known. We therefore investigated the effect of p53 on the ability
of HMG1 to recognize damaged DNA.
Cells--
MCF-7 cells were grown in Dulbecco's modified
Eagle's medium (Nissui, Tokyo, Japan) supplemented with 10% fetal
calf serum.
Antibodies--
Antibodies to p53 (Do-1) and Sp1 (PEP 2) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to
p53 (Pab421) was purchased from Calbiochem (San Diego, CA). Anti-Thio
antibody was purchased from Invitrogen (San Diego, CA). Antibodies to
HMG1 and HMG2 were generated from synthetic peptides KGETKKKFKDPNAP (K
plus amino acids 83-95), and KSEAGKKGPGRPTG (K plus amino acids 168-180) respectively, as described previously (10). Antibodies to
HMG1 and HMG2 were purified using protein A/G-agarose (Qiagen, Hilden,
Germany) (11).
Plasmid Preparation--
A full-length cDNA for human p53
was amplified from total RNA from a human epidermoid cancer lines, KB
cells, by reverse transcription-polymerase chain reaction using the
primer pairs 5'-CCATGGAGGAGCCGCAGTCAGATCC-3' and
5'-GAAGTGGAGAATGTCAGTCTGAGTCAGGCCC-3'. The polymerase chain reaction
product was cloned into the pGEM-T Easy vector (Promega., Madison, WI).
The cDNA fragment then was gel purified after digestion with
NotI and cloned into the pThioHis vector (Invitrogen) for a
ThioHis fusion construct or the pGEX-4T vector (Amersham Pharmacia Biotech) for a GST fusion construct. GST-p53 deletion mutants were
prepared as follows. For construction of GST-p53 N124, GST-p53 plasmid
was digested with BsaAI, filled in, and self-ligated. For
GST-p53 160C, GST-p53 plasmid was digested with NcoI and
circularized by self-ligation. ThioHis-p53 deletion mutants were
constructed from a ThioHis-p53 plasmid by digestion with
AccI for N376, BanII for N362, and Eco81I for
224C. Plasmids for GST-HMG1 and GST-Y box-binding protein 1 were
described previously (10). GST-HMG1 deletion mutants were prepared as
follows. For construction of GST-HMG1 Pull-down Assay with 35S-Labeled Nuclear
Extract--
For metabolic labeling, MCF-7 cells in a 100-mm tissue
culture dish were cultured with in Dulbecco's methionine and cysteine free modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% dialyzed fetal calf serum and were labeled with 50 µCi/ml of
[35S]methionine and cysteine labeling mixture (Amersham
Pharmacia Biotech) (12) with or without 20 µM cisplatin
(Sigma) for 24 h. After washing the cells twice with ice-cold
phosphate-buffered saline, the nuclear fraction was obtained as
described previously (10) and then was eluted with buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol, after which the NaCl
concentration of the nuclear fraction was changed to 120 mM
by adding the same buffer without NaCl. GST fusion proteins were
induced by isopropyl-1-thio- Coimmunoprecipitation Assay--
MCF-7 cells growing in 100-mm
tissue culture dishes were treated with 20 µM cisplatin
(Sigma) for 24 h. The nuclear fraction was obtained as described
above. Tubes containing the nuclear fraction were incubated for 2 h at 4 °C with 20 µl of protein A/G-agarose. Then 10 µl of
preimmune rabbit IgG, anti-HMG1 antibody, or anti-p53 antibody were
added to each tube and mixed. The mixture was incubated further for
2 h at 4 °C. Immunoprecipitates were washed three times with
buffer containing 20 mM HEPES, pH 7.9, 120 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol
followed by centrifugation. The immunoprecipitates mixed with 20 µl
of sampling buffer were boiled for 5 min and subjected to
SDS-polyacrylamide gel electrophoresis. The samples were
transferred to polyvinylidene difluoride membranes, immunoblotted with
anti-HMG1 antibody or anti-p53 antibody, and developed for visualization by chemiluminescence according to the ECL protocol (Amersham Pharmacia Biotech).
