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J Biol Chem, Vol. 273, Issue 45, 29586-29591, November 6, 1998
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From the Chugai Research Institute for Molecular
Medicine, 153-2 Nagai, Niihari, Ibaraki 300-41, Japan, the
¶ National Institute of Bioscience and Human Technology, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, and the Children's
Medical Research Institute, 214 Hawkesbury Road, Westmead Sydney, New
South Wales 2145, Australia
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The mortalin genes, mot-1 and
mot-2, are hsp70 family members that were originally cloned
from normal and immortal murine cells, respectively. Their proteins
differ by only two amino acid residues but exhibit different
subcellular localizations, arise from two distinct genes, and have
contrasting biological activities. We report here that the two proteins
also differ in their interactions with the tumor suppressor protein
p53. The pancytosolic mot-1 protein in normal cells did not show
colocalization with p53; in contrast, nonpancytosolic mot-2 and p53
overlapped significantly in immortal cells. Transfection of
mot-2 but not mot-1 resulted in the repression
of p53-mediated transactivation in p53-responsive reporter assays.
Inactivation of p53 by mot-2 was supported by the down-regulation of
p53-responsive genes p21WAF-1 and
mdm-2 in mot-2-transfected cells only.
Furthermore, NIH 3T3 cells transfected with expression plasmid encoding
green fluorescent protein-tagged
mot-2 but not mot-1 showed an abrogation of
nuclear translocation of wild-type p53. These results demonstrate a
novel mechanism of p53 inactivation by mot-2 protein.
Evidence has been accumulating that inactivation of p53, a tumor
suppressor and cellular transcription factor (1), is involved in
cellular transformation and immortalization (2-5). Extensive analyses
of p53 have defined at least four functional domains, including an
amino terminus transactivation domain (amino acids 1-44), a
sequence-specific DNA-binding domain (amino acids 100-300), a carboxyl
terminus oligomerization domain, and a regulatory domain (amino acids
319-393; Ref. 6), and shown that the conformation of p53 and its
interactions with other proteins have key roles in its various cellular
activities (7, 8). Several cellular proteins, including some of the
hsp70 family members, have been shown to interact with p53 (9-12).
Although mutational or mdm-2-mediated inactivation of p53 is a common
event involved in cellular transformation (1), p53 is inactivated in a
considerable number of tumors and transformed cells by an unknown mechanism(s).
We initially cloned mortalins mot-1 and mot-2,
which code for pancytosolically and perinuclearly distributed members
of the hsp70 family of proteins, from normal and immortal murine cells, respectively (13, 14). The open reading frames of the two types of
murine mortalins differ in two nucleotides, encode proteins differing
in two amino acids, arise from distinct genes, and have contrasting
biological activities (13-16). RNA in situ hybridization and immunohistochemical studies on mortalin in normal murine tissues showed a higher level of expression in nondividing cell populations than in dividing cells. However, tumor tissues were seen to have a high
intensity of mortalin staining by an antibody that reacts with both the
mot-1 and mot-2 proteins (17, 18). Mortalin was also identified as
PBP-74, mtHSP70, and Grp75 and has been assigned roles in antigen
processing, in vivo nephrotoxicity, and radioresistance in
independent studies from other groups (19, 20).
In the present study, we demonstrate functional interactions of mot-2
protein with wild-type p53. Colocalization of wild-type p53 and
mortalin protein was observed in transformed (nonpancytosolic mortalin)
but not in normal (pancytosolic mortalin) cell types. The
transcriptional activation function of p53 was impaired by transfections of mot-2 but not mot-1.
Consistently, transfections of mot-2, but not those of
mot-1, resulted in the down-regulation of p53-responsive
genes p21WAF-1 and mdm-2.
Furthermore, G1-associated nuclear translocation of p53 was
abrogated by mot-2 but not by mot-1. These results demonstrated that
mot-2 protein inactivates wild-type p53 function.
Cell Culture--
Cells were cultured as described previously
(Refs. 13, 14, and 21; American Type Culture Collection catalogue).
