From the Derald H. Ruttenberg Cancer Center, Mount
Sinai School of Medicine, New York, New York 10029, the
¶ Department of Biochemistry and Molecular Biology, Oregon Health
Sciences University, Portland, Oregon 97201, and the
§ Huffington Center on Aging and Department of Molecular and
Cellular Biology, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, August 30, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Regulation of the stability of p53 is key to its
tumor-suppressing activities. mdm2 directly binds to the
amino-terminal region of p53 and targets it for degradation through the
ubiquitin-proteasome pathway. The coactivator protein
TAFII31 binds to p53 at the amino-terminal region that is
also required for interaction with mdm2. In this report, we demonstrate
that expression of TAFII31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels.
TAFII31-mediated p53 stabilization results in activation of
p53-mediated transcriptional activity and leads to
p53-dependent growth arrest in fibroblasts. UV-induced
stabilization of p53 coincides with an increase in p53-associated
TAFII31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAFII31 fails to
stabilize p53. Our results suggest that direct interaction of
TAFII31 and p53 not only mediates p53 transcriptional
activation but also protects p53 from mdm2-mediated degradation,
thereby resulting in activation of p53 functions.
Mutation of the p53 tumor suppressor gene is frequently associated
with different forms of human cancers. The ability of p53 protein to
suppress tumor growth is attributed by two major biological processes
induced by p53: cell cycle arrest and apoptosis (reviewed in Refs.
1-3). Regulation of p53 protein stability plays a pivotal role in
modulating p53 functions (4, 5). Under normal conditions, the half-life
of p53 is limited to minutes. Cellular stress or DNA damage leads to a
rapid stabilization of p53 protein and activation of p53-mediated
checkpoint functions. Several proteins are known to affect p53
stability through protein-protein interactions, including HPV16 E6 (6),
WT-1 (7), E1B 55K/E4orf6 (8), SV40 large T antigen (9, 10), JNK (11),
HIF (12), p19ARF (13), p300 (14), and the cellular oncoprotein mdm2
(15, 16). Whereas mdm2, JNK, and E6 have been implicated in targeting p53 for degradation, WT1, E1B 55K, and SV40T antigen stabilize p53
leading to increases in p53 levels. mdm2 functions as a pivotal p53
regulator and inhibits p53 functions by dual mechanisms. Interaction of
mdm2 with p53 at its transactivation domain directly inhibits p53-mediated transcriptional activity (17, 18). Binding of mdm2 to p53
also targets p53 for degradation through ubiquitin-mediated pathways
(19, 20). As one of p53 targets, expression of mdm2 is
transcriptionally regulated by p53 (21), resulting in a feedback loop
that regulates the level and activity of p53 (21). The biological
implication of this regulatory loop is evident in that amplification of
mdm2 gene has been found in about 30% of human soft tissue
sarcomas and in other human cancers (22, 23), suggesting that negative
regulation of p53 by mdm2 is important in the development of
those tumors. Loss of mdm2, on the other hand, results in an
up-regulation of p53 activity and subsequent abrogation in cell cycle
control and lethality in mice (24, 25).
As a transcription factor, the p53 protein activates
transcription of target genes by binding RNA polymerase II
complex. Two of the TATA-binding polypeptide (TBP)-associated
factors (TAFs)1 within the
general transcription factor TFIID complex, TAFII31 and
TAFII70, have been shown to interact with the
amino-terminal activation domain of p53 directly in vitro
(26, 27). It is generally believed that TBP mediates only basal
transcription, whereas the TAF-TBP complex (TFIID) can mediate specific
transcription factor-directed transactivation (28). Although the
in vivo requirement for TAFs in p53-mediated transcription
activation remains unclear, the fact that TAFII31 and
TAFII70 support p53-mediated transcriptional activation in
reconstituted transcription reactions suggests a critical role for TAFs
in p53-mediated transactivation in vivo.
The observation that both TAFII31 and mdm2 bind to the same
amino-terminal region of p53 suggests that TAFII31 may play
an important role in regulating p53 protein stability and functions. To
test this hypothesis, we have measured the effect of
TAFII31 on ubiquitination and stability of p53 both
in vivo and in vitro using either purified
protein components or whole cell extracts (11). Our results showed that
TAFII31 enhances p53 stability by inhibiting mdm2-mediated
p53 ubiquitination and degradation. Expression of TAFII31
also induces p53- dependent transcriptional activation and
cell cycle inhibition, suggesting that TAFII31 plays an
active role in regulating p53 stability and activity in cells.
Cell Lines, Plasmids, and Transfection--
All cells were
maintained in Dulbecco's modified Eagle's medium, supplemented with
10% fetal bovine serum in 5% CO2 incubators at 37 °C.
The human TAFII31 expression vector was constructed by
cloning human TAFII31 cDNA into the pMTN expression
vector under the control of SV40 promoter. The HA tag was engineered at
the amino terminus. Mutant TAFII31 was generated by
polymerase chain reaction and constructed into pMTN with HA tag. The
mdm2 and p53 expression plasmids have been described elsewhere (11). The p53 reporter plasmids were described previously (29). Transfections were carried out using Fugene or
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium methyl sulfate transfection reagent according to the manufacturer's protocol (Roche Molecular Biochemicals). In transient transfection experiments, cells were harvested 48-72 h after transfection.