Expression of GST and ThioHis Fusion Proteins and Pull-down
Assay--
GST fusion proteins and ThioHis fusion proteins were
induced by isopropyl-1-thio- EMSA--
Oligonucleotides of a 32-mer
(5'-TCGGGGCGGGGCGATCGATCGGGGCGGGGCGA-3') or a 63-mer
(5'-CGCCCCCACCCTGCGCGCGCGATCCAAGGTTCTCACCTGGAGCGCTCAGGTAGGGCTGGGGCG-3') were annealed with the complementary strands. Both
single-stranded and double-stranded oligonucleotides were end-labeled
with [ HMG1 Interacts with p53--
HMG1 has been shown to interact
directly with p53 to enhance p53 DNA binding in vitro (4).
We initially examined the spectrum of nuclear proteins bound to a GST
protein fused to either HMG1 or p53 using MCF-7 cells.
35S-Labeled nuclear extracts prepared from MCF-7 cells
treated with or without cisplatin were incubated with GST fusion
proteins immmobilized on glutathione-Sepharose. A comparison of the
profile of proteins bound to the GST fusion proteins is shown in Fig.
1A (left panel). A
53-kDa protein likely to correspond to p53 was observed by pull-down with GST-HMG1. On the other hand, a 28-kDa protein likely to correspond to HMG1 was observed by pull-down with GST-p53. No significant difference of protein profile was seen between MCF-7 cells treated with
cisplatin and those treated without cisplatin. To determine whether
these proteins respond to treatment with cisplatin, extracts from
nuclei and cytoplasm were prepared from logarithmically growing MCF-7
cells and analyzed by Western blotting in Fig. 1A
(right panel). When cells were treated with cisplatin, p53
was rapidly up-regulated. Both HMG1 and p53 were localized mainly in
nuclei. Sp1, a nuclear transcription factor, showed no change in amount after cisplatin treatment. Sp1 could not be detected in the cytosol, indicating that the cytosolic fraction was not contaminated with nuclear protein. Basal expression of wild type of p53 was relatively high in MCF-7 cells. When MCF-7 cells were treated with cisplatin for
24 h, nuclear levels of p53 increased 4-fold compared with untreated MCF-7 cells. Cellular amounts of HMG1 and p53 were
essentially similar at base lines, but relative in vivo
levels of p53 following cisplatin treatment significantly exceeded
levels of HMG1. To assess the intracellular association of p53 with
HMG1, coimmunoprecipitation was performed using either anti-HMG1- or
anti-p53 antibodies with MCF-7 cells treated with cisplatin.
Immunoblotting analysis showed that p53 interacted with HMG1 and the
interaction of HMG1 with p53 was reproducible (Fig. 1B). To
further confirm this association, we performed a pull-down assay using
immobilized GST fusion proteins and nuclear extracts. Immunoblotting of
the bound nuclear proteins demonstrated that p53 interacted with HMG1
but not with GST alone (Fig. 1C). Y box-binding protein 1 also has been found to interact with p53 (13) (Fig. 1C).
We mapped the domain of the HMG protein required for interaction with
p53 by a pull-down assay using GST fusion proteins containing deletion
mutants of HMG1 and ThioHis fusion proteins with p53 (Fig.
2A). Immobilized GST fusion
proteins were shown by staining. Fusion protein deleting the A box of
HMG1 was unable to bind p53, whereas fusion protein lacking the B box
of HMG1 showed intact binding (Fig. 2A). To exclude the
possibility that p53 and HMG1 might associate to form a ternary complex
via genomic DNA, we also tested the fusion proteins after treatment
with DNaseI, which completely destroys bacterial DNA under the
conditions (data not shown). As shown in Fig. 2B, an
association between HMG1 and p53 could be demonstrated when
DNaseI-treated fusion protein was assayed, indicating that HMG1
interacted with p53 directly and that the HMG A box of HMG1 was
sufficient for interaction with p53.
We next mapped the domain of p53 responsible for interaction with HMG1,
using GST fusion proteins to capture ThioHis-p53 or its deletion
derivatives (Fig. 3A). This
assay demonstrated that the C-terminal region of p53 participated in
the interaction with HMG1 (Fig. 3B). A region containing 14 amino acids (residues 363-376) in this region of p53 is critical for
the interaction.
p53 Enhances Cisplatin-modified DNA Binding by HMG1--
HMG1 can
stimulate sequence-specific DNA binding of p53 (4). We examined the
converse, the influence of p53 on DNA binding of HMG1, because HMG1 is
well known to bind preferentially to cisplatin-modified DNA (7). We
previously have shown that HMG1 cannot bind effectively to
cisplatin-modified oligonucleotides of a length limited to 20 base
pairs (10). We prepared oligonucleotides of two different lengths, a
32-mer and a 63-mer, and examined HMG1 binding. As shown in Fig.