Immunofluorescence and Fluorescence Digital Imaging
Microscopy--
Cells were double-stained with monoclonal anti-p53
(PAb421; Calbiochem) and polyclonal anti-mortalin antibodies (13) and visualized by secondary staining with fluorescein
isothiocyanate-conjugated sheep anti-mouse IgG and Texas Red-conjugated
donkey anti-rabbit IgG (Amersham Corp.). Three-dimensional images with
enhanced fluorescence were obtained using laser digital imaging
microscopy with a ×40 Plan-NEOFLUAR objective on a Zeiss Axiophot
microscope (Carl Zeiss, Germany) equipped with a CELLscan system
(Scanalytics, Billerica, MA; Ref. 22). The extent to which the two
proteins were similarly distributed was assessed by combining the two
images using computer graphics software. The individual mortalin and
p53 images were seen as red and green fluorescence, respectively, and
the colocalized proteins appeared yellow under this program.
Transfections--
Transient and stable transfections were
performed using LipofectAMINETM (Life Technologies, Inc.). Typically, 3 µg of plasmid DNA were used per 6-cm dish. All assays were performed
after 48 h of transfections. Stable clones of NIH 3T3 cells with
temperature-sensitive p53 expression were isolated by cotransfections
of pMSVp53Val135 (a kind gift from Dr. Paul Jackson; Ref. 23) and a
pSR In Vivo Coimmunoprecipitation--
Nonidet P-40 lysates (500 µg) from NIH 3T3 and NIH 3T3/p53.4 cells were precleared by
incubating with 40 µl of protein A-agarose for 2 h. The
supernatant was incubated with slow agitation overnight at 4 °C with
2-5 µg of control (isotype-matched IgG) or anti-p53 antibody
(PAb421) that was cross-linked to protein A-agarose beads with
dimethylpimelimidate as described previously (24). Immunocomplexes were
pelleted by centrifugation, washed with Nonidet P-40 lysis buffer,
separated by SDS-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose membrane by semidry transfer. The membrane was probed
with an anti-mortalin antibody raised against a peptide (amino acids
469-487; Ref. 13).
Reporter Assays--
Cells grown in 12-well plates were
transfected with 0.5 µg of p53-responsive reporter plasmid, pWWP-luc
or PG13-luc (kindly provided by Dr. Bert Vogelstein; Ref. 25), and 0.5 µg of empty vector (pSR Western Blot Analysis--
The protein sample (20-40 µg)
separated on a SDS-polyacrylamide gel was electroblotted onto
nitrocellulose membrane using a semidry transfer blotter. Immunoassays
were performed with anti-mortalin, anti-p53 (PAb421), anti-mdm-2
(SMP14; Santa Cruz Biotechnology), anti-p21WAF-1 (C-19;
Santa Cruz Biotechnology),
anti-GFP1
(CLONTECH), and anti-actin (Boehringer Mannheim) antibodies.
Double Immunolocalization of p53 and Mortalin--
Normal and
immortal murine fibroblasts were double-stained for mortalin and p53.
Whereas cytoplasmic staining of p53 was barely visible, nuclear p53
could be detected easily with an epifluorescence microscope (Fig.
1A). Next, the
immunofluorescence was observed under a high-resolution
three-dimensional digital laser microscope (Fig. 1B).
Negative controls, including the staining of p53 In Vivo Interactions of Mot-2 and Wild-Type p53--
To determine
whether the colocalized proteins actually interact, p53 immunocomplexes
from NIH 3T3 cells (which contain mot-2 protein) were Western blotted
with anti-mortalin antibody. A faint band of mortalin was detected in
these immunocomplexes. Control precipitations with isotype-matched IgG
did not bring down mortalin protein. Quantitation of the input and
immunoprecipitated mortalin signals revealed that approximately 0.7%
of the total mortalin present in the lysate was coprecipitated with
p53. This was consistent with the low amounts of p53 (undetectable by
Western blotting) present in these cells. To substantiate these
findings, we stably transfected NIH 3T3 cells with a
temperature-sensitive p53 expression plasmid, pMSVp53Val135. A clone
(NIH 3T3/p53.4) isolated from these transfections expressed wild-type
p53 at 32.5 °C and mutant p53 at 37 °C, as demonstrated by the
accumulation of the latter on Western blotting with the p53-specific
antibody PAb421 (Fig. 2A).
Furthermore, p53-responsive reporter assays were performed on NIH
3T3/p53.4 cells maintained at 32.5 °C and 37 °C (Fig.
