From the § Institute of Biomedical Sciences, Academia
Sinica, Taipei 115, Taiwan, the ¶ Graduate Institute of Life
Sciences, National Defense Medical Center, Taipei 100, Taiwan, the
School of Medical Technology, China Medical College, Taichung
404, Taiwan, the ** Institute of Pathology, College of Medicine,
National Taiwan University, Taipei 100, Taiwan, and the
Graduate Institute of Clinical Medicine,
College of Medicine, National Taiwan University,
Taipei 100, Taiwan
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ABSTRACT |
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We previously identified a movable and regulable
inactivation function within the central region
(CRts247) of a temperature-sensitive p53
(p53ts) mutant, p53N247I. Here we showed that
central regions from several p53ts mutants behaved
similarly, i.e. they repressed a neighboring activation
domain only when existing in the mutant status. Using chimeric protein
GAL4VP16-CRts247 as an example, we demonstrated that
de novo protein synthesis was not required for the
reactivation of the chimeric protein, indicating that a
post-translational mechanism was involved in the control of
CRts247 activity. The CRts247-conferred
thermo-regulability did not work via a mechanism demanding either an
alteration of the subcellular compartmentalization of or the
inactivation of DNA-binding activity of the GAL4 chimera. Further,
CRts247 did not function in trans, eliminating
the possibility that the observed repression was because of the
competition for a putative factor(s) by the mutant p53 domain. Rather,
CRts247 bestowed temperature-dependent
interaction with hTAFII32 to the VP16 activation domain. In
a parallel experiment, CRts247 also caused a large
reduction in the affinity of hTAFII32 to the p53 activation
domain at the nonpermissive temperature. These results strongly
suggested that inhibition of hTAFII32 binding could be one
of the mechanisms responsible for the transcriptional repression by
mutant p53 central regions.
The tumor suppressor p53 protein is an important negative
regulator of cell proliferation (1). Although the half-life of the p53
protein is quite short, in response to DNA damage the protein
accumulates and turns on the expression of genes that are involved in
growth arrest, DNA repair, and apoptosis (2, 3). Thus, the biological
activities of p53 are mainly attributed to its function as a
transcription factor. In structure, the human p53 protein consists of
393 amino acids and is organized into three functional domains: an
N-terminal region involved in transcriptional activation, a central
region mediating specific DNA binding, and a C-terminal region
responsible for oligomerization, transcriptional repression, and
nonspecific DNA binding (4, 5).
The natural forms of cancers almost universally contain mutations in
the p53 gene. Mostly, they are single missense point mutations located
within the central region, that is, approximately residues 100-300 of
the p53 protein. These mutations not only lead to the inactivation of
the tumor suppressor function of p53 but also transform p53 into an
oncogene through a dominant negative effect (6). It has also been
demonstrated that wild-type and mutant p53 proteins appear to exhibit
distinctive phenotypes (7, 8), and the two phenotypes represent
distinctive conformations of the p53 polypeptide (7, 9).
Biochemically, mutations in the central region of p53 typically result
in the loss of the specific DNA-binding activity of the protein (4).
Moreover, we have previously demonstrated that the central region
(CRts247)1
encompassing residues 101-318 of p53N247I, a
p53ts mutant with a substitution of isoleucine for
asparagine at residue 247, functions as a movable and regulable
inactivation cassette (10). It represses several activation domains
present on the same polypeptide chain at the nonpermissive temperature
(37 °C) but not at the permissive temperature (30 °C). These
observations suggest that CRts247 has the potential to be
used as a desirable tool for the construction of temperature-sensitive
derivatives of transcription factors (10). Nevertheless, how the mutant
p53 central region gives rise to the repression activity in general and
how CRts247 confers thermo-regulability to a neighboring
activation domain in particular remain largely unknown. Here we
exploited the thermo-regulability of CRts247 to investigate
the mechanism(s) responsible for the aforementioned transcriptional repression.
