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Mol Cell Biol, March 1998, p. 1517-1524, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Physical Association of Multiple Molecular Chaperone Proteins
with Mutant p53 Is Altered by Geldanamycin, an hsp90-Binding
Agent
Luke
Whitesell,1,*
Patrick D.
Sutphin,1
Elizabeth J.
Pulcini,1
Jesse D.
Martinez,2 and
Paul H.
Cook1
Department of Pediatrics and Steele Memorial
Children's Research Center1 and
Department of Radiation Oncology, Arizona Cancer
Center,2 University of Arizona, Tucson,
Arizona 85724
Received 14 July 1997/Returned for modification 19 September
1997/Accepted 25 November 1997
 |
ABSTRACT |
Wild-type p53 is a short-lived protein which turns over very
rapidly via selective proteolysis in the ubiquitin-proteasome pathway.
Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by
which mutation leads to impairment of ubiquitination and
proteasome-mediated degradation is unknown, but it has been noted that
many transforming p53 mutants are found in stable physical association
with molecular chaperones of the hsp70 class. To explore a possible
role for aberrant chaperone interactions in mediating the altered
function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a
temperature-sensitive murine p53 mutant. In lysate prepared from A1-5
cells grown under mutant temperature conditions, hsp70 coprecipitated
with p53Val135 as previously reported by others, but in
addition, other well-recognized elements of the cellular chaperone
machinery, including hsp90, cyclophilin 40, and p23, were detected.
Under temperature conditions favoring wild-type p53 conformation, the
coprecipitation of chaperone proteins with p53 was lost in conjunction
with the restoration of its transcriptional activating activity.
Chaperone interactions similar to those demonstrated in A1-5 cells
under mutant conditions were also detected in human breast cancer cells
expressing two different hot-spot mutations. To examine the effect of
directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been
shown to alter the chaperone associations regulating the function of
unliganded steroid receptors. GA treatment of cells altered
heteroprotein complex formation with several different mutant p53
species. It increased p53 turnover and resulted in nuclear
translocation of the protein in A1-5 cells. GA did not, however, appear
to restore wild-type transcriptional activating activity to mutant p53
proteins in either A1-5 cells or human breast cancer cell lines.
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INTRODUCTION |
The wild-type p53 transcription
factor is a nuclear tumor suppressor involved in cell cycle regulation,
and loss of its normal function through mutation results in genetic
instability and abnormalities in the induction of apoptotic cell death
(14). Many p53 mutations are also associated with
positive tumor-promoting activity, and their protein products are found
to display markedly increased intracellular levels. Wild-type p53 is a
very short lived protein which turns over rapidly via selective
proteolysis in the ubiquitin-proteasome pathway (22). We
have recently shown, however, that for several common p53 mutants, the
normal processing of the protein is impaired, which results in the
marked accumulation of dysfunctional molecules with a prolonged
intracellular half-life (40). The mechanism by which
mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown at this time, but it has been noted that many
transforming p53 mutants are found in stable physical association with
hsc70, a member of the hsp70 class of molecular chaperones (9). Studies of both yeast and vertebrate cells have
suggested a role for heat shock proteins in modulating the transit of
target proteins through proteolytic processing pathways (6,
32). Consequently, we hypothesized that mutant p53 molecules,
presumably due to alterations in conformation, might be retained within
the molecular chaperone machinery and protected from ubiquitination and
subsequent degradation.
To examine such a possible role for aberrant chaperone interactions in
mediating mutant p53's intracellular accumulation, we have examined
the chaperone proteins which physically associate with a
temperature-sensitive murine p53 mutant (p53Val135) in A1-5
fibroblasts which stably overexpress the protein. This protein behaves
like other p53 mutants at 39°C, but when cells are maintained at
32°C, most of the protein assumes wild-type p53 activity, including
the ability to transactivate gene expression and induce cell cycle
arrest (23). Using this system, we were able to examine the
pattern of chaperone proteins which coprecipitated with mutant versus
wild-type p53 within the same cellular background. Next, using a panel
of human breast cancer cell lines, we were able to demonstrate that
clinically relevant p53 mutants also physically associate with multiple
chaperones. Finally, we examined the effects of geldanamycin (GA), a
selective hsp90-binding agent (37, 39) which recent work has
shown restores the ubiquitination and proteasome-mediated degradation
of mutant p53 in tumor cells (40). We found that drug
interaction with hsp90 altered heteroprotein complex formation with
several mutant p53 species and increased their turnover. Unlike a
temperature shift in A1-5 cells, however, GA treatment did not appear
to restore function as a transcription factor to mutant p53 in any of
the cell lines tested.
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MATERIALS AND METHODS |
Cells, antibodies, and reagents.
The rat embryo fibroblast
cell line A1-5 (26) was routinely cultured at 37°C in an
atmosphere of 10% CO2 in air in Dulbecco modified Eagle
medium supplemented with 10% fetal bovine serum. The breast cancer
cell lines MCF-7, SkBr3, MDA-MB-468, and T47D were obtained from the
American Type Culture Collection (ATCC, Rockville, Md.) and cultured in
RPMI 1640 supplemented with 10% fetal bovine serum. Cells were
passaged when 80% confluent, and all experiments were performed on
cells within 10 serial passages. Mouse monoclonal anti-53 antibodies
(PAb421, PAb242, and DO-1) were obtained from Oncogene Science.
Anti-p23 monoclonal antibody JJ3 was a gift from D. Toft (Mayo Clinic,
Rochester, Minn.). Monoclonal anti-hop (hsp-organizing protein;
previously called p60) antibody F5 and anti-hsp70 antibody BB70 were
provided by D. Smith (University of Nebraska, Omaha). Polyclonal rabbit
antiserum to cyclophilin 40 (Cyp40) (PA3-022) was purchased from
Affinity BioReagents (Golden, Colo.); polyclonal antiserum to human
WAF-1 was from Pharmingen (San Diego, Calif.; catalog no. 15431E).
Monoclonal antibodies to hsp90 (AC88) and hsp70 (N27F3-4) were
purchased from StressGen (Vancouver, British Columbia, Canada). GA was
obtained from the Drug Synthesis and Chemistry Branch, National Cancer
Institute. It was prepared as a 2-mg/ml stock in dimethyl sulfoxide and
maintained at 
20°C in the dark. All other chemical reagents were
purchased from Sigma unless otherwise stated.
Analysis of p53-chaperone protein complexes.
Cell monolayers
were rinsed twice with cold Tris-buffered saline (pH 7.4) and scraped
into ice-cold lysis buffer containing Tris-HCl (pH 7.4, 10 mM),
MgCl2 (1 mM), Tween 20 (0.2%, vol/vol), sodium molybdate
(10 mM), aprotinin (20 µg/ml), leupeptin (20 µg/ml), and
phenylmethylsulfonyl fluoride (1.0 mM). Cells were sonicated for 5 s two times with 1 min of cooling on ice between bursts, followed by
centrifugation at 16,000 × g for 30 min at 4°C. The
protein content of the supernatant fraction was determined with
bicinchoninic acid reagent (Pierce), and immunoprecipitation (IP) was
routinely performed with 1 to 2 mg of total protein in a final volume
of 300 to 400 µl. Incubation with primary antibody was performed at
4°C for 60 min with gentle agitation followed by addition of protein
G-Sepharose (15-µl resin pellet; Pharmacia) and further incubation
for 60 min. For IP experiments, bead pellets were then washed four
times in lysis buffer and extracted into 1× Laemmli sample loading
buffer by heating to 95°C for 5 min. For immunodepletion studies,
supernatants were collected from bead pellets and subjected to a second
round of IP to fully deplete the target chaperone. Supernatants were
then collected, and aliquots were added to Laemmli sample buffer and
heated as described above. All samples were then fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on either
7.5 or 12.5% polyacrylamide gels, and proteins were transferred to
nitrocellulose by electroblotting. Membranes were blocked in 3% nonfat
powdered milk and probed with primary antibodies followed by
species-appropriate peroxidase-conjugated secondary antibody.