2B). PG13-luc containing a synthetic wild-type
p53-responsive promoter that contains 13 repeats of wild-type p53
binding consensus sequence was transfected into these cells. A
3-4-fold higher activity was observed in NIH 3T3/p53.4 cells as
compared with untransfected NIH 3T3 controls when the cells were
maintained at 32.5 °C after the transfections. In contrast, the
cells maintained at 37 °C showed p53 activity comparable to that of
the control. This confirmed the wild-type and mutant conformations of
exogenous p53 in the NIH 3T3/p53.4 clone at 32.5 °C and 37 °C,
respectively. The clone was next used for p53 immunoprecipitation.
Western blotting of the p53 immunocomplexes from NIH 3T3/p53.4 cells
(grown at 32.5 °C) with anti-mortalin antibody revealed the presence
of mortalin (mot-2) at a level that was much higher than that in NIH
3T3 cells (Fig. 2C). Quantitation of the input and
immunoprecipitated mot-2 signals revealed that approximately 3.2% of
the total mortalin present in the NIH 3T3/p53.4 lysate was
immunoprecipitated. These data demonstrated a nearly 4.5-fold
enrichment of the coimmunoprecipitated mot-2 protein in cells
overexpressing p53 and therefore suggest that mot-2 and p53 bind
in vivo.
Functional Inactivation of p53 by Mot-2--
We next examined
whether mot-2 can functionally inactivate p53 by carrying out
p53-responsive reporter assays. NIH 3T3, COS7, and embryonic
fibroblasts from a p53-null (p53
Inactivation of p53 by mot-2 but not mot-1 was supported by the
analysis of p53-responsive genes in cells that were transiently transfected with expression plasmids encoding mot-1 or -mot-2 proteins
tagged with GFP. The expression of exogenous mortalins was first
analyzed by Western blotting with anti-GFP antibody (data not shown)
and anti-mortalin antibody (Fig. 3C). The cell lysates were
subsequently analyzed for p53 and p53-responsive genes mdm-2
and p21WAF-1 by Western blotting with specific
antibodies. Cells transfected with mot-2 exhibited a lower level of
steady-state expression of p53, mdm-2, and p21WAF-1 as
compared with those transfected with the empty vector or mot-1 (Fig.
3C). These data were consistent with the inactivation of p53
by mot-2 but not mot-1 seen in the abovementioned reporter assays. The
data also suggest that mot-2 acts, at least in part, by reducing the
p53 steady-state levels.
Abrogation of Nuclear Translocation of p53 by Mot-2--
Wild-type
p53 has been reported to translocate to the nucleus at the
G1 stage of the cell cycle. It exhibits bright
immunofluorescence in the nucleus as compared with the cytoplasm, where
it is barely detected by epifluorescence microscopy (Fig.
1A). Coimmunolocalization of mot-2 and p53 in transformed
cells prompted us to investigate whether the mot-2-mediated
inactivation of p53 was accompanied by its retention in cytoplasm.
Expression plasmids encoding fusion proteins containing GFP and either
mot-1 or mot-2 were transiently transfected into NIH 3T3 cells grown on
coverslips. Expression of the fusion proteins was confirmed by Western
analysis with anti-GFP and anti-mortalin antibodies. The transfected
cells were serum-starved for 48 h and stained for p53. Cytoplasmic
p53 (detected as a very faint staining with Texas Red-conjugated
secondary antibody) translocated to the nucleus (brighter staining)
upon serum starvation in untransfected background cells. Of 155 cells
that exhibited green fluorescence for mot-2-GFP, 139 (approximately
90%) were seen to have no nuclear staining of p53. On the contrary, of
89 mot-1-GFP-transfected cells, 78 (87.6%) were seen to have nuclear p53 similar to that of the untransfected cells in the same cultures. Thus, the transfected cells that expressed mot-2 (green fluorescence) were significantly devoid of nuclear p53 (red fluorescence; Fig. 4), demonstrating the abrogation of its
translocation. Parallel experiments with mot-1-GFP did not reveal an
equivalent effect in three independent experiments in which Double immunolocalization studies with laser digital microscopy
showed the colocalization of p53 and mot-2 in NIH 3T3 cells that have
wild-type p53 (26-29).2 The
faint staining of p53 observed by epifluorescence microscopy (Fig.