Plasmid Constructions--
Construction of plasmids
pG5E1bCAT, pL6E1bCAT, pSG424, pSGVP, pGAL4VP16-CRwt
(previously referred to as pGAL4VP16-p53(101-318)),
pGAL4VP16-CRts247 (previously referred to as pGAL4VP16-CR),
and pLex-VP was described previously (10).
pGAL4VP16-CRts143 and pGAL4VP16-CRts173 were
constructed by inserting the corresponding p53 fragment between the
EcoRI and BamHI sites of pSGVP. pL6EG5CAT was
created by inserting the PstI/XbaI fragment of
pG5E1bCAT into the SmaI site of pL6EC-BSK (10).
pGEX3X-hTAFII32 was constructed by inserting a DNA fragment
encoding residues 1-140 of hTAFII32, which is soluble and
sufficient for interaction with VP16 (11-13), between the
BamHI and EcoRI sites of pGEX-3X (Amersham
Pharmacia Biotech). pH8HMKGAL4VP16-CRwt,
pH8HMKGAL4VP16-CRts247, pH8HMKp53-(1-318) and
pH8HMKp53N247I-(1-318) were cloned by inserting the DNA
fragment encoding GAL4VP16-CRwt,
GAL4VP16-CRts247, p53-(1-318), and
p53N247I-(1-318) proceeded by a consensus motif for heart
muscle kinase (HMK), respectively, between the NdeI and
BamHI sites of 8His-pET11d (14). pRK5F-CRts247
was constructed by inserting CRts247 between the
EcoRI and BamHI sites of pRK5F (15).
Transient Expression Assays and CAT Protein and RNA
Analyses--
Saos-2 cells were maintained in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum. Unless specified, calcium phosphate-mediated DNA transfections were performed as described previously (10, 16). CAT activity was measured and quantified according
to the method by Carey et al. (17). For
temperature-sensitive assays, the incubation temperature was switched
to 30 °C after a 12-h transfection at 37 °C. To block protein
synthesis, cycloheximide was added to the medium to a final
concentration of 10 µg/ml. RNA preparation and primer extension were
performed as described previously (18, 19).
Western Blotting Analysis--
The anti-GAL4 antibody was
purchased from Upstate Biotechnology, Inc., New York. The anti-FLAG
antibody was purchased from Kodak, New York. Immunoblotting was
performed as described previously (10, 16).
Immunostaining--
Cells were plated on acid-washed cover
slides and transfected as described above. Cells were fixed for 10 min
with 3% formaldehyde in phosphate-buffered saline (PBS) 36 h
after transfection. The fixed cells were washed twice with PBS,
permeabilized for 10 min in a mixture containing an equal volume of
acetone and ethanol at 4 °C, and then washed twice with PBS. For
immunofluorescence, slides were first incubated with 10% normal goat
serum in PBS at room temperature for 30 min to block nonspecific
binding. The slides were then incubated at room temperature for 12 h with the polyclonal anti-GAL4-(1-147) (Upstate Biotechnology, Inc.)
diluted (1:1000) in PBS containing 0.1% saponin and 0.05% sodium
azide. The slides were washed twice for 10 min with PBS at room
temperature and incubated for another 1 h with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG antibody. Finally, the
slides were washed extensively with PBS containing 0.02% Tween 20 and
mounted on a microscope.
GST Fusion Proteins, His-tagged Proteins, and Pull-down
Assay--
Glutathione S-transferase (GST) and
GST-hTAFII32 were expressed in and purified from
Escherichia coli XA90 strain according to standard protocols
(18, 20, 21). The ligand concentration, using bovine serum albumin as a
standard, was adjusted to approximately 0.125 mg per ml of resin for
GST and GST-hTAFII32. The His-tagged GAL4VP16-CRwt, GAL4VP16-CRts247, p53-(1-318),
and p53N247I-(1-318) were expressed in and purified from
E. coli JM109(DE3) strain according to the suggestions of
the manufacturer (QIAGEN, Inc.). In vitro phosphorylation of
the His-tagged proteins was performed as follows: 16 µg of the
His-tagged protein were incubated with 8 pmol of
[ The Central Regions of p53ts Mutants All Possess a
Thermo-regulable Inactivation Function--
We have shown that
CRts247 represses a neighboring transactivation domain only
at the nonpermissive temperature (10), suggesting that the p53 central
region, when existing in the mutant conformation, gains an inactivation
function. It is not clear, however, whether the gain of function is a
general phenomenon for mutant p53 central regions or just a special
property of CRts247. To address this question, we analyzed
the central regions of other mutants. Individual CRts from
the three p53ts mutants identified to date (22) was fused
to GAL4VP16, and the transcriptional activity of the resultant fusion
protein was measured. As shown in Fig.