4A, a single band with
retarded migration was detected when the 32-mer was modified with
cisplatin, whereas two retarded bands were observed with the
cisplatin-modified 63-mer. These results indicated that the 32-mer
length is sufficient for strong binding of HMG1, whereas with the
63-mer, the slowly migrating bands appear to represent attachment of
two HMG1 molecules. We therefore used the 32-mer for the following
experiments. First we analyzed the DNA-binding activity of GST fusion
protein using synthetic oligonucleotides with or without cisplatin
modification. As shown in Fig. 4B, HMG1 and HMG1 We demonstrated here that p53 directly interacts with HMG1 in cell
extracts and in intact cells to enhance the cisplatin-modified DNA
binding activity of HMG1. Both HMG1 and HMG2 are nonhistone chromosomal
proteins that are abundant and highly conserved in eukaryotic cells
(14). Although the function of these proteins is not fully understood,
they play an important role in chromatin structure and function
including DNA replication, DNA repair, transcription, and chromatin
assembly. The most distinctive feature of the HMG box is a basic domain
of about 70 amino acids that contains three Interestingly from that viewpoint, the HMG A box is involved in p53
binding, but the HMG B box is not. The lack of p53 binding of the HMG B
box may reflect a difference in protein structure between this region
and the HMG A box. Interaction between the HMG1 A box and p53 required
amino acids 363-376 of p53. This region of the highly basic C terminus
of p53 is particularly "sticky" and appears to associate with many
cellular proteins. The HMG A box also is highly basic, suggesting that
interaction is not of a simple electrostatic nature. This view of
interactions is supported by the finding that DNA binding by HMG1 also
is enhanced by addition of anti-HMG1 antibody (Fig. 5C).
Participation of the DNA-binding domain in the HMG1-p53 interaction
implies that the mode of DNA binding and the binding specificity of
resulting heterodimer might be altered.
Normal cells contain p53 protein in a latent form that can be activated
by DNA damage. Post-translational modifications of the C-terminal
domain of p53 have been shown to be important in p53-specific DNA
binding such as phosphorylation, antibody binding, and acetylation
(18-20). HMG1 has been shown to significantly stimulate the
sequence-specific DNA binding of p53 (4). Small peptides derived from
the negative regulatory domain can activate the latent sequence-specific DNA binding function of p53 (21). In the present study, we found that the C-terminal negative regulatory domain of p53
is involved in HMG protein binding (Fig. 3). Interaction of HMG1 with
p53 may be modulated by the post-translational modifications of p53.
HMG proteins have been further shown to enhance the sequence-specific
DNA binding of a wide variety of transcription factors such as
sex-hormone receptor (22, 23), HOX proteins (24), POU domain-containing
factors (25), and p53, functioning as a coactivator for transcription
of their target genes (4). However, no evidence has been reported to
indicate whether cisplatin-modified DNA binding activity of HMG
proteins is modulated by these cellular proteins. Therefore, we
examined whether damaged DNA binding of HMG1 was modulated by
interaction with p53. This is the first report to demonstrate that p53
enhances the cisplatin-modified DNA binding of HMG1. This enhancement
was observed when p53, which can bind to HMG1, was added in an EMSA
(Fig. 5). The mobility of HMG1 complexed with cisplatin-modified DNA
was not altered by p53, and p53-stimulated HMG1 DNA complex could not
be supershifted by the addition of the anti-p53 antibody, Pab421.
Binding to HMG1 may disturb the binding of p53 antibody because the
interacting region of p53 is near the Pab421 epitope. Another
possibility is that this interaction is unstable, and p53 may
dissociate from the HMG1-DNA complex during electrophoresis. However,
DNA-HMG1 complex can be enhanced and supershifted by the addition of
affinity-purified polyclonal antibody directed against HMG1, which
indicates that either antibody or p53 association may induce the
conformational change in the HMG1 A box that optimizes interaction with
cisplatin-modified DNA.