1A) and the undetectable amounts of p53 in these cells on Western blots (Fig. 2A) were consistent with a low-level
expression of the wild-type protein. A perinuclear staining pattern for
p53 has also been reported in immortalized but untransformed Balb/3T3 cells (30). The colocalization of mot-2 and p53 suggested that these
proteins might interact, and this was confirmed by in vivo coimmunoprecipitations of the two proteins from NIH 3T3 and its transfected derivative that overexpressed wild-type p53.
COS7 cells showed detectable amounts of p53 by Western blotting, and
p53 immunocomplexes from these cells were seen to have mortalin by
Western blot analysis (data not shown). This supported the in
vivo interactions of the two proteins. p53-responsive reporter assays in COS7 cells revealed that p53 is transcriptionally active in
these cells. Several other studies have detected the presence of p53
that is unbound to SV40 T antigen in SV40-transformed cells (31-34).
Furthermore, similar to NIH 3T3 cells, transfections of mot-2 but not
mot-1 were seen to repress p53 activity in COS7 cells. Reporter assays
performed on p53 Immunolocalization studies on serum-deprived NIH 3T3 cells transfected
with GFP-tagged mot-1 and mot-2 proteins showed abrogation of its
nuclear translocation by mot-2. The differential effects of mot-1 and
mot-2 in this respect were consistent with the results obtained in the
above-described p53 transactivation and p53-responsive gene analysis.
Such functional impairment of wild-type p53 may abrogate some or all of
its normal tumor suppressor activity and therefore may contribute, at
least in part, to the malignant transformation of mot-2-overexpressing
NIH 3T3 cells (16). An elevated level of mortalin has been observed in
rat and human brain tumors (17, 18), suggesting that up-regulation of
mot-2 may be an event that contributes to tumor growth and/or
progression. In addition, a reduced amount of steady-state p53 was
detected in NIH 3T3 (data not shown) and COS7 (Fig. 3C)
cells that were transfected with the mot-2 expression plasmid. The data
suggested that mot-2 may cause either p53 degradation or
transcriptional repression. Direct interactions of p53 and mot-2 (Fig.
2C), along with the predicted chaperonin function of the
latter, suggested that it is more likely to be the result of
mot-2-mediated p53 degradation.
Functional inactivation of wild-type p53 by abnormal sequestration in
the cytoplasm has been reported in a subset of neuroblastomas (35). The
mechanism of this sequestration has not been elucidated. Our
immunofluorescence results (as described above) demonstrated that
mortalin was colocalized with p53 in the cytoplasm of neuroblastoma (SY-5Y and YKG-1), glioblastoma (A172), teratocarcinoma (NT-2), cervical carcinoma (HeLa), bladder carcinoma (A2182), and osteosarcoma (U2OS) cells, all of which have wild-type p53, and suggested that mot-2
may be involved in the inactivation of wild-type p53 function in these
cells. Ostermeyer et al. (36) have reported that the carboxyl terminus of p53 is involved in cytoplasmic aggregates in
neuroblastomas. Interestingly, in in vitro pull down assays of mot-2 and in vitro-translated p53, a fragment of p53
protein that lacks the carboxyl terminus was not coimmunoprecipitated with mortalin in in vitro binding
assays,3 suggesting that the
carboxyl terminus of p53 may be involved in its interactions with
mortalin. These observations are particularly interesting in the
context of the presence of three nuclear localization signals in the
carboxyl terminus of p53 (37) and may provide an explanation, at least
in part, to the mot-2-mediated abrogation of the nuclear localization
of p53.
In summary, mot-2 was shown to colocalize with wild-type p53 in
vivo and to inhibit p53-mediated exogenous and endogenous transactivation. The mot-2-mediated reduction in the steady-state level
and the abrogation of nuclear translocation of p53 suggested a novel
mechanism of p53 inactivation that may contribute to tumorigenesis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
mammalian expression vector containing the hybrid SV40-human
immunodeficiency virus promoter/enhancer and the
neoR gene and analyzed for p53 expression by
Western blotting with anti-p53 antibody (PAb421).