1A, fusion of CRwt
to GAL4VP16, consistent with previous results (10), exhibited little
effect on the transactivation activity of the hybrid protein regardless
of the assaying temperature (compare lanes 3 and
9 to lanes 2 and 8). In contrast, all
three of the CRts repressed the transactivation activity of
the GAL4 fusion protein at 37 °C and not at 30 °C (compare
lanes 10-12 to lanes 4-6). The rationale to
work with p53ts mutants was as follows. First,
p53N247I, p53V143A, and p53V173L
all acquire a mutant-specific conformation at the nonpermissive temperature (22, 23). Second, it was aimed to take advantage of their
thermo-regulability so that the repression activity could be directly
related to their mutant conformation. However, the mutant
conformation-specific repression activity was not unique to the three
CRts because central regions from other p53 denaturing
mutants (23) all repressed the GAL4 fusion
protein.2 Nonetheless, the
three CRts differed quantitatively. Their ability to
restore the activity of the fusion protein at the permissive
temperature was repeatedly observed to obey the following order:
CRts247 De Novo Protein Synthesis Is Not Required for CRts247
Activity--
Next, using GAL4VP16-CRts247 as an example,
we set out to address the underlying molecular mechanism(s) responsible
for the observation that CRts-chimeric proteins turned into
transcriptional activators after the temperature was shifted from 37 to
30 °C (Fig. 1; also see Ref. 10). Conceivably, an increase in the
transactivation activity of GAL4VP16-CRts247 at the
permissive temperature might be because of the reactivation of the
hybrid molecules already present in the cell. Alternatively, de
novo synthesis of additional hybrid molecules was the underlying mechanism for the increase of transactivation activity after the temperature shift. To differentiate these possibilities, we examined the effect of cycloheximide, a potent inhibitor of translation, on the
transactivation activity of the hybrid protein. Because protein
synthesis was shut down in the presence of cycloheximide (data not
shown), the transactivation activity of GAL4VP16-CRts247
was determined by quantifying the CAT mRNA rather than the CAT protein. As shown in Fig. 2, at 37 °C
and in the presence of GAL4VP16-CRts247, the CAT transcript
was barely detectable by primer extension (lane 5). After
the assaying temperature was shifted to 30 °C, the level of CAT
transcript dramatically increased (lane 3). Importantly, the
thermo-regulated increase of CAT transcript was still observed in the
presence of cycloheximide (lane 4), demonstrating that de novo protein synthesis was not required for the
restoration of the activity of the fusion protein at the permissive
temperature; i.e. a post-translational mechanism was
involved in the reactivation of GAL4VP16-CRts247.
CRts247 Does Not Interfere with the Subcellular
Distribution of the GAL4 Chimera--
Because nuclear localization is
a prerequisite for a transcription factor to exert its regulatory role,
one potential mechanism responsible for the inactivity of
GAL4VP16-CRts247 at the nonpermissive temperature is that
the fusion protein is sequestered in the cytoplasm, especially in light
of the report that some mutant p53 proteins are found to be distributed
abnormally (4). To test this possibility, we performed an
immunostaining study to inspect the subcellular distribution of the
fusion protein. Fig. 3 shows that
GAL4VP16-CRts247 was predominately localized to the
nucleus, regardless of the assaying temperature. Thus, the inactivity
of GAL4VP16-CRts247 in transactivating gene expression at
37 °C could not be attributed to the exclusion of the protein from
the nucleus.