On the other hand, HMG1 can stimulate DNA binding by p53 Four classes of damage detector proteins have been identified that bind
preferentially to damaged sites: repair-related proteins, HMG
box-containing proteins, transcription factors, and linker histones
(7). Both the p53 protein and a damage detector protein possibly might
bind at sites of DNA damage and repair. In this context, determination
of whether other damage detector proteins can interact directly with
p53 is important. The p53 protein not only recognizes structurally
altered DNA such as single-stranded DNA (26) and mismatching DNA (27)
but also exhibits 3' to 5' exonuclease activity (3). Increased p53
protein after DNA damage might be associated with active participation
in a DNA repair process, because p53 can associate with a number of
proteins involved in excision repair. Thus, interaction of p53 with HMG proteins may have an important role in p53-dependent DNA
repair. Overexpression of HMG1 in response to the steroid hormones have been recently shown to sensitize MCF-7 cells to cisplatin and carboplatin (28). HMG1 often is up-regulated in cisplatin-resistant cell lines (data not shown). These observations indicate that HMG
proteins could be important in modulating the cellular toxicity of
cisplatin. Further study is needed to explore the role of HMG1 in
cisplatin resistance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A and GST-HMG1
B,
StuI-NotI and BamHI fragments of
GST-HMG1 were cloned into a pGEX-4T vector, respectively.
-D-galactopyranoside as
described previously (10). GST fusion proteins binding to 15 µl of
glutathione-Sepharose 4B in a 50% slurry were mixed with 0.25 mg of
35S-labeled nuclear extract from MCF-7 cells treated with
or without cisplatin. The mixtures were incubated for 2 h at
4 °C and washed three times with binding buffer. Pull-down samples
representing 1% of input were electrophoresed and autoradiography.
-D-galactopyranoside as
described previously (10). Cell extracts were treated with or without
75 units/ml DNaseI (Worthington, Lakewood, NJ) in pull-down
buffer containing 1 mM CaCl2 at 37 °C for
1 h. GST fusion proteins binding to 15 µl of
glutathione-Sepharose 4B in a 50% slurry were mixed with 0.5 mg of
MCF-7 nuclear extract treated with 20 µM cisplatin for 24 h or ThioHis fusion proteins in binding buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 120 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride. The mixtures were incubated for 2 h
at 4 °C and washed three times with binding buffer. Pull-down
samples representing 10% of input were electrophoresed and analyzed by
immunoblotting by anti-Thio antibody as described above.
-32P]ATP (Amersham Pharmacia Biotech), and
purified from the gel. Half of each labeled oligonucleotides was
treated with 0.3 mM cisplatin at 37 °C for 12 h and
then was purified by ethanol precipitation. Numbers of platinum atoms
bound to oligonucleotides (32-mer) were determined by atomic absorption
spectroscopy (polarized Zeeman atomic absorption spectrophotometer
Z-8200; Hitachi). The mean amount of platinum bound to DNA was about
6.7 platinum atoms/oligonucleotide under our conditions. GST fusion
proteins binding glutathione-Sepharose 4B were eluted with 50 mM Tris-HCl, pH 8.0, and 20 mM reduced glutathione according to the manufacturer's protocol (Amersham Pharmacia Biotech). GST fusion proteins were used directly for EMSA.
Reaction mixtures contained 4 µl of 5× EMSA buffer (25% glycerol,
50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 5 mM dithiothreitol), 2 µl of bovine serum albumin (1 mg/ml), 32P-labeled probe DNA (4 ng), GST fusion protein,
and antibodies as indicated, and water in a total volume of 20 µl.
Binding reactions were incubated for 5 min at room temperature.
Products were analyzed on 4% polyacrylamide gels in 0.5× Tris borate
EDTA buffer followed by BAS 2000 (10).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of HMG1 with p53 in
vitro and in vivo. A,
interaction of HMG1 or p53 with MCF-7 cell nuclear extract. GST,
GST-HMG1 or p53 on glutathione-Sepharose beads was incubated with
35S-labeled nuclear extract from MCF-7 cells treated with
or without cisplatin. Bound proteins were detected by
SDS-polyacrylamide gel electrophoresis and autoradiography. Molecular
mass markers are indicated as well as positions of proteins that likely
correspond to p53 (open arrowhead) and HMG1 (closed
arrowhead). HMG1 and p53 expression in vivo is shown.
Western blots of fractionated protein (150 µg/lane) extracted from
MCF-7 cells treated with 20 µM of cisplatin for 0, 12, or
24 h are shown. p53 was detected with the monoclonal antibody
Do-1. HMG1 and Sp1 were detected with polyclonal antibody.