) or mot-1 (pSR/mot-1) or mot-2
(pSR/mot-2) expression plasmids. pMSVp53Val135 was cotransfected
into p53
/ cells. Cotransfections of pRL-CMV were performed as an
internal control to determine the efficiency of transfections. 48 h after transfection, luciferase assays (Dual-LuciferaseTM
Reporter Assay System; Promega) were performed. Luciferase values were
calculated per microgram of the protein as determined by Bradford protein assay.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/ fibroblasts and
the staining of NIH 3T3 cells with secondary antibodies alone (data not
shown), confirmed the specificity of the p53 immunostaining. Fluorescence digital imaging analysis revealed the colocalization of
p53 and mortalin in the perinuclear region of NIH 3T3 cells that
express mot-2; however, no colocalization was observed in normal mouse
fibroblasts (CMEF cells expressing pancytosolically distributed mot-1
protein; Fig. 1B). Normal and transformed human cells
containing wild-type p53 were also examined for p53 and mortalin
immunolocalization. Nonpancytosolic mortalin staining was apparent in
all of the immortalized cell lines examined. Notably, coimmunolocalization of the two proteins was observed in HeLa, U2OS,
and A172 cells (Fig. 1B) and also in NT-2, SY-5Y, and YKG-1 cells (data not shown). In contrast, normal human fibroblasts TIG-3
(Fig. 1C) and MRC-5 (data not shown), which express
wild-type p53 and pancytosolic mortalin, did not show any
colocalization.
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Fig. 1.
Immunolocalization studies for p53 and
mortalin. A, p53-related immunofluorescence in an
unsynchronized culture of NIH 3T3 cells is shown as observed by
epifluorescence microscopy. Cytoplasmic p53 (arrowheads) and
nuclear p53 were observed as faint and bright staining, respectively.
B, double immunolocalization of p53 (a and
d) and mortalin (b and e) in murine
immortal (NIH 3T3) and normal (CMEF) fibroblasts.
Colocalization of p53 and mortalin is visible as yellow
staining in NIH 3T3 (c) but not in CMEF cells
(f). C, coimmunolocalization of p53 and mortalin
(yellow staining) was seen in human transformed
(A172, glioblastoma; U2OS, osteosarcoma;
HeLa, cervical carcinoma) cells but not in normal
fibroblasts (TIG-3).
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Fig. 2.
Coimmunoprecipitation of p53 and mot-2.
A, exogenous expression of temperature-sensitive p53 in NIH
3T3 cells. A Western blot of NIH 3T3 and NIH 3T3/p53.4 cells (grown at
32.5 °C and 37 °C) with p53-specific antibody PAb421 and
anti-actin antibody is shown. NIH 3T3/p53.4 cells grown at 37 °C
showed the expected accumulation of mutant p53 at 37 °C.
B, p53-responsive reporter assays in NIH 3T3 cells and its
derivative cell line, NIH 3T3/p53.4. Cells were transfected with
PG13-luc and pRL-CMV (containing Renilla luciferase reporter, an
internal control for transfection efficiency) and maintained at
32.5 °C or 37 °C. A 3-4-fold higher reporter activity was
detected when cells were maintained at 32.5 °C. Bars and
error bars represent the mean ± S.D.
(n = 3). Similar results were obtained in four
experiments. C, coimmunoprecipitation of mot-2 with p53 in
NIH 3T3 and NIH 3T3/p53.4 cell extracts grown at 32.5 °C. p53
immunocomplexes from the indicated cells were Western blotted with
anti-mortalin antibody. Quantitation of the input (10% of the amount
used for immunoprecipitation is seen) and the immunoprecipitated
mortalin was performed by using Micro Computer Imaging Device MCID-M2
(FUJIX). 0.7 and 3.2% of total mot-2 were seen to coimmunoprecipitate
with p53 in NIH 3T3 and NIH 3T3/p53.4 cells, respectively.
/) mouse were used for p53-mediated
reporter assays with PG13-luc plasmid. Both NIH 3T3 and COS7 cells
showed the presence of transcriptionally active p53 (Fig.