CRts247 Does Not Inactivate the DNA-binding Activity of
the GAL4 Fusion Protein--
Another possible mechanism for the
observed inactivity was that GAL4VP16-CRts247, although
present in the nucleus at the nonpermissive temperature, failed to bind
its cognate site so that a promoter driven by GAL4 sites was, of
course, not activated. To investigate this possibility, we determine
the in vivo DNA-binding activity of the GAL4 chimeric proteins using the well established blocking assay (10, 24). Briefly,
five copies of GAL4-binding sites were placed immediately downstream of
the TATA box of a reporter. In this way, binding of GAL4 derivatives
would block, via steric hindrance, assembly of the transcription
initiation complex on the promoter and therefore reduce CAT activity.
GAL4-(1-147), a derivative providing only GAL4 DNA-binding activity,
as well as GAL4VP16-CRwt and GAL4VP16-CRts247,
all inhibited transcription from the reporter containing GAL4 binding
sites at 37 °C (Fig. 4, compare
lanes 3-5 to lane 2). The reduction was specific
because transcription of a corresponding reporter lacking GAL4 binding
sites was not affected (Fig. 4, compare lanes 8-10 to
lane 7). It was concluded that the GAL4 module of the fusion
protein was still able to bind its cognate site even at the
nonpermissive temperature.
CRts247 Does Not Function in Trans--
Because
neither the nuclear localization (Fig. 3) nor the DNA-binding activity
(Fig. 4) of the chimeric protein was altered, only the possibility that
the VP16 activation domain of the fusion protein was the target of
regulation by CRts247 remained viable. It is possible that
CRts247 inhibits the VP16 domain by competing for a
putative factor(s) that is required for the transactivation by VP16. In
other words, this hypothesis predicts that CRts247 should
function in trans as well; i.e. the
transactivator and CRts247 co-exist as two individual
molecules. Accordingly, experiments were performed to examine whether
activator GAL4VP16 was repressed at 37 °C by
CRts247 in trans. As shown in Fig.
5A, CRts247 was
found to reproducibly repress GAL4VP16 in cis (lane
3). In contrast, the transactivation activity of GAL4VP16 remained
constant (Fig. 5A, compare lanes 5-8 to
lane 4) in the presence of a broad range of
CRts247 (Fig. 5B, lanes 1-5).
Moreover, the fact that CRts247 was unable to inhibit
activator GAL4VP16 in trans was not because of the
possibility that CRts247 itself failed to enter into the
nucleus because CRts247, a protein of about 30 kDa, was
observed to be homogeneously distributed in both the cytoplasm and the
nucleus by immunofluorescence (data not shown). Thus, two conclusions
could be drawn from the above study. First, CRts247 did not
work via competing for a putative transcription factor(s). Second, and
consequently, CRts247 could not be exploited as a
regulatory cassette in trans.
CRts247 Confers Temperature-dependent
Binding of hTAFII32 to Activation Domains Present on the
Same Polypeptide Chain--
Because CRts247 only
functioned in cis (Figs. 1 and 5; also see Ref. 10),
i.e. it repressed a transactivation domain present on the
same polypeptide chain only at the nonpermissive temperature (37 °C)
and not at the permissive temperature (30 °C), an alternative mechanism was proposed to explain how CRts247 functions.
Perhaps, a spreading of the temperature-dependent structural transition from CRts247 into the neighboring
region may account for the distinct behavior of CRts247. In
other words, the conformation of a neighboring activation domain could
be influenced by CRts247 so that the affinity of the
activation domain to its target(s) is greatly reduced. In this regard,
it was worthy to note that the activation domains of VP16 and p53 share
the ability both to be regulated by CRts247 (Ref. 10) and
to interact with a common co-activator, hTAFII32 (11, 25).
Thus, it was compelling to question whether CRts247
inhibits the VP16 and p53 activation domains via interference of their
interaction with hTAFII32 at the nonpermissive temperature. To test this idea, a GST pull-down assay was performed. In brief, hTAFII32 was expressed and purified from bacteria as a GST
fusion protein, GST-hTAFII32. Binding of
32P-labeled GAL4 derivatives to hTAFII32 was
measured by their retention on glutathione beads preloaded with the GST
fusion protein. As expected, fusion of CRwt to the VP16
activation domain showed little effect on the affinity of the chimeric
protein toward hTAFII32, regardless of the assaying temperature (Fig. 6A, compare
lane 2 to lane 4). In contrast, binding of the
CRts247-fusion protein to hTAFII32 became
temperature-dependent: GAL4VP16CRts247
interacted with hTAFII32 at the permissive temperature
(Fig. 6A, lane 8) but not at the nonpermissive
one (Fig. 6A, lane 6). Besides, neither of the
hybrid activators was retained by control GST beads (Fig.