Reactivity was detected by using chemiluminescent substrate and
exposure to Kodak XAR-5 film as previously described (40).
Multiple exposure times were evaluated for each blot to ensure that the
band intensities observed were within the dynamic response range of the
film.
Metabolic labeling.
A1-5 cells were incubated for 1 h
at 39°C in methionine-free RPMI 1640 containing 5% dialyzed,
heat-inactivated fetal bovine serum. [35S]methionine
(11,750 Ci/mmol; NEN) was added to yield 100 µCi/ml in the medium,
and incubation was continued for an additional hour. Dishes were rinsed
three times with cold phosphate-buffered saline and lysed in nonionic
detergent-containing buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet
P-40, 2 mM EDTA, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 20 µg of leupeptin per ml, 20 µg of aprotinin per ml). p53 IP was
performed with PAb421 from equal amounts of trichloroacetic
acid-precipitable material (108 cpm) in 400 µl (total
volume) as described above, and immunoprecipitates were fractionated by
SDS-PAGE (7.5% gel). After enhancement (Enlightening; NEN),
radiolabeled proteins were visualized by autoradiography.
Immunocytochemistry.
The intracellular localization of p53
in A1-5 cells growing on glass coverslips under various experimental
conditions was visualized by immunocytochemistry as previously
described (23) except that cells were reacted with purified
primary antibody (PAb421) at a dilution of 1:100. Detection was
achieved by using biotinylated goat anti-mouse and
streptavidin-peroxidase reagents supplied in kit form as recommended by
the manufacturer (HistoMark; Kirkegaard & Perry Laboratories,
Gaithersburg, Md.).
Reporter construct transfection.
The p53-responsive reporter
constructs PG13/
gal and WAF1/gal were a generous gift of P. Abarzua (Roche Research Center, Nutley, N.J.). PG13/gal contains 13 copies of oligonucleotide PG and the polyomavirus promoter, while
WAF1/gal contains a 2.4-kb HindIII WAF1
promoter fragment cloned upstream of the -galactosidase gene
(1). A1-5 cells were cotransfected by electroporation with
each of these reporter constructs plus a vector encoding G-418
resistance (pRcCMV; Invitrogen) at a 3:1 molar ratio. G-418 was added
48 h after electroporation at a concentration of 500 µg/ml, and
selection continued for 14 days, at which time both individual clones
and a pool of colonies were isolated and subcultured for subsequent
experimentation. Following a temperature shift or drug exposure
overnight, -galactosidase activity was assayed in 0.5% Nonidet
P-40-containing cytosolic extracts, using
o-nitrophenyl--D-galactopyranoside as a
substrate and measuring absorbance at 450 nm. The protein content of
each lysate was also measured by using the bicinchoninic acid reagent,
and specific -galactosidase activity was calculated as optical
density units per milligram of cellular protein. All determinations
were performed in triplicate.
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RESULTS |
Mutant p53 exists in multiprotein chaperone complexes.
We made use of A1-5 cells, which stably overexpress a
p53Val135 mutant that is temperature sensitive for
conformation, localization, and function. In this way, the chaperone
proteins which physically associate with mutant versus wild-type p53
could be examined within identical cellular backgrounds. We found in
this system that under conditions favoring mutant localization and
function, p53 coprecipitated with multiple chaperone proteins in
addition to hsp70. Demonstration of these novel associations was
dependent on the presence of millimolar concentrations of the
transition metal molybdate (Fig. 1).
hsp70 coprecipitation, however, did not appear molybdate sensitive, which may explain why its stable association with many mutant p53
species has long been appreciated. The finding that hsp90, as well as
the large immunophilin Cyp40 and the recently cloned accessory
chaperone p23, coprecipitated with mutant p53 in a
molybdate-sensitive fashion was intriguing given that unliganded
steroid receptors display similar patterns of chaperone protein
associations which are also stabilized by molybdate. Unlike
steroid receptors, however, reducing agents such as monothioglycerol
were found to enhance complex stability under the lysis and
precipitation conditions used in this study. The failure to
coprecipitate hsp90 or the other chaperones examined in Fig. 1 in
buffers which did not contain molybdate was not due to degradation or
inadequate lysis, as all chaperones were readily detected in total
cytostolic extract prepared in buffer which contained neither molybdate
nor monothioglycerol (lane T).
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FIG. 1.
Mutant p53 is physically associated with multiple
molecular chaperones. A1-5 cells were grown at 39°C (mutant
conditions), and lysates were prepared in buffer containing sodium
molybdate (10 mM) and/or monothioglycerol (10 mM) as indicated.
Anti-p53 IP was performed with PAb421, and precipitates were analyzed
by immunoblotting for the presence of the indicated chaperone proteins.
Total lysate (lane T) was analyzed to verify the presence and migration
position of each chaperone. LC, antibody light chain.
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To assess the proportion of p53 in A1-5 cells found in complex with
chaperone proteins, we performed immunodepletion studies
using anti-p23
antibody or anti-hsp70 antibody. As seen in Fig.
2A, this approach was able to selectively
deplete either the cochaperone
p23 (compare p23 signals in lanes 1 and
2) or hsp70 (compare hsp70
levels in lanes 3 and 4) from A1-5 lysates.
As expected from the
data in Fig.
1, depletion of these chaperones
resulted in significant
reductions in the p53 contents of the lysates
(compare p53 bands
in lanes 1 and 2 and in lanes 3 and 4). To
quantitate the extent
of p53 reduction, a standard curve relating p53
level to band
intensity was generated by simultaneously blotting
various amounts
of A1-5 protein onto the membrane used for the
immunodepletion
samples. Figure
2B demonstrates visually the expected
titration
of p53 signal as less protein is loaded, while Fig.
2C
depicts
optical density measurements of the bands in Fig.
2B plotted
against
various amounts of cell protein. Using this standard curve as
a
basis for interpolation, we have indicated the optical densities
of the
p53 bands seen in Fig.
2A by arrows numbered to correspond
to the
relevant lanes of Fig.
2A. Based on this analysis, it appears
that 70%
of p53 existed in a complex or complexes containing p23,
while 30%
could be coprecipitated with hsp70. Whether hsp70- and
p23-containing
complexes with p53 represent distinct entities
as appears to be the
case with the progesterone receptor (
36)
cannot be
determined definitively from the data presented. The
finding
illustrated by Fig.
2A that immunodepletion of p23 did
not result in
much decline in hsp70 signal and that depletion
of hsp70 did not
deplete p23 suggests, however, that distinct
complexes do exist.
Finally, care must be taken in the interpretation
of these results
because immunodepletion is by nature performed
under nonequilibrium
conditions. Some dissociation or alteration
of intrinsically dynamic
complexes may have occurred during the
procedure. As a result, the
percentages cited above are best viewed
as minimal estimates of mutant
p53 involvement in chaperone complexes
rather than absolute
determinations.
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FIG. 2.