B, coimmunoprecipitation assay. A nuclear extract prepared
from MCF-7 cells treated with cisplatin (20 µM) for
24 h was incubated with preimmune serum or antibodies to HMG1 or
p53. The immune complexes and 10% of input were electrophoresed and
analyzed by immunoblotting with antibody to p53 or HMG1. C,
pull-down assay using nuclear extract. A nuclear extract from
cisplatin-treated MCF-7 cells was incubated with immobilized GST fusion
proteins. Bound protein samples representing 10% of input were
electrophoresed and analyzed by immunoblotting with antibody to p53
(middle panel) or antibody to HMG1 (right panel).
Purified GST fusion proteins used in this assay were stained with
Coomassie Brilliant Blue (left panel). Asterisks
indicate full-length GST fusion protein.
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Fig. 2.
Direct interaction of HMG1 with p53 and
localization of the portion of the HMG1 molecule that binds to
p53. A, schematic representation of GST-HMG1 fusion
protein and pull-down assay using ThioHis-p53 fusion protein. The
upper panel provides a schematic illustration of the HMG1
protein and their deletion mutants used in the assay. Purified GST
fusion proteins were stained with Coomassie Brilliant Blue (right
panel). Asterisks indicate full-length GST fusion
protein. Immobilized fusion proteins were incubated with ThioHis-p53
expressed in bacteria, and the bound protein samples representing 10%
of input were electrophoresed and analyzed by immunoblotting with
anti-Thio antibody (bottom panel). B, effect of
DNaseI treatment on pull-down assay. GST fusion or ThioHis fusion
protein was treated with (+) or without ( ) DNaseI. Immobilized GST
fusion proteins were incubated with ThioHis-p53, and bound proteins
were analyzed by immunoblotting with anti-Thio antibody.
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Fig. 3.
Mapping of the HMG1 binding sites of
p53. A, schematic representation of the ThioHis-p53 and
its deletion mutants used in this assay. A schematic representation of
the functional domains of p53 also is shown (top panel).
B, pull-down assay using ThioHis-p53 and its deletion
mutants. Full-length and truncated forms of ThioHis-p53 were expressed
in bacteria and used for pull-down experiments with GST or GST-HMG1.
Bound protein samples representing 10% of input were electrophoresed
and analyzed by immunoblotting with anti-Thio antibody. ThioHis tag was
used alone as a negative control.
B can
significantly bind to double-stranded oligonucleotides modified with
cisplatin but not to single-stranded oligonucleotides. On the other
hand, p53 alone could not bind to these probes. We next examined the
effect of p53 on cisplatin-modified DNA binding of HMG1 by EMSA (Fig.
5). Cisplatin-modified DNA binding by
HMG1 was significantly enhanced by p53 in a dose-dependent
manner (Fig. 5A). To avoid HMG1 aggregation in the EMSA, we
diluted the HMG1 protein and examined the effect of p53 on DNA binding
activity. Mobility of the HMG1-DNA complex was not affected by the
concentration of HMG1. Under our conditions, addition of p53 to the
HMG1-DNA binding reaction resulted in 5-10-fold activation of the DNA
binding activity of HMG1. Mutant p53, which cannot interact with HMG1,
was unable to stimulate DNA binding by HMG1 or GST alone (Fig.
5B). However, p53 did not alter the electrophoretic mobility
of the HMG1 complex formed with cisplatin-modified DNA, and p53-induced
HMG1-DNA complex could not be supershifted by the anti-p53 antibody
Pab421 (data not shown). Further, we found that HMG1-specific antibody
also promoted formation of the cisplatin-modified DNA HMG1 complex but
HMG2- or p53-specific antibody did not (Fig. 5C, left
panel). Addition of anti-HMG1 antibody did not stimulate formation
of DNA-protein complex when HMG1
B was used, because the HMG1
antibody epitope is not present in HMG1
B. Addition of anti-HMG1
antibody to the binding reaction resulted in a further shift in
mobility (Fig. 5C, right panel).
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Fig. 4.
EMSA using GST fusion proteins.
A, the effect of the length of oligonucleotides on GST-HMG1
binding. Both double-stranded 32-mer (left panel) and 63-mer
(right panel) are platinated and used as probes.