3A). Cotransfections of the
expression plasmid (pSR/mot-2) encoding mot-2 resulted in the
decline of p53-responsive reporter activity. In contrast, mot-1
cotransfections did not show any effect. Next, the p53/ cells were
transfected with a temperature-sensitive p53 expression plasmid,
pMSVp53Val135, together with a wild-type p53-responsive reporter
plasmid, either pWWP-luc containing the native p53-responsive promoter
or PG13-luc. As expected, the cells maintained at 37 °C showed no
transactivation (data not shown) of the reporter plasmids.
Cotransfections of the p53 and mot-2 expression plasmids resulted in a
significant decline of pWWP-luc (Fig. 3B) and PG13-luc (data
not shown) activities at 32.5 °C. Furthermore, a
dose-dependent effect of mot-2 plasmid concentration on
reporter repression was observed. Cotransfections of the mot-1
expression plasmid, pSR/mot-1, did not affect p53-mediated transactivation (Fig. 3B).
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Fig. 3.
Transcriptional repression of p53 by mot-2
protein. A, p53-responsive reporter assays in NIH 3T3 and
COS7 cells. Cells were cotransfected with PG13-luc and mortalin
expression plasmid pSR /mot-1 or pSR/mot-2. Transfection
efficiency was monitored by cotransfections of pRL-CMV containing
Renilla luciferase reporter (Promega). Transcriptionally active p53 was
detected in NIH 3T3 and COS7 cells; p53/ cells were the negative
control. Cotransfection of mot-2 but not mot-1 resulted in the
repression of p53-mediated transactivation in NIH 3T3 and COS7 cells.
Bars and error bars represent the mean ± S.D. (n = 3). B, cotransfection of mot-2 but
not mot-1 resulted in the repression of p53-mediated transactivations
in p53/ mouse embryonic fibroblasts (MEF). A dose-dependent effect on
p53 repression was observed by transfections of increasing amounts of
mot-2 (0.5 µg/µl). Transfection efficiency was monitored by pRL-CMV
cotransfections. Bars and error bars represent
the mean ± S.D. (n = 3). C, Western
blotting for mortalin, p53, mdm-2, p21WAF-1, and actin in
empty vector-, GFP-mot-1-, and -mot-2-transfected COS7 cells. Exogenous
GFP-tagged (
110-kDa) and endogenous (70-kDa) mortalins, p53
(53 kDa), mdm-2 (90 kDa), p21WAF-1 (21 kDa), and
-actin (43 kDa) were detected with the respective
antibodies.
100-150
transfected cells were examined. The data were consistent with the
abovementioned analysis on p53-responsive exogenous reporter assays and
endogenous gene expression in mot-2- and mot-1-transfected cells and
suggested that mot-2 interacts with wild-type p53 and causes its
inactivation both by a reduction in its steady-state level and by
nuclear exclusion.
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Fig. 4.
Abrogation of nuclear translocation of p53 by
mot-2. mot-GFP-transfected cells (visible as green
fluorescence) were stained for p53 (red) after serum
starvation. The mot-2-transfected cells were devoid of nuclear staining
for p53. Untransfected and mot-1-transfected cells showed the
translocation of p53 (red) to the nucleus.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/ mouse embryonic fibroblasts revealed repression
of exogenous p53-mediated reporter activity by cotransfections of
mot-2. In parallel experiments with mot-1, no effect on p53 reporter
activity was observed. Analysis on p53-responsive genes
p21WAF-1 and mdm-2 exhibited
down-regulation in mot-2-transfected cells, but not in
mot-1-transfected cells. These data demonstrated the inactivation of
wild-type p53 by mot-2. Consistent with the double immunolocalization
data and the p53-responsive reporter assays, mot-1 did not show any
effect on p53-responsive endogenous gene expression.
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ACKNOWLEDGEMENTS |
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We thank Bert Vogelstein and Paul Jackson for the kind gifts of pWWP-luc and PG13-luc and pMSVp53Val135 plasmids, respectively.
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FOOTNOTES |
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* This work was supported in part by the Center of Excellence Grant, Japan (to S. C. K. and Y. M.) and the Carcinogenesis Fellowship of the New South Wales Cancer Council (to R. R. R.).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: Chugai Research Institute for Molecular Medicine, 153-2 Nagai, Niihari, Ibaraki 300-41, Japan. Tel.: 81-298-30-6211; Fax: 81-298-30-6270; E-mail: renu{at}tk.chugai-pharm.co.jp.