Most of the mutant p53 in A1-5 cells is complexed with
molecular chaperones. Cells were grown at 39°C, and lysates were
prepared in molybdate-containing buffer. (A) IPs were performed on
replicate aliquots by using control mouse IgG (lanes 1 and 3), anti-p23
antibody (JJ3; lane 2), or anti-hsp70 antibody (BB70; lane 4) in order
to deplete the lysate of the relevant chaperone. Aliquots of
immunodepleted supernatant were then analyzed for remaining hsp70, p53,
and p23 content by Western blotting. HC and LC indicate the positions
of residual antibody heavy chain and light chain, respectively,
remaining in some of the supernatants after IP. (B) Aliquots of cell
lysate containing the indicated amounts of total protein were
immunoblotted on the same membrane as the samples depicted in panel A
in order to generate a standard curve for the estimation of p53 levels.
The position of p53 as detected by PAb421 is indicated. (C) Scanning
densitometry (Bio-Rad GS-700 instrument and Molecular Analyst software)
was performed to quantitate the p53 signals displayed in panels A and
B. The optical densities (arbitrary units) of the bands depicted in
panel B are plotted on the y axis, while the corresponding
amounts of total cellular protein are plotted on the x axis.
A linear curve fit (r2 = 0.98) for the
data points is shown as a solid line. Numbered arrows indicate the
optical densities of the p53 bands visible in each of the corresponding
lanes displayed in panel A.
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Using hsp90 as a representative of the novel associations demonstrated
in Fig.
1 and
2, we next examined the effects of temperature
shift and
the hsp90-binding agent GA on the composition of p53-chaperone
protein
complexes. As seen in Fig.
3, a shift to
the wild-type
temperature for 6 h prior to preparation of cytosol
resulted in
loss of hsp90 coprecipitation with p53 as well as a
substantial
reduction in hsp70 (compare lanes 3 and 2). The declines in
hsp70
and hsp90 were not due to obvious differences in p53 levels in
the total lysate (Fig.
3B, lanes 2 and 3). The relative amount
of p53
actually present in the immune complexes analyzed in Fig.
3A could not
be readily determined in this experiment because
the heavy chain of the
immunoglobulin used for IP comigrated with
p53 and obscured its
detection (data not shown). Unlike the temperature
shift, exposure of
A1-5 cells maintained at 39°C to GA for 10
min resulted in loss of
hsp90 but not hsp70 coprecipitation (compare
lanes 3 and 4).
Interestingly, GA treatment also induced the association
with p53 of
another chaperone formerly termed p60 and now known
as hop (lane 4).
This protein is a well-recognized component of
progesterone receptor
heteroprotein complexes, where its association
with the hormone-binding
protein is also stimulated by GA (see
below for discussion).
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FIG. 3.
GA treatment of cells alters the composition of
chaperone complexes coprecipitating with p53 in a manner distinct from
temperature shift. A1-5 cells were incubated at 32°C (lanes 2) or
39°C (lanes 3 to 5) for 6 h, followed by the addition of 1.8 µM GA (lanes 4) or an equal volume of dimethyl sulfoxide vehicle
(lanes 2, 3, and 5) for 10 min prior to lysis in molybdate-containing
buffer. As a control, PC-3M carcinoma cells, which do not express p53,
were lysed in the same buffer (lane 1). (A) IP with PAb421 (lanes 1 to
4) or irrelevant isotype-matched mouse IgG (lanes 5) was performed, and
precipitates were analyzed by immunoblotting for the presence of the
indicated chaperones. (B) Total proteins (25 µg) from the lysates
analyzed in panel A were analyzed by immunoblotting for the level of
p53 present.
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Our findings that manipulations such as temperature shift and exposure
of intact cells to GA prior to cytosol preparation
resulted in
alterations in the composition of the heteroprotein
complexes
coprecipitating with p53 suggest that these complexes
are biologically
significant and not formed artifactually during
cytosol extraction and
IP. Likewise, the detection of the chaperone
proteins demonstrated in
Fig.
1 and
3 did not result from nonspecific
cross-reactivity of the IP
reagents used. Specificity controls
presented in Fig.
3 include IP from
cells which do not express
p53 (lane 1) and control IP from cytosol
derived from A1-5 cells
maintained at 39°C with isotype-matched mouse
immunoglobulin (Ig)
(lane 5). No chaperone protein precipitation was
detected under
these conditions.
The binding of monoclonal antibody PAb421 near the carboxy terminus of
some missense p53 mutants has been reported to alter
their function and
restore their DNA binding activity (
17).
To determine
whether the novel coprecipitation of chaperones with
p53 seen in our
system was dependent on PAb421 interaction with
the Val135 mutant, we
used alternate IP targets and evaluated
the pattern of coprecipitating
proteins. For the data presented
in Fig.
4A, cytosol was prepared from A1-5 cells
shifted to wild-type
(lanes 1 and 2) or mutant (lanes 3 and 4)
temperature conditions
for 6 h with (lanes 2 and 4) or without
(lanes 1 and 3) the addition
of GA 10 min prior to lysis. IP was
performed with monoclonal
antibody JJ3, which recognizes p23, a
component of the mutant
p53 heteroprotein complexes demonstrated in
Fig.
1. Using this
alternate target, we found that p53 coprecipitated
efficiently
with p23 only in cytosol from cells grown at 39°C (Fig.
4A, lane
3). hsp90 and Cyp40 were seen to coprecipitate with p23 at
both
32 and 39°C, and as previously reported, GA treatment of cells
prior to lysis disrupted this coprecipitation (
18,
36). As
an additional means to ensure that precipitation of mutant p53
with
chaperone proteins was not epitope specific, we also performed
IPs with
the anti-p53 monoclonal antibody PAb242, which recognizes
a
conformation-insensitive epitope at the amino-terminal end of
the
molecule (Fig.
4B, lanes 1 and 4). Although PAb242 was not
as efficient
as PAb421 (lanes 2 and 5), it did coprecipitate hsp70
and hsp90.
Coprecipitation of hsp90 with p53 by this antibody
was disrupted by
pretreatment of cells with GA in a fashion similar
to that observed in
assays using PAb421.
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FIG. 4.
IP of p53 with chaperone complexes is not epitope
dependent. (A) A1-5 cells were incubated at 32°C (lanes 1 and 2) or
39°C (lanes 3 and 4) for 6 h, followed by the addition of 1.8 µM GA (lanes 2 and 4) or control vehicle (lanes 1 and 3) for 10 min
prior to lysis as described in the legend to Fig. 3. Anti-p23 IP with
monoclonal antibody JJ3 was performed, and precipitates were analyzed
by immunoblotting for the presence of the indicated proteins. HC refers
to the position of the immunoglobulin heavy chain, which was used for
IP and detected by the secondary antibody used in the immunoblotting
procedure. (B) A1-5 cells were incubated at 39°C for 6 h
followed by the addition of GA (lanes 4 and 5) or control vehicle
(lanes 1 to 3) for 10 min and lysis as described for panel A. p53 IP
was performed with PAb242 (lanes 1 and 4), PAb421 (lanes 2 and 5), or
no primary antibody (lane 3). Precipitates were analyzed by
immunoblotting for the presence of hsp70 and hsp90 as indicated.
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To demonstrate the biologic relevance of the chaperone interactions
with p53 shown above, we examined complex formation with
several
clinically relevant p53 hot-spot mutants in human breast
cancer cells.
As seen in Fig.
5, the chaperones p23 and
hsp90
were found to specifically coprecipitate with the mutant p53
species
expressed in the cell lines T47D (mutated codon 194/other
allele
deleted) and SkBr3 (mutated codon 175/other allele deleted).
This
coprecipitation was disrupted by brief exposure of cells to GA
prior to lysis, as was observed with the Val135 mutant in A1-5
cells.
No coprecipitation of chaperones was detected in lysate
of MCF-7 cells,
which express wild-type p53 alleles, and no coprecipitation
was
observed in MDA-MB-468 cells, which overexpress p53 from an
allele
mutated at codon 273. These findings demonstrate that simple
overexpression of p53 is not sufficient for detection of the stable
complexes that we have observed but rather requires mutation within
specific regions of the protein as previously described for hsp70
association with p53 in a panel of human breast tumor specimens
(
7).