6A, lanes 1 and 5), indicating that
the observed binding was specific for hTAFII32. Also, in
consistence with previous reports (11, 25), the VP16 domain was
required for the observed binding of GAL4VP16-CRts247 to
hTAFII32 because GAL4-CRts247, a derivative
with the VP16 domain removed, was not retained by the column at
30 °C (data not shown). In a parallel experiment, wild-type
p53-(1-318) was also retained by the beads regardless of the assaying
temperature (Fig. 6B, lanes 2 and 4).
However, the analogous region from p53N247I bound to the
beads much better at the permissive temperature than at the
nonpermissive one (Fig. 6B, compare lane 8 to
lane 6). For the sake of simplicity, the C-terminal
truncated p53 proteins were used rather than the full-length ones to
exclude the bacterial heat shock 70-kDa protein, which has been shown
to interact with the C terminus of p53 (26, 27), from complicating the
interpretation of the experiment. Using the C-terminal truncated
proteins also excluded the repression domain of the C terminus of p53
(5, 16), further simplifying the experiment. Taken together, the observation that CRts247 conferred temperature-sensitive
binding of hTAFII32 to the VP16 and p53 activation domains
not only was consistent with the conformation-spreading hypothesis but
also strongly suggested that the temperature-dependent interaction of these domains with hTAFII32 was one of the
underlying mechanisms responsible for the activity of
CRts247 in vivo.
It has been shown that the central region of p53N247I
gains a thermo-regulable repression activity toward several
transcriptional activation domains (10). In this report, we demonstrate
that the central regions of p53ts mutants all behave
similarly (Fig. 1). The thermo-regulability of CRts247
requires neither new protein synthesis (Fig. 2), nor an alteration of
the subcellular localization of the chimeric protein (Fig. 3). In
addition, CRts247 does not inactivate the DNA-binding
activity of the chimeric protein (Fig. 4). Further, CRts247
does not work via the competition for a putative transcription factor(s) that is required for the activity of a neighboring activation domain (Fig. 5). Rather, a temperature-dependent binding of
hTAFII32 to the activation domains of chimeric proteins may
account for the thermo-regulability of CRts247-fusion
proteins (Fig. 6). However, both the VP16 and the p53 activation domain
can bind several transcription factors (4, 28, 29). It is thus
conceivable that, to achieve the observed repression,
CRts247 may also interfere with the interaction of VP16 and
p53 activation domains with transcription factors other than
hTAFII32. This possibility is strengthened by the study
that the activation domain of adenovirus E1a protein is likewise
subjected to the thermo-regulation by a juxtaposing CRts247
(10). To our knowledge, there is no evidence supporting that the E1a
activation domain augments gene expression by interacting with
hTAFII32. Taken together, these observations strongly argue that the interference of hTAFII32 binding to neighboring
activation domains is probably not the only mechanism responsible for
the repression function of CRts247.
p53 is the most frequently mutated gene found in human cancer. Mutant
p53 is believed to adopt a conformation that is different from that of
wild-type p53 (7, 9). Because of their increased stability, mutant p53
proteins accumulate to a high concentration in cancer cells, and in
many cases, they are found to be localized to the nucleus (30, 31). The
strength of p53 activation domain (32-34) is comparable with that of
VP16, the strongest one known to date. Theoretically, it should cause a
problem described as transcriptional squelching (35) in cancer cells
harboring a mutant p53 if the activation domain of such a mutant p53 is
still active. In light of this, the finding that p53 mutants usually lose not only the DNA-binding but also the transactivation activities (this work and see Ref.10) seems biologically relevant. However, it is
not clear how p53 mutants inactivate their transactivation domain.