Arrowheads indicate HMG1-DNA complexes. B, the
DNA-binding activity of purified GST fusion proteins to four different
types of oligonucleotides. GST-p53 fusion protein and its deletion
mutants were electrophoresed and stained with Coomassie Brilliant Blue
(upper panel). Asterisks indicate
full-length GST fusion protein. Binding of purified GST
fusion proteins (100 ng) to either 32P-labeled
single-stranded or double-stranded oligonucleotides (32-mer) with or
without cisplatin treatment were analyzed by EMSA (lower
panel).
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Fig. 5.
Stimulation of HMG-1 binding to
cisplatin-modified DNA by p53 or anti-HMG1 antibody. A,
effect of p53 protein on cisplatin-modified DNA binding by HMG1
protein. Various amounts of purified GST-HMG1 bound to
32P-labeled cisplatin-modified oligonucleotides in the
presence of purified GST-p53 were analyzed by EMSA.
Arrowhead indicates DNA-protein complex. B,
effect of deletion of p53 protein on cisplatin-modified DNA binding by
HMG1 protein. 10 ng of purified GST-HMG1 bound to
32P-labeled cisplatin-modified oligonucleotides in the
presence of either purified GST-p53, 160C, or N124 were analyzed by
EMSA. GST was used alone as a negative control. Arrowhead
indicates DNA-protein complex. C, effect of anti-HMG1
antibody on cisplatin-modified DNA binding by HMG1 protein. 10 ng of
purified GST, GST-HMG1, or GST-HMG1 B bound to
32P-labeled cisplatin-modified oligonucleotides in the
presence of 100 ng of antibodies were analyzed by EMSA (lane
1, control rabbit IgG; lane 2, anti-HMG1 antibody;
lane 3, anti-HMG2 antibody; lane 4, anti-p53 antibody). The
dose-dependent effect of antibody on DNA binding of HMG1 is
shown in the right panel (lane 1, 0 ng of
antibody; lane 2, 20 ng of antibody; lane 3, 60 ng of antibody; lane 4, 180 ng of antibody; and lane
5, 540 ng of antibody). Arrowhead indicates DNA-protein
complex. Arrow indicates supershifted complex.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical regions
including well conserved hydrophobic amino acids. The HMG A box and the
HMG B box are about 30% identical in amino acid sequence. A
phenylalanine residue at position 37 is critical for binding to
cisplatin-modified oligonucleotides (15). However, no phenylalanine
residue is present in the HMG B box, so the two linked HMG boxes are
not functionally equivalent in DNA binding. Under our conditions, only
the HMG A box can bind to cisplatin-modified DNA, whereas the HMG B box
cannot. Although the conditions under which EMSA is performed can
greatly influence the results obtained, our result is consistent with
the previous report that the HMG1 A box has a higher affinity than the
HMG1 B box for cisplatin-modified DNA (16). Further, the HMG A box has
been proposed to be involved mainly in structure-specific DNA binding,
whereas the B box may be a target for protein-protein interactions
(17).
30, which
lacks the 30 C-terminal amino acids (4). This region is necessary for
interaction with HMG1, indicating that DNA binding of p53 is
independent of direct interaction between p53 and HMG1. Structural
change of target DNA by HMG1 has been suggested to activate and
stabilize p53 DNA binding, given that HMG1 is a well characterized
DNA-binding protein (26).
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FOOTNOTES |
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* This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan, by Grant 99-23106 from the Princess Takamatsu Cancer Research Fund, by the Fukuoka Anticancer Research Fund, and by the Ground Research Announcement for Space Utilization Fund.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 Molecular Biology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan. Tel.: 81-93-691-7423; Fax: 81-93-692-2766; E-mail: k-kohno@med. uoeh-u.ac.jp.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008143200
2 K. Kohno, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HMG, high mobility group; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay.
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1. | Jamieson, E. R., and Lippard, S. J. (1999) Chem. Rev. 99, 2467-2498[CrossRef][Medline] [Order article via Infotrieve] |
2. | Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve] |
3. | Mummenbrauer, T., Janus, F., Müller, B., Wiesmüller, L., Deppert, W., and Grosse, F. (1996) Cell 85, 1089-1099[Medline] [Order article via Infotrieve] |
4. |
Jayaraman, L.,
Moorthy, N. C.,
Murthy, K. G. K.,
Manley, J. L.,
Bustin, M.,
and Prives, C.
(1998)
Genes Dev.