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FIG. 5.
Certain p53 mutants are stably associated with molecular
chaperones in human breast cancer cells. Subconfluent cultures of the
cell lines MCF-7 (wild-type p53), T47D (mutant p53, codon 194),
MDA-MB-468 (mutant, codon 273), and SkBr3 (mutant, codon 175) were
lysed in molybdate-containing buffer with or without prior incubation
at 37°C in medium containing GA (1.8 µM) for 15 min as indicated.
Anti-p53 IP was performed with antibody DO-1. Control IP consisted of
mouse IgG at the same concentration and lysate from non-GA-treated T47D
cells. Precipitates were analyzed by immunoblotting for the presence of
the indicated chaperones. LC refers to the position of the antibody
light chain used for IP and detected by the secondary antibody used in
the blotting procedure.
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Chaperone complex disruption alters mutant p53 stability and
localization.
Having demonstrated that chaperone protein complexes
involving mutant p53 are disrupted by GA, we examined the consequences of this disruption on p53 function in A1-5 cells. Consistent with our
findings for cells carrying other p53 mutants (40), we found that drug treatment of A1-5 cells maintained at 39°C resulted in a
marked decline in the total cellular p53 level (Fig.
6A). This decline resulted from enhanced
turnover and not decreased synthesis of the protein. In Fig. 6B, cells
were pulse-labeled with [35S]methionine for 1 h
after an overnight exposure to GA. Immunoprecipitation of p53 from
these cells revealed comparable levels of newly synthesized p53 despite
the markedly lower level of total p53 seen in Fig. 6A. Such a finding
is consistent with restoration of the normally rapid turnover
characteristic of wild-type p53 following disruption of chaperone
protein-mutant p53 complex formation by GA.
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FIG. 6.
GA treatment of A1-5 cells decreases p53 levels without
altering its rate of synthesis. Cells were cultured overnight at 39°C
in the presence or absence of GA (1.8 µM) as indicated. (A) Cells
were lysed, and the level of p53 in equal amounts of total protein was
evaluated by immunoblotting. (B) Cells were metabolically labeled with
[35S]methionine for 1 h, followed by lysis and IP of
p53 with PAb421 from equal amounts of trichloroacetic acid-precipitable
material. Precipitates were fractionated by SDS-PAGE and visualized by
autoradiography.
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In addition to destabilizing the mutant protein, GA treatment of cells
induced nuclear translocation of the protein in a manner
similar to
that observed with the temperature shift. Figure
7A
demonstrates the clear cytoplasmic
immunolocalization of mutant
p53 characteristic of A1-5 cells at
39°C. As previously reported
(
24), a temperature shift to
32°C for 6 h resulted in a dramatic
shift to nuclear
localization (Fig.
7B). Interestingly, GA treatment
of cells maintained
at 39°C resulted in a nuclear pattern of p53
localization that was
indistinguishable from that observed with
a temperature shift (Fig.
7C). Localization associated with concurrent
temperature shift plus GA
treatment appeared the same as after
either manipulation alone (Fig.
7D). The specificity of the immunostaining
protocol used was confirmed
by the results in Fig.
7E, where isotype-matched
control antibody was
used to stain A1-5 cells maintained at 39°C
and minimal background
reactivity was observed.
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FIG. 7.
GA treatment of A1-5 cells induces nuclear localization
of mutant p53. Cells growing on coverslips were incubated at 39°C (A,
C, and E) or 32°C (B and D) for 6 h in the presence (C and D) or
absence (A, B, and E) of GA. Following fixation in cold
methanol-acetone, p53 localization was visualized by indirect
immunocytochemistry using PAb421 except for panel E, where an
irrelevant control antibody was applied. The dark diaminobenzidine
signal represents p53 immunoreactivity. All panels were photographed at
the same magnification and exposure settings.
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GA does not restore transcriptional activating activity to mutant
p53.
To assess the transcriptional activating activity of mutant
p53 relocalized to the nucleus by GA treatment, we made use of A1-5
cells stably transfected with reporter constructs under the control of
two different p53 response elements. Plasmid PG13/gal carries the
-galactosidase gene driven by the mouse polyomavirus promoter and a
multimer of a genomic p53 binding site from the ribosome gene cluster
(1). As seen in Fig. 8A, this
plasmid displays very low basal activity at 39°C in A1-5 cells and
robust induction following a shift to 32°C. Although GA treatment of cells at 39°C clearly induced nuclear translocation of p53 (Fig. 7),
it did not confer significant transactivating activity for the PG13
response element, presumably for reasons to be discussed below.
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FIG. 8.
GA treatment of A1-5 cells does not restore wild-type
p53 transcriptional activating activity. Cells were stably transfected
with the p53-responsive reporter construct PG13/gal (A) or
WAF1/gal (B). The level of -galactosidase activity was
quantitated in cell lysates following overnight incubation with or
without GA at the indicated temperatures. BKGD refers to the level of
-galactosidase activity measured in nontransfected A1-5 cells. All
determinations were performed in triplicate, and mean values are
depicted, with the standard deviations of the means indicated by error
bars. The results presented are derived from analysis of a clonal
isolate derived from transfections with each of the two vectors, but
similar results were obtained in assays using pooled colonies. ODU,
optical density units.
|
|
To be certain that the negative results obtained with PG13/
gal were
not restricted to this response element alone, we also
examined the
effect of drug treatment on the activity of the native
WAF-1
promoter, which drives
-galactosidase expression in the
vector
WAF1/
gal. Substantial basal activity at 39°C was observed
with
this plasmid, consistent with a previous report (
1), but
approximately fourfold-higher induction was observed following
temperature shift to wild-type conditions (Fig.
8B). The effect
of GA
treatment on
-galactosidase activity was more complex in
this
reporter system, however, than with the PG13/
gal vector.
Here drug
treatment of cells at 39°C resulted in a small but reproducible
increase in
-galactosidase activity, while treatment at 32°C
actually resulted in a small decline. Given the complexity of
the
native
WAF-1 promoter, it seems most likely that these
conflicting
results represent p53-independent effects of GA on
activation
of this response element in A1-5 cells (
27).
As a final approach to demonstrating GA effects on the transactivating
activity of mutant p53, we examined cellular levels
of the endogenous,
p53-regulated gene products mdm-2 in A1-5 cells
and WAF-1 in human
breast cancer cells. The expression of mdm-2
is tightly regulated by
p53, and it appears to serve as a negative
regulator of p53 function
(
28). As seen in the immunoblot of
whole-cell lysates
presented in Fig.
9, temperature shift
from
mutant to wild-type conditions increased cellular mdm-2 to readily
detectable levels in A1-5 cells as previously reported for other
cells
carrying a p53
Val135 mutant (
2). Consistent with
our findings in assays using the
PG13 reporter construct, GA treatment
of A1-5 cells did not induce
detectable levels of mdm-2 in cells
maintained at 39°C. Likewise,
as evident in Fig.
10, we did not observe GA-induced
increases
in the level of WAF-1 protein in human tumor cells expressing
two different mutant p53s, one of which we have shown above
coprecipitates
in a chaperone complex which is disrupted by GA (Fig.
5,
T47D).
Consistent with its demonstrated target of action (
11,
31)
and lack of DNA-damaging activity, GA did not increase WAF-1
levels
in MCF-7 cells. These cells were used as a control because they
express wild-type p53 and were able to respond to doxorubicin-mediated
damage by increases in both p53 and WAF-1 levels as expected.