Using temperature-sensitive p53 mutants as a model, we demonstrate that
the central region, when trapped in the mutant conformation, gains a
repression function by preventing, at least, hTAFII32 from
interacting with the p53 activation domain. In support of this
hypothesis, destruction of the intactness of the central region of
wild-type p53 by deletion, which is thought to cause a conformational
change by disrupting the compact packing of the region (36) so that a
repression domain approximately encompassing amino acids 101-170 is
activated (16), also renders the resultant p53 fragments with a
repression activity (16, 37). In contrast, the central regions of p53
mutants that retain the wild-type conformation, such as
p53R248W, p53R273H, and p53R280K
(23), behaved like that of wild-type p53 and could not repress the p53
and VP16 activation domains (22).2
The ligand-binding domain (LBD) of steroid receptors can also function
as a movable and regulable inactivation cassette (38). There exist,
however, differences between CRts247 and LBD. First of all,
the activity of CRts247 is regulated by temperature (Ref.
10 and this work), in contrast to the case of LBD which is controlled
by its cognate steroid hormone (38). Second, whereas it is presumed
that steric hindrance via the interaction of heat shock protein 90 (HSP90) with the unliganded LBD is mainly responsible for the
inactivation of fusion proteins (39), several lines of evidence
indicate that CRts247 does not require the interaction with
a putative bulky protein to function as an autonomous regulatory
cassette. First, although HSP70 and HSC70 are known to bind to p53,
they do not bind to the central region (4). In fact, we could not
detect any interaction between CRts247 and HSP70, HSC70, or
HSP90 by various biochemical and genetic approaches (data not shown).
Second and more importantly, recombinant hTAFII32 and
CRts247-fusion proteins, expressed in and purified from
bacteria, directly interact with each other in a
temperature-dependent manner (Fig. 6). Accordingly, our
experimental results do not favor the bulky protein model to explain
how CRts247 functions. Instead, available data are
consistent with the conformation-spreading hypothesis to explain the
molecular mechanism by which CRts247 represses the
neighboring transactivation domain present on the same polypeptide
chain. Nevertheless, because the partial digestion patterns of
GAL4VP16-CRts247 by several proteases, including V8
protease, endoproteinase Arg-C,
INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES
MATERIALS AND METHODS
-32P]ATP and 20 units of heart muscle kinase (Sigma)
in 60 µl of a buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, and 12 mM MgCl2 at 37 °C for 1 h. The reaction
sample was passed through a NucTrap column (Stratagene) to remove free [
-32P]ATP. Aliquots (40 µl) of the GST protein beads
were incubated for 2 h at either 30 °C or 37 °C as indicated
in the figure legends with 2 µg of 32P-labeled GAL4 and
p53 derivatives in 500 µl of a buffer (NETN buffer) containing 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.2% Nonidet P-40. After being washed five
times with 1 ml of NETN buffer containing either 200 mM
NaCl and 0.2% Nonidet P-40 (for derivatives of GAL4VP16) or 500 mM NaCl and 0.01% Nonidet P-40 (for derivatives of p53)
prewarmed to the indicated temperature, bound proteins were eluted from
beads and analyzed by electrophoresis on a 10% SDS-polyacrylamide gel.
RESULTS
CRts173
CRts143
(Fig. 1A, lanes 4-6), suggesting that they were
somewhat dissimilar in the extent of recovery to the wild-type
conformation at the permissive temperature. An immunoblotting analysis
(Fig. 1B) showed that the difference in the transcriptional
activity of the fusion proteins could not be explained by variations in
their levels of expression. For example, whereas the concentration of
GAL4VP16-CRts143 appeared to be equal to, if not higher
than, that of other fusion proteins (Fig. 1B, compare
lane 4 to lanes 2, 3, and 5), its
transactivation activity was the lowest (Fig. 1A, compare
lane 5 to lanes 3, 4, and 6). Another
example was that, although all three CRts-fusion proteins
were synthesized to a level similar to that of GAL4VP16-CRwt at the nonpermissive temperature (Fig.