12,
462-472 |
5. | Brown, R., Clugston, C., Burns, P., Edlin, A., Vasey, P., Vojtesek, B., and Kaye, S. B. (1993) Int. J. Cancer 55, 678-684[Medline] [Order article via Infotrieve] |
6. | Landsman, D., and Bustin, M. (1993) Bioassays 15, 539-546[Medline] [Order article via Infotrieve] |
7. | Pil, P. M., and Lippard, S. J. (1992) Science 256, 234-237[Medline] [Order article via Infotrieve] |
8. | Brown, S. J., Kellett, P. J., and Lippard, S. J. (1993) Science 261, 603-605[Medline] [Order article via Infotrieve] |
9. | Chao, J. C., Wan, X. S., Engelsberg, B. N., Rothblum, L. I., and Billings, P. C. (1996) Biochim. Biophys. Acta 1307, 213-219[Medline] [Order article via Infotrieve] |
10. |
Ise, T.,
Nagatani, G.,
Imamura, T.,
Kato, K.,
Takano, H.,
Nomoto, M.,
Izumi, H.,
Ohmori, H.,
Okamoto, T.,
Ohga, T.,
Uchiumi, T.,
Kuwano, M.,
and Kohno, K.
(1999)
Cancer Res.
59,
342-346 |
11. | Harlow, E., and Lane, D. (1999) Using Antibodies: A Laboratory Manual , pp. 74-75, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
12. | Sefton, M. B., Beemon, K., and Hunter, T. (1978) J. Virol. 28, 957-971[Medline] [Order article via Infotrieve] |
13. | Okamoto, T., Izumi, H., Imamura, T., Takano, H., Ise, T., Uchiumi, T., Kuwano, M., and Kohno, K. (2000) Oncogene 19, 6194-6202[CrossRef][Medline] [Order article via Infotrieve] |
14. | Grosschedl, R., Giese, K., and Pagel, J. (1994) Trends Genet. 10, 94-100[CrossRef][Medline] [Order article via Infotrieve] |
15. | Ohndorf, U. M., Rould, M. A., He, Q., Pabo, C. O., and Lippard, S. J. (1999) Nature 399, 708-712[CrossRef][Medline] [Order article via Infotrieve] |
16. | Dunham, S. U., and Lippard, S. J. (1997) Biochemistry 36, 11428-11436[CrossRef][Medline] [Order article via Infotrieve] |
17. | Webb, M., and Thomas, J. O. (1999) J. Mol. Biol. 294, 373-387[CrossRef][Medline] [Order article via Infotrieve] |
18. | Hupp, T. R., Meek, D. W., Midgley, C. A., and Lane, D. P. (1992) Cell 71, 875-876[Medline] [Order article via Infotrieve] |
19. |
Takenaka, I.,
Morin, F.,
Seizinger, B. R.,
and Kley, N.
(1995)
J. Biol. Chem.
270,
5405-5411 |
20. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
21. | Hupp, T. R., Sparks, A., and Lane, D. P. (1995) Cell 83, 237-245[Medline] [Order article via Infotrieve] |
22. |
Verrier, C. S.,
Roodi, N.,
Yee, C. J.,
Bailey, L. R.,
Jensen, R. A.,
Bustin, M.,
and Parl, F. F.
(1997)
Mol. Endocrinol.
11,
1009-1019 |
23. | Onate, S. A., Prendergast, P., Wagner, J. P., Nissen, M., Reeves, R., Pettijohn, D. E., and Edwards, D. P. (1994) Mol. Cell. Biol. 14, 3376-3391[Abstract] |
24. | Zappavigna, V., Falciola, L., Citterich, M. H., Mavilio, F., and Bianchi, M. E. (1996) EMBO J. 15, 4981-4991[Abstract] |
25. | Zwilling, S., König, H., and Wirth, T. (1995) EMBO J. 14, 1198-1208[Abstract] |
26. |
Zlatanova, J.,
and van Holde, K.
(1998)
FASEB. J.
12,
421-431 |
27. |
Selivanova, G.,
Iotsova, V.,
Kiseleva, E.,
Ström, M.,
Bakalkin, G.,
Grafström, R. C.,
and Wiman, K. G.
(1996)
Nucleic Acids Res.
24,
3560-3567 |
28. |
He, Q.,
Liang, C. H.,
and Lippard, S. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5768-5772 |