GA
treatment decreased the level of mutant p53 in T47D cells (and
to a
lesser extent in MDA-MB-468 cells), as anticipated based
on our
previous findings (
40) and data presented above. Thus,
although GA treatment decreased mutant p53 levels (Fig.
6 and
10) and
induced its nuclear translocation in A1-5 cells (Fig.
7),
GA-mediated
disruption of chaperone complex formation did not
appear sufficient to
confer wild-type transactivating activity
to several different mutant
species, as monitored by the level
of p53-regulated gene products in
drug-treated cells.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 9.
GA treatment does not induce mdm-2 expression in A1-5
cells. Cells were incubated overnight at the indicated temperatures
with or without the addition of GA (1.8 µM). Lysates were prepared,
and the induction of mdm-2 was detected by immunoblotting with
monoclonal antibody 2A10.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 10.
GA treatment does not induce WAF-1 expression in human
breast cancer cells. Subconfluent cultures of the indicated cell lines
were treated overnight with control vehicle, doxorubicin (Dox; 0.2 or
1.0 µM), or GA (1.8 µM). Lysates were fractionated by SDS-PAGE, and
proteins were transferred to nitrocellulose. The upper half of the
membrane was probed with anti-p53 antibody DO-1, while the lower half
was probed with antiserum to human WAF-1.
|
|
| |
DISCUSSION |
It has long been appreciated that many mutant p53 species display
unusually stable physical association with the heat shock protein hsc70
both in tissue culture cells and in human tumor specimens
(7). This association has been reported to regulate p53
conformation in vitro (12) and correlates with increased transforming activity in intact cells (13, 16). In the
present study, we made use of a temperature-sensitive p53 mutant and
the selective hsp90-binding agent GA to establish a role for multiple molecular chaperones, not just hsc70, in modulating the stability, localization, and function of mutant and wild-type p53 within a
conserved cellular background. The biologic relevance of these findings
was then confirmed by demonstrating a similar pattern of chaperone
interactions with the mutant p53 species expressed by some human breast
cancer cell lines.
Although the biochemical activity of many molecular chaperones is well
established, much less is known about how chaperones act together in
large heteromeric complexes to regulate the posttranslational function
of diverse kinases, receptors, and transcription factors (29). The best-studied example of chaperone-mediated
conformational regulation in eukaryotic cells is that of steroid
hormone receptors, which require the interaction of multiple chaperones
to acquire or maintain a state competent to bind hormone (30,
35). Through a series of ATP-dependent reactions, an immature
hormone receptor complex that contains hsp90, hsp70, and at least the
two cochaperones hip (p48) and hop (p60) is maintained in dynamic
equilibrium with a more favored mature complex which is competent to
bind hormone (4). This mature complex lacks hsp70, hip, and
hop but contains at least two new proteins, p23 and one of the three
large immunophilins FKBP52, FKBP54, and Cyp40. Upon hormone binding,
the receptor is no longer found in association with chaperone proteins
and becomes active as a transcription factor (8). Treatment
with GA has been shown to block the transition of steroid receptors from immature to mature complexes, thus preventing hormone binding (36) and resulting in enhanced ubiquitination and
proteasome-mediated degradation of the hormone-binding protein
(38).
We now report that analogous to steroid receptors, p53 coprecipitated
in lysate with multiple molecular chaperones in addition to hsp70 when
cells were maintained under conditions favoring mutant p53 conformation
and function (Fig. 1). As recently reported by Sepehrnia et al. for
A1-5 cells (34) and Selkirk et al. for T47D breast cancer
cells (33), we also found hsp90 coprecipitating with mutant
p53. In addition, however, we detected coprecipitation of p23 and the
large immunophilin Cyp40, components characteristic of more mature
steroid receptor complexes. Immunodepletion studies indicated that at
least 70% of the p53 in A1-5 cells grown under mutant temperature
conditions was associated with a p23-containing chaperone complex (Fig.
2), while a significantly smaller fraction appeared to be associated
with hsp70. Such a pattern is consistent with that reported for
unliganded progesterone receptors (35). Upon a temperature
shift, coprecipitation of all of these chaperones was markedly reduced
(Fig. 3), yielding a p53 species capable of acting as a transcription
factor (Fig. 8 and 9).
Treatment of A1-5 cells with GA resulted in an effect on p53 similar to
that observed with steroid receptors, namely, loss of mature complex
components and enhancement of intermediate components, as indicated by
the increased hop signal seen in Fig. 3A, lane 4. Despite these
similarities, however, differences also were apparent. Specifically,
hsp90 is present in both intermediate and mature progesterone and
glucocorticoid receptor complexes (36, 38), while with
mutant p53, we found that hsp90 was lost in complexes from GA-treated
A1-5 and breast cancer cells even though p53 appeared to be trapped in
intermediate, hop-containing complexes (Fig. 3 to 5). In this respect,
mutant p53 appeared to behave more like transforming tyrosine kinases
such as v-Src. With these targets, association with hsp70 and hsp90 is
detectable under control conditions and hsp90 association is disrupted
by GA (39). While chaperone interactions appear relatively
well conserved, differences do exist in the associations seen with specific targets, the functional significance of which is unclear at
this time (29).
As with the glucocorticoid receptor, trapping of p53 in an apparently
intermediate complex by GA stimulated its degradation (Fig. 6 and 10)
but did not render it active as a transcription factor (Fig. 8 to 10).
The concept that association with other proteins can modulate the
degradation of p53 is supported by recent studies demonstrating such a
role for mdm-2 (15, 19). GA-stimulated degradation of mutant
p53 in our system, however, does not appear to be mediated through
mdm-2, as no induction of the protein could be detected following GA
treatment of A1-5 cells. Our finding that GA failed to restore
transcription factor activity to mutant p53 species agrees with a
previous study reporting that although GA treatment alters the
conformation of mutated p53 as measured by IP with
conformation-specific antibody, it only partially restores its ability
to bind a consensus DNA sequence (3). It seems most likely
that GA's failure to restore p53 function as a transcription factor
results from the continued association of p53 with chaperones such as
hsp70 and hop in GA-treated cells. These persistent associations may
impair oligomerization or posttranslational modifications such as
phosphorylation which are required for p53 activity as a transcription
factor (21, 24). It is also possible that GA interferes
directly with the function of some of the various kinases which have
been proposed to be involved in activating p53 such as casein kinase II
or raf-1 (reviewed in reference 25).
At this point, we do not know whether the chaperone components that we
have identified coprecipitate with p53 as a single complex or several
distinct complexes (Fig. 2), but in vitro reconstitution experiments in
reticulocyte lysate may be able to address this issue directly in
future studies. It is not possible to comment on the stoichiometry of
the components observed in our coprecipitation experiments for two
reasons. First, complexes are isolated by IP under nonequilibrium
conditions, which may allow for their gradual dissociation during the
procedure. Second, inherent variation in the affinity of the antibodies
used to detect components by Western blotting makes it impossible to
directly compare the absolute amounts of each protein detected.
It is interesting to speculate that the extended chaperone interactions
we have observed with mutant p53 actually represent a pathologic
exaggeration of normal, physiologic interactions of wild-type p53 with
the chaperone machinery. Due to its intrinsic conformational lability,
the turnover and function of wild-type p53 could be regulated to a
significant extent by ongoing posttranslational interactions with
components of the chaperone machinery. Such interactions may become
detectable by coimmunoprecipitation only when they become
pathologically extended as a consequence of mutation of the target.
Under normal conditions, their transient nature may serve to modulate
the presentation of p53 for degradation by the ubiquitin-proteasome
system. Because they involve heat shock proteins, these same
interactions could also serve as sensors of cell stress or damage.