1B, compare lanes 8-10 to lane 7),
CRts-fusion proteins were inactive in stimulating
transcription (Fig. 1A, compare lanes 10-12 to
lane 9). Thus, it was concluded that all of the
CRts possessed a movable and thermo-regulable inactivation
function and that, among CRts of the p53ts
mutants, CRts247 was optimal in terms of such an activity.
In addition, these data suggested that the closer the mutation site was
positioned to the N-terminal end of the central region of a
p53ts mutant, the less the corresponding CRts
functioned as a movable regulatory cassette.
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Fig. 1.
The central regions of temperature-sensitive
p53 mutants possess a thermo-regulable inactivation function.
A, transactivation of reporter pG5E1bCAT by GAL4
chimeras at 30 °C (lanes 1-6) and at 37 °C
(lanes 7-12). Activator GAL4VP16 was included as a positive
control for transactivation (lanes 2 and 8).
Diagrams of reporter and activator structure are shown below
the autoradiogram. Experiments were repeated three times. An
autoradiogram of a typical experiment is shown. The relative CAT
activity (± S.D.) was 1 (± 0), 47.5 (± 6.3), 30.3 (± 4.1), 25.6 (± 3.6), 8.9 (± 1.4), 21.3 (± 4.0), 1 (± 0), 64.0 (± 10.6), 41.9 (± 6.5), 1.5 (± 0.2), 1.1 (± 0.1), and 1.2 (± 0.1) for lanes
1-12, respectively. B, expression levels of
GAL4VP16-CR derivatives. The chimeras are the same as in panel
A. The GAL4 derivative is indicated above each
track of the immunoblot. Lane M, molecular mass markers
in kilodaltons are indicated on the left. The position of
GAL4 derivatives is shown on the right
(arrowhead). Approximately 30 µg of proteins (using bovine
serum albumin as a standard) from transfected Saos-2 cells were
fractionated on a 10% SDS-polyacrylamide electrophoresis gel. GAL4
derivatives expressed at 30 °C (lanes 2-5) and at
37 °C (lanes 7-10) were detected with an
anti-GAL4-(1-147) antibody.
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Fig. 2.
Cycloheximide fails to prevent the
reactivation of GAL4VP16-CRts247.
Upper panel, analysis of CAT mRNA by primer extension.
Transfection was performed at 37 °C for 36 h. Cycloheximide was
added to the medium to a final concentration of 10 µg/ml 1 h
before the incubation temperature was shifted to 30 °C. Total RNA
from 2 × 106 transfected cells 20 h after the
temperature shift was isolated and subjected to the primer extension
analysis. The presence (+) or absence ( ) of activator, reporter, and
cycloheximide is indicated above each track of the
autoradiogram. Lower panel, an equivalent amount of total
RNA as used for the primer extension was loaded in each lane and was
then illustrated by the ethidium bromide staining of the agarose
gel.
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Fig. 3.
CRts247 does not alter
the subcellular distribution of the GAL4 chimera at the nonpermissive
temperature. Immunostaining of transfected
GAL4VP16-CRts247 protein in Saos-2 cells grown at 30 °C
(left panel) and at 37 °C (right panel).
GAL4VP16-CRts247 was accumulated in the nucleus regardless
of the assaying temperature. Magnification, ×100.
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Fig. 4.
CRts247 does not
inactivate the DNA-binding activity of the GAL4 chimera at the
nonpermissive temperature. One microgram of the illustrated
reporter DNA, 1 µg of the activator DNA, and 13 µg of the specified
blocker DNA were used for calcium phosphate-mediated DNA transfection.
A GAL4 derivative, GAL4-(1-147), which contains residues 1-147 of
yeast GAL4 and possesses only DNA-binding activity, was used as a
positive control. Cells were harvested 36 h after transfection.
The absence ( ) or presence (+) of activator is indicated above each
track of the autoradiogram, as is the blocker. The reporter was
pL6EG5C (lanes 1-5) or
pL6EC (lanes 6-10). Diagrams of the structure
of activator, reporters, and blockers are shown below the
autoradiogram. Otherwise as in Fig. 1A. The relative CAT
activity (± S.D.) was 1 (± 0), 12.3 (± 1.7), 2.2 (± 0.4), 1.7 (± 0.2), 2.1 (± 0.4), 1 (± 0), 6.4 (± 1.0), 5.7 (± 0.9), 5.8 (± 0.7),
and 5.8 (± 0.8) for lanes 1-10, respectively.