The abbreviation used is: GFP, green fluorescent protein.
2 Unpublished data.
3 Unpublished observations.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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 D. Pilzer and Z. Fishelson Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis Int. Immunol., September 1, 2005; 17(9): 1239 - 1248. [Abstract] [Full Text] [PDF]
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G. J. Bhat, T. Samikkannu, J. J. Thomas, and T. J. Thekkumkara {alpha}-Thrombin Rapidly Induces Tyrosine Phosphorylation of a Novel, 74-78-kDa Stress Response Protein(s) in Lung Fibroblast Cells J. Biol. Chem., November 19, 2004; 279(47): 48915 - 48922. [Abstract] [Full Text] [PDF]
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 G. Saretzki, L. Armstrong, A. Leake, M. Lako, and T. von Zglinicki Stress Defense in Murine Embryonic Stem Cells Is Superior to That of Various Differentiated Murine Cells Stem Cells, November 1, 2004; 22(6): 962 - 971. [Abstract] [Full Text] [PDF]
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 J.-F. Lo, M. Hayashi, S. Woo-Kim, B. Tian, J.-F. Huang, C. Fearns, S. Takayama, J. M. Zapata, Y. Yang, and J.-D. Lee Tid1, a Cochaperone of the Heat Shock 70 Protein and the Mammalian Counterpart of the Drosophila Tumor Suppressor l(2)tid, Is Critical for Early Embryonic Development and Cell Survival Mol. Cell. Biol., March 15, 2004; 24(6): 2226 - 2236. [Abstract] [Full Text] [PDF]
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 S. Taurin, V. Seyrantepe, S. N. Orlov, T.-L. Tremblay, P. Thibault, M. R. Bennett, P. Hamet, and A. V. Pshezhetsky Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na+]i/[K+]i Ratio in Cultured Vascular Smooth Muscle Cells Circ. Res., November 15, 2002; 91(10): 915 - 922. [Abstract] [Full Text] [PDF]
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D. L. Smith, C. A. Evans, A. Pierce, S. J. Gaskell, and A. D. Whetton Changes in the Proteome Associated with the Action of Bcr-Abl Tyrosine Kinase Are Not Related to Transcriptional Regulation Mol. Cell. Proteomics, November 1, 2002; 1(11): 876 - 884. [Abstract] [Full Text] [PDF]
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S. Benvenuti, R. Cramer, C. C. Quinn, J. Bruce, M. Zvelebil, S. Corless, J. Bond, A. Yang, S. Hockfield, A. L. Burlingame, et al. Differential Proteome Analysis of Replicative Senescence in Rat Embryo Fibroblasts Mol. Cell. Proteomics, April 1, 2002; 1(4): 280 - 292. [Abstract] [Full Text] [PDF]
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R. Wadhwa, T. Sugihara, A. Yoshida, H. Nomura, R. R. Reddel, R. Simpson, H. Maruta, and S. C. Kaul Selective Toxicity of MKT-077 to Cancer Cells Is Mediated by Its Binding to the hsp70 Family Protein mot-2 and Reactivation of p53 Function Cancer Res., December 1, 2000; 60(24): 6818 - 6821. [Abstract] [Full Text]
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 R. R. Reddel The role of senescence and immortalization in carcinogenesis Carcinogenesis, March 1, 2000; 21(3): 477 - 484. [Abstract] [Full Text] [PDF]
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M. Grigorian, S. Andresen, E. Tulchinsky, M. Kriajevska, C. Carlberg, C. Kruse, M. Cohn, N. Ambartsumian, A. Christensen, G. Selivanova, et al. Tumor Suppressor p53 Protein Is a New Target for the Metastasis-associated Mts1/S100A4 Protein. FUNCTIONAL CONSEQUENCES OF THEIR INTERACTION J. Biol. Chem., June 15, 2001; 276(25): 22699 - 22708. [Abstract] [Full Text] [PDF]
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S. Akakura, M. Yoshida, Y. Yoneda, and S. Horinouchi A Role for Hsc70 in Regulating Nucleocytoplasmic Transport of a Temperature-sensitive p53 (p53Val-135) J. Biol. Chem., April 27, 2001; 276(18): 14649 - 14657. [Abstract] [Full Text] [PDF]
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