Altering their levels and availability by insults such as ionizing
radiation or alkylating agents could lead to p53 stabilization and
provide a mechanism for its rapid activation as a transcription factor
in response to cellular damage. Consistent with this proposal, cellular
stresses that do not involve DNA damage have been shown to induce p53
activation (20). In addition, salicylate concentrations
which inhibit the heat shock response have been shown to inhibit p53
activation in response to UV irradiation and the chemotherapeutic agent
adriamycin (5).
In summary, we have shown that several mutant p53 species, but not
wild-type p53, are stably associated with a conserved group of
molecular chaperones. Mutant p53 molecules, presumably due to specific
alterations in conformation, appeared to be retained within this
molecular chaperone machinery, leading to their mislocalization and
protection from degradation. Alteration of specific chaperone interactions by GA treatment resulted in destabilization of mutant proteins, supporting the view that posttranslational interaction with
certain chaperone heteroprotein complexes may stabilize a target while
interaction with others may actually stimulate its degradation
(10). Taken together, our findings demonstrate that chaperone proteins play an important role in modulating the function of
many mutant p53 species and suggest that they could be involved in
regulating the activity of wild-type protein in response to cellular
stress.
| |
ACKNOWLEDGMENTS |
We thank B. Dyczweski for expert secretarial assistance and D. Toft and D. Smith for supplying antibody reagents.
This work was supported in part by funds from the Caitlin Robb
Foundation and by Public Health Service grant CA69537-02 from the
National Cancer Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, AHSC Room 3336, 1501 N. Campbell Ave., Tucson, AZ 85724. Phone: (520) 626-6527. Fax: (520) 626-4220. E-mail:
whitelj{at}peds.arizona.edu.
| |
REFERENCES |
| 1.
|
Abarzua, P.,
J. E. LoSardo,
M. L. Gubler, and A. Neri.
1995.
Microinjection of monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the transcriptional activation function to mutant p53.
Cancer Res.
55:3490-3494[Abstract/Free Full Text].
|
| 2.
|
Barak, Y.,
T. Juven,
R. Hafner, and M. Oren.
1993.
MDM2 expression is induced by wild type p53 activity.
EMBO J.
12:461-468[Medline].
|
| 3.
|
Blagosklonny, M. V.,
J. Toretsky, and L. Neckers.
1995.
Geldanamycin selectively destabilizes and conformationally alters mutated p53.
Oncogene
11:933-939[Medline].
|
| 4.
|
Chen, S.,
V. Prapapanich,
R. A. Rimerman,
B. Honore, and D. F. Smith.
1996.
Interactions of p60, a mediator of progesterone receptor assembly, with heat shock proteins hsp90 and hsp70.
Mol. Endocrinol.
10:682-693[Abstract/Free Full Text].
|
| 5.
|
Chernov, M. V., and G. Stark.
1997.
The p53 activation and apoptosis induced by DNA damage are reversibly inhibited by salicylate.
Oncogene
14:2503-2510[Medline].
|
| 6.
|
Craig, E. A.,
B. K. Baxter,
J. Becker,
J. Halladay, and T. Ziegelhoffer.
1994.
Cytosolic hsp70s of Saccharomyces cerevisiae: roles in protein synthesis, protein translocation, proteolysis and regulation, p. 31-52. In
R. I. Morimoto, A. Tissieres, and C. Georgopoulos (ed.), The biology of heat shock proteins and molecular chaperones, vol. 26.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 7.
|
Davidoff, A. M.,
J. D. Iglehart, and J. R. Marks.
1992.
Immune response to p53 is dependent upon p53/HSP70 complexes in breast cancers.
Proc. Natl. Acad. Sci. USA
89:3439-3442[Abstract/Free Full Text].
|
| 8.
|
Dittmar, K. D.,
K. A. Hutchison,
J. K. Owens-Grillo, and W. B. Pratt.
1996.
Reconstitution of the steroid receptor-hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.
J. Biol. Chem.
271:12833-12839[Abstract/Free Full Text].
|
| 9.
|
Finlay, C. A.,
P. W. Hinds,
T.-H. Tan,
D. Eliyahu,
M. Oren, and A. J. Levine.
1988.
Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.
Mol. Cell. Biol.
8:531-539[Abstract/Free Full Text].
|
| 10.
|
Freeman, B. C.,
D. O. Toft, and R. I. Morimoto.
1996.
Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.
Science
274:1718-1720[Abstract/Free Full Text].
|
| 11.
|
Grenert, J. P.,
W. P. Sullivan,
P. Fadden,
T. A. J. Haystead,
J. Clark,
E. Mimnaugh,
H. Krutzsch,
H.-J. Ochel,
T. W. Schulte,
E. Sausville,
L. M. Neckers, and D. O. Toft.
1997.
The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.
J. Biol. Chem.
272:23843-23850[Abstract/Free Full Text].
|
| 12.
|
Hainaut, P., and J. Milner.
1992.
Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.
EMBO J.
11:3513-3520[Medline].
|
| 13.
|
Halevy, O.,
D. Michalovitz, and M. Oren.
1990.
Different tumor-derived p53 mutants exhibit distinct biological activities.
Science
262:113-116.
|
| 14.
|
Hartwell, L. H., and M. B. Kastan.
1994.
Cell cycle control and cancer.
Science
266:1821-1828[Abstract/Free Full Text].
|
| 15.
|
Haupt, Y.,
R. Maya,
A. Kazaz, and M. Oren.
1997.
Mdm-2 promotes the rapid degradation of p53.
Nature
387:296-299[Medline].
|
| 16.
|
Hinds, P. W.,
C. A. Finlay,
R. S. Quartin,
S. J. Baker,
E. R. Fearon,
B. Vogelstein, and A. J. Levine.
1990.
Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes.
Cell Growth Differ.
1:571-580[Abstract].
|
| 17.
|
Hupp, T. R.,
D. W. Meek,
C. A. Midgley, and D. P. Lane.
1993.
Activation of the cryptic DNA binding function of mutant forms of p53.
Nucleic Acids Res.
21:3167-3174[Abstract/Free Full Text].
|
| 18.
|
Johnson, J. L., and D. O. Toft.
1995.
Binding of p23 and hsp90 during assembly with the progesterone receptor.
Mol. Endocrinol.
9:670-678[Abstract/Free Full Text].
|
| 19.
|
Kubbutat, M. H. G.,
S. N. Jones, and K. H. Vousden.
1997.
Regulation of p53 stability by Mdm2.
Science
387:299-303.
|
| 20.
|
Linke, S. P.,
K. C. Clarkin,
A. Di Leonardo,
A. Tsou, and G. M. Wahl.
1996.
A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage.
Genes Dev.
10:934-947[Abstract/Free Full Text].
|
| 21.
|
Lohrum, M., and K. H. Scheidtmann.
1996.
Differential effects of phosphorylation of rat p53 on transactivation of promoters derived from different p53 responsive genes.
Oncogene
13:2527-2539[Medline].
|
| 22.
|
Maki, C. G.,
J. M. Huibregtse, and P. M. Howley.
1996.
In vivo ubiquitination and proteasome-mediated degradation of p53.
Cancer Res.
56:2649-2654[Abstract/Free Full Text].
|
| 23.
|
Martinez, J.,
I. Georgoff,
J. Martinez, and A. J. Levine.
1991.
Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein.
Genes Dev.
5:151-159[Abstract/Free Full Text].
|
| 24.
|
Martinez, J. D.,
M. T. Craven,
E. Joseloff,
G. Milczarek, and G. T. Bowden.
1997.
Regulation of DNA binding and transactivation in p53 by nuclear localization and phosphorylation.