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Fig. 5.
CRts247 does not
inhibit GAL4VP16 in trans at the nonpermissive
temperature. A, transactivation of reporter
pG5E1bCAT by GAL4VP16 in the absence (lane 4) or
presence of increasing amounts of CRts247 (lanes
5-8) at 37 °C. Fusion proteins GAL4VP16-CRwt and
GAL4VP16-CRts247 were included as controls (lanes
2 and 3). Diagrams of reporter and activator structure
are shown below the autoradiogram. The activator is
indicated above each track of the autoradiogram. For the expression of
CRts247, lanes 5-8 were co-transfected with 1, 2, 5, and 10 µg, respectively, of pRK5F-CRts247, a
plasmid expressing FLAG-tagged CRts247; otherwise, as in
Fig. 1A. The relative CAT activity (± S.D.) was 1 (± 0),
31.3 (± 5.6), 1.1 (± 0.2), 32.3 (± 6.4), 30.9 (± 4.9), 31.7 (± 5.2), 31.0 (± 5.5), and 30.8 (± 5.9) for lanes 1-8,
respectively. B, expression levels of CRts247.
The transfection conditions for lanes 1-5 were the same as
in lanes 4-8 of panel A. Lane M, molecular mass
markers in kilodaltons are indicated on the left. The
position of CRts247 is shown on the right
(arrowhead). Approximately 30 µg of proteins (using bovine
serum albumin as a standard) from transfected Saos-2 cells were
fractionated on a 12% SDS-PAGE gel. CRts247 was detected
with an anti-FLAG antibody.
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Fig. 6.
CRts247 confers
temperature-dependent binding of
hTAFII32 to a neighboring activation
domain. A, GAL4VP16-CRts247 binds to the
GST-hTAFII32 bead only at the permissive temperature.
Left panel, binding of GAL4VP16-CRwt at 37 °C
(lanes 1-2) and at 30 °C (lanes 3-4).
Right panel, binding of GAL4VP16-CRts247 at
37 °C (lanes 5-6) and at 30 °C (lanes
7-8). The GST protein bead used is indicated above each track of
the autoradiogram. Input, one tenth of the input protein
directly loaded onto the gel. Molecular mass markers in kilodaltons are
shown on the left. B, interaction of
p53N247I-(1-318) with the GST-hTAFII32 bead is
also temperature-dependent. Left panel, binding
of p53-(1-318) at 37 °C (lanes 1-2) and at 30 °C
(lanes 3-4). Right panel, binding of
p53N247I-(1-318) at 37 °C (lanes 5-6) and
at 30 °C (lanes 7-8). The GST protein bead used is
indicated above each track of the autoradiogram.
Input, one-twentieth of the input protein directly loaded
onto the gel. Molecular mass markers in kilodaltons are shown on the
left.
DISCUSSION
-chymotrypsin, m-calpain,
and alkaline protease, in the presence or absence of
hTAFII32 at either 30 °C or 37 °C were not
informative (data not shown), future studies are required to directly
test the hypothesis.
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ACKNOWLEDGEMENT |
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We acknowledge Dr. J. Y. Chen for p53N247I, p53V143A, and p53V173L clones.
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FOOTNOTES |
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* This work was supported by grants from the Academia Sinica and the National Science Council of Taiwan (to C. T. L., M. Y. L., C. W. W., and Y. S. L.).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.
These authors contributed equally to the study.
§§ To whom correspondence should be addressed. Tel.: 886-2-26523074; Fax: 886-2-27829142; E-mail: bmyslin{at}ccvax.sinica.edu.tw.
2 S.-F. Tung, J.-Y. Chuang, C.-T. Lin, M.-Y. Lai, C.-W. Wu, and Y.-S. Lin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CRts, temperature-sensitive mutant of the central region; CRwt, wild-type mutant of the central region; PBS, phosphate-buffered saline; GST, glutathione S-transferase; LBD, ligand-binding domain.
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REFERENCES |
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