Oncogene
14:2511-2520[Medline].
|
| 25.
|
Meek, D. W.
1994.
Post-translational modification of p53.
Semin. Cancer Biol.
5:203-210[Medline].
|
| 26.
|
Michalovitz, D.,
O. Halevy, and M. Oren.
1990.
Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53.
Cell
62:671-680[Medline].
|
| 27.
|
Michieli, P.,
M. Chedid,
D. Lin,
J. H. Pierce,
W. E. Mercer, and D. Givol.
1994.
Induction of WAF1/CIP1 by a p53-independent pathway.
Cancer Res.
54:3391-3395[Abstract/Free Full Text].
|
| 28.
|
Momand, J.,
G. P. Zambetti,
D. C. Olson,
D. George, and A. J. Levine.
1992.
The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.
Cell
69:1237-1245[Medline].
|
| 29.
|
Nair, S. C.,
E. J. Toran,
R. A. Rimerman,
S. Hjermstad,
T. E. Smithgall, and D. F. Smith.
1996.
A pathway of multi-chaperone interactions common to diverse regulatory proteins: estrogen receptor, Fes tyrosine kinase, heat shock transcription factor Hsf1 and the aryl hydrocarbon receptor.
Cell Stress Chap.
1:237-250.
[Medline] |
| 30.
|
Pratt, W. B.
1993.
The role of heat shock proteins in regulating the function, folding and trafficking of the glucocorticoid receptor.
J. Biol. Chem.
268:21455-21458[Free Full Text].
|
| 31.
|
Prodromou, C.,
S. M. Roe,
R. O'Brien,
J. E. Ladbury,
P. W. Piper, and L. H. Pearl.
1997.
Identification and structural characterization of the ATP/ADP-binding site in the hsp90 molecular chaperone.
Cell
90:65-75[Medline].
|
| 32.
|
Schneider, C.,
L. Sepp-Lorenzino,
E. Nimmesgern,
O. Ouerfelli,
S. Danishefsky,
N. Rosen, and F. U. Hartl.
1996.
Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90.
Proc. Natl. Acad. Sci. USA
93:14536-14541[Abstract/Free Full Text].
|
| 33.
|
Selkirk, J. K.,
B. A. Merrick,
B. L. Stackhouse, and C. He.
1994.
Multiple p53 protein isoforms and formation of oligomeric complexes with heat shock proteins hsp70 and hsp90 in the human mammary tumor, T47D, cell line.
Appl. Theor. Electrophoresis
4:11-18[Medline].
|
| 34.
|
Sepehrnia, B.,
I. B. Paz,
G. Dasgupta, and J. Momand.
1996.
Heat shock protein 84 forms a complex with mutant p53 protein predominantly within a cytoplasmic compartment of the cell.
J. Biol. Chem.
271:15084-15090[Abstract/Free Full Text].
|
| 35.
|
Smith, D. F.
1993.
Dynamics of heat shock protein 90-progesterone receptor binding and the disactivation loop model for steroid receptor complexes.
Mol. Endocrinol.
7:1418-1429[Abstract/Free Full Text].
|
| 36.
|
Smith, D. F.,
L. Whitesell,
S. C. Nair,
S. Chen,
V. Prapapanich, and R. A. Rimerman.
1995.
Progesterone receptor structure and function altered by geldanamycin, an Hsp90-binding agent.
Mol. Cell. Biol.
15:6804-6812[Abstract].
|
| 37.
|
Stebbins, C. E.,
A. A. Russo,
C. Schneider,
N. Rosen,
F. U. Hartl, and N. P. Pavletich.
1997.
Crystal structure of an hsp90-geldanamycin complex: targeting of a protein chaperone by an anti-tumor agent.
Cell
89:239-250[Medline].
|
| 38.
|
Whitesell, L., and P. Cook.
1996.
Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells.
Mol. Endocrinol.
10:705-712[Abstract/Free Full Text].
|
| 39.
|
Whitesell, L.,
E. G. Mimnaugh,
B. De Costa,
C. E. Myers, and L. M. Neckers.
1994.
Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.
Proc. Natl. Acad. Sci. USA
91:8324-8328[Abstract/Free Full Text].
|
| 40.
|
Whitesell, L.,
P. Sutphin,
W. G. An,
T. Schulte,
M. V. Blagosklonny, and L. Neckers.
1997.
Geldanamycin-stimulated destabilization of mutated p53 is mediated by the proteasome in vivo.
Oncogene
14:2809-2816[Medline].
|
Mol Cell Biol, March 1998, p. 1517-1524, Vol. 18, No. 3
0270-7306/98/$04.00+0
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-
Burch, A. D., Weller, S. K.
(2005). Herpes Simplex Virus Type 1 DNA Polymerase Requires the Mammalian Chaperone Hsp90 for Proper Localization to the Nucleus. J. Virol.
79: 10740-10749
[Abstract]
[Full Text]
-
Adjei, A. A., Hidalgo, M.
(2005). Intracellular Signal Transduction Pathway Proteins As Targets for Cancer Therapy. JCO
23: 5386-5403
[Abstract]
[Full Text]
-
Esser, C., Scheffner, M., Hohfeld, J.
(2005). The Chaperone-associated Ubiquitin Ligase CHIP Is Able to Target p53 for Proteasomal Degradation. J. Biol. Chem.
280: 27443-27448
[Abstract]
[Full Text]
-
Banerji, U., O'Donnell, A., Scurr, M., Pacey, S., Stapleton, S., Asad, Y., Simmons, L., Maloney, A., Raynaud, F., Campbell, M., Walton, M., Lakhani, S., Kaye, S., Workman, P., Judson, I.
(2005). Phase I Pharmacokinetic and Pharmacodynamic Study of 17-Allylamino, 17-Demethoxygeldanamycin in Patients With Advanced Malignancies. JCO
23: 4152-4161
[Abstract]
[Full Text]
-
Tsvetkov, P., Asher, G., Reiss, V., Shaul, Y., Sachs, L., Lotem, J.
(2005). Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin. Proc. Natl. Acad. Sci. USA
102: 5535-5540
[Abstract]
[Full Text]
-
Grem, J. L., Morrison, G., Guo, X.-D., Agnew, E., Takimoto, C. H., Thomas, R., Szabo, E., Grochow, L., Grollman, F., Hamilton, J. M., Neckers, L., Wilson, R. H.
(2005). Phase I and Pharmacologic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients With Solid Tumors. JCO
23: 1885-1893
[Abstract]
[Full Text]
-
Muller, P., Ceskova, P., Vojtesek, B.
(2005). Hsp90 Is Essential for Restoring Cellular Functions of Temperature-sensitive p53 Mutant Protein but Not for Stabilization and Activation of Wild-type p53: IMPLICATIONS FOR CANCER THERAPY. J. Biol. Chem.
280: 6682-6691
[Abstract]
[Full Text]
-
Walerych, D., Kudla, G., Gutkowska, M., Wawrzynow, B., Muller, L., King, F. W., Helwak, A., Boros, J., Zylicz, A., Zylicz, M.
(2004). Hsp90 Chaperones Wild-type p53 Tumor Suppressor Protein. J. Biol. Chem.
279: 48836-48845
[Abstract]
[Full Text]
-
Muller, L., Schaupp, A., Walerych, D., Wegele, H., Buchner, J.
(2004). Hsp90 Regulates the Activity of Wild Type p53 under Physiological and Elevated Temperatures. J. Biol. Chem.
279: 48846-48854
[Abstract]
[Full Text]
-
Bagatell, R., Whitesell, L.
(2004). Altered Hsp90 function in cancer: A unique therapeutic opportunity. Molecular Cancer Therapeutics
3: 1021-1030
[Abstract]
[Full Text]
-
Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., Pratt, W. B.
(2004). Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to the Nucleus. J. Biol. Chem.
279: 22483-22489
[Abstract]
[Full Text]
-
Beliakoff, J., Bagatell, R., Paine-Murrieta, G., Taylor, C. W., Lykkesfeldt, A. E., Whitesell, L.
(2003). Hormone-Refractory Breast Cancer Remains Sensitive to the Antitumor Activity of Heat Shock Protein 90 Inhibitors. Clin. Cancer Res.
9: 4961-4971
[Abstract]
[Full Text]
-
Saydam, N., Steiner, F., Georgiev, O., Schaffner, W.
(2003). Heat and Heavy Metal Stress Synergize to Mediate Transcriptional Hyperactivation by Metal-responsive Transcription Factor MTF-1. J. Biol. Chem.
278: 31879-31883
[Abstract]
[Full Text]
-
Ficker, E., Dennis, A. T., Wang, L., Brown, A. M.
(2003). Role of the Cytosolic Chaperones Hsp70 and Hsp90 in Maturation of the Cardiac Potassium Channel hERG. Circ. Res.
92
: e87-e100
[Abstract]
[Full Text]
-
Chiosis, G., Huezo, H., Rosen, N., Mimnaugh, E., Whitesell, L., Neckers, L.
(2003). 17AAG: Low Target Binding Affinity and Potent Cell Activity--Finding an Explanation. Molecular Cancer Therapeutics
2: 123-129
[Abstract]
[Full Text]
-
Wang, C., Chen, J.
(2003). Phosphorylation and hsp90 Binding Mediate Heat Shock Stabilization of p53. J. Biol. Chem.
278: 2066-2071
[Abstract]
[Full Text]
-
Dugyala, R. R., Claggett, T. W., Kimmel, G. L., Kimmel, C. A.
(2002). HSP90{alpha}, HSP90{beta}, and p53 Expression following in Vitro Hyperthermia Exposure in Gestation Day 10 Rat Embryos. Toxicol Sci
69: 183-190
[Abstract]
[Full Text]
-
Strano, S., Fontemaggi, G., Costanzo, A., Rizzo, M. G., Monti, O., Baccarini, A., Del Sal, G., Levrero, M., Sacchi, A., Oren, M., Blandino, G.
(2002). Physical Interaction with Human Tumor-derived p53 Mutants Inhibits p63 Activities. J. Biol. Chem.
277: 18817-18826
[Abstract]
[Full Text]
-
Bonvini, P., Gastaldi, T., Falini, B., Rosolen, A.
(2002). Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK), a Novel Hsp90-Client Tyrosine Kinase: Down-Regulation of NPM-ALK Expression and Tyrosine Phosphorylation in ALK+ CD30+ Lymphoma Cells by the Hsp90 Antagonist 17-Allylamino,17-demethoxygeldanamycin. Cancer Res.
62: 1559-1566
[Abstract]
[Full Text]
-
Asher, G., Lotem, J., Kama, R., Sachs, L., Shaul, Y.
(2002). NQO1 stabilizes p53 through a distinct pathway. Proc. Natl. Acad. Sci. USA
10.1073/pnas.052706799v1
[Abstract]
[Full Text]
-
Wiesgigl, M., Clos, J.
(2001). Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani. Mol. Biol. Cell
12: 3307-3316
[Abstract]
[Full Text]
-
Bagatell, R., Khan, O., Paine-Murrieta, G., Taylor, C. W., Akinaga, S., Whitesell, L.
(2001). Destabilization of Steroid Receptors by Heat Shock Protein 90-binding Drugs: A Ligand-independent Approach to Hormonal Therapy of Breast Cancer. Clin. Cancer Res.
7: 2076-2084
[Abstract]
[Full Text]
-
Shiotsu, Y., Neckers, L. M., Wortman, I., An, W. G., Schulte, T. W., Soga, S., Murakata, C., Tamaoki, T., Akinaga, S.
(2000). Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G1 phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex. Blood
96: 2284-2291
[Abstract]
[Full Text]
-
Bagatell, R., Paine-Murrieta, G. D., Taylor, C. W., Pulcini, E. J., Akinaga, S., Benjamin, I. J., Whitesell, L.
(2000). Induction of a Heat Shock Factor 1-dependent Stress Response Alters the Cytotoxic Activity of Hsp90-binding Agents. Clin. Cancer Res.
6: 3312-3318
[Abstract]
[Full Text]
-
Cheung, J., Smith, D. F.
(2000). Molecular Chaperone Interactions with Steroid Receptors: an Update. Mol. Endocrinol.
14: 939-946
[Full Text]
-
Kelland, L. R., Sharp, S. Y., Rogers, P. M., Myers, T. G., Workman, P.
(1999). DT-Diaphorase Expression and Tumor Cell Sensitivity to 17-Allylamino,17-demethoxygeldanamycin, an Inhibitor of Heat Shock Protein 90. JNCI J Natl Cancer Inst
91: 1940-1949
[Abstract]
[Full Text]
-
Liu, X.-D., Morano, K. A., Thiele, D. J.
(1999). The Yeast Hsp110 Family Member, Sse1, Is an Hsp90 Cochaperone. J. Biol. Chem.
274: 26654-26660
[Abstract]
[Full Text]
-
Russell, L. C., Whitt, S. R., Chen, M.-S., Chinkers, M.
(1999). Identification of Conserved Residues Required for the Binding of a Tetratricopeptide Repeat Domain to Heat Shock Protein 90. J. Biol. Chem.
274: 20060-20063
[Abstract]
[Full Text]
-
Soga, S., Neckers, L. M., Schulte, T. W., Shiotsu, Y., Akasaka, K., Narumi, H., Agatsuma, T., Ikuina, Y., Murakata, C., Tamaoki, T., Akinaga, S.
(1999). KF25706, a Novel Oxime Derivative of Radicicol, Exhibits in Vivo Antitumor Activity via Selective Depletion of Hsp90 Binding Signaling Molecules. Cancer Res.
59: 2931-2938
[Abstract]
[Full Text]
-
Smith, D. F., Whitesell, L., Katsanis, E.
(1998). Molecular Chaperones: Biology and Prospects for Pharmacological Intervention. Pharmacol. Rev.
50: 493-514
[Abstract]
[Full Text]
-
Trentin, G. A., Yin, X., Tahir, S., Lhotak, S., Farhang-Fallah, J., Li, Y., Rozakis-Adcock, M.
(2001). A Mouse Homologue of the Drosophila Tumor Suppressor l(2)tid Gene Defines a Novel Ras GTPase-activating Protein (RasGAP)-binding Protein. J. Biol. Chem.
276: 13087-13095
[Abstract]
[Full Text]
-
Akakura, S., Yoshida, M., Yoneda, Y., Horinouchi, S.
(2001). A Role for Hsc70 in Regulating Nucleocytoplasmic Transport of a Temperature-sensitive p53 (p53Val-135). J. Biol. Chem.
276: 14649-14657
[Abstract]
[Full Text]
-
Peng, Y., Chen, L., Li, C., Lu, W., Chen, J.
(2001). Inhibition of MDM2 by hsp90 Contributes to Mutant p53 Stabilization. J. Biol. Chem.
276: 40583-40590
[Abstract]
[Full Text]
-
Asher, G., Lotem, J., Kama, R., Sachs, L., Shaul, Y.
(2002). NQO1 stabilizes p53 through a distinct pathway. Proc. Natl. Acad. Sci. USA
99: 3099-3104
[Abstract]
[Full Text]
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Abstract
Introduction