Immunoprecipitation, Western, and Northern Blotting--
Cells
were lysed in lysis buffer (20 mM HEPES, pH 7.5, 350 mM NaCl, 25% glycerol, 0.25% Nonidet P-40, 1 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, pepstatin, and leupeptin) by sonication
on ice. Lysates were clarified by centrifugation at 14,000 × g for 15 min at 4 °C. The protein concentrations were
determined, and aliquots were stored at
For immunoprecipitation, equal amount of lysates were
immunoprecipitated using a monoclonal antibody against p53, pAb421, and
protein A-agarose (Life Technologies, Inc.) for 16-24 h. The complex
was washed three times with lysis buffer and subjected to
SDS-polyacrylamide gel electrophoresis analysis. The proteins were
transferred to a Hybond-C membrane (Amersham Pharmacia Biotech), blotted by a rabbit polyclonal antibody against p53 (Santa Cruz), and
developed using ECL (Amersham Pharmacia Biotech). Endogenous TAFII31 was detected using a monoclonal antibody against
TAFII31. mdm2 was detected by monoclonal antibody 2A10.
For Northern analysis, equal quantities of total RNA isolated by RNAzol
(Life Technologies, Inc.) at 10-20 µg/sample were subjected to
electrophoresis on a denaturing 1% formaldehyde agarose gel. The RNA
was transferred to a nylon membrane and hybridized with
[32P]dCTP-labeled probes using the Rapid Hybridization
System (Amersham Pharmacia Biotech).
Pulse-Chase Labeling--
10.1 cells growing in 10-cm plates
were transfected with p53, mdm2, and TAFII31. A protein
labeling mixture containing [35S]methionine (50 µCi;
Amersham Pharmacia Biotech) was added to the plates 48 h after
transfection and incubated for 10 min. The label was removed, and fresh
media were added. The cells were harvested at 0, 1, and 2 h. The
p53 protein was detected as described (11).
In Vitro and in Vivo Ubiquitination
Assays--
Baculovirus-expressed mdm2 and histidine-tagged
p53 proteins were obtained as described (19). The TAFII31
protein was produced by in vitro transcription and
translation (Promega). Ubiquitination was performed using reticulocyte
lysates depleted of mdm2 and JNK that provide the necessary
components. The mdm2 and/or TAFII31 (in 1-ng
quantities) were incubated on ice with bacterially expressed p53 (1-5
µg) bound to nickel-nitrilotriacetic acid beads for 45 min.
After extensive washes (four times with 1 ml of kinase buffer), the
substrate-bound beads were equilibrated with 1× ubiquitination buffer
(50 mM Tris-HCl, pH8.0, 5 mM MgCl2,
0.5 mM dithiothreitol, 2 mM NaF, and 3 mM okadaic acid) and incubated in the same buffer supplemented with 2 mM ATP, 10 mM creatine
phosphate, 0.02 unit creatine phosphokinase, 2 µg of Ub HA, 1.5 mM ATP Reporter Assay--
Saos-2 cells were transfected using the
N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium methyl
sulphate liposomal transfection reagent (Roche Molecular Biochemicals)
as follows. 100,000 cells were plated in each well of 6-well dishes
and incubated overnight. Cells were fed with Dulbecco's modified
Eagle's medium containing 10% FBS and incubated for an additional
3 h. N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium
methyl sulphate/DNA mixtures containing appropriate amounts of reporter
constructs and expression plasmids were prepared according to the
manufacturer's instructions and incubated at room temperature for 15 min. Serum-free medium was then added to the mixtures and used to
replace the medium in the wells. The dishes were incubated at 37 °C
for 3 h, after which the transfection mix was removed and replaced
with Dulbecco's modified Eagle's medium containing 10% FBS. After
48 h, the 6-well plates were placed on ice and washed once with
PBS. The cells were then lysed by scraping into 120 µl of reporter
buffer (Promega Luciferase Assay System), and samples were spun for 1 min at 14,000 rpm at 4 °C. The total protein concentration was
determined using a commercially available assay (Bio-Rad). Of each
sample, 40 µl were warmed to room temperature and mixed with
luciferase assay substrate reconstituted with Luciferase Assay Buffer
(Promega). Light emission was determined in a TD-20e luminometer (Turner).
Colony Forming Assay--
10.1 and 12.1 cells were plated in 60 cm plates. The cells were transfected with 2 µg of neo vector
(pCMV-BM), p53, TAFII31, and 5 µg of salmon sperm DNA as
carrier using the calcium phosphate precipitation procedure.
Transfected cells were selected by Geneticin (50 µg/ml, Life
Technologies, Inc.) for 2-3 weeks. The colony was stained with Giemsa
stain (Sigma).
Flow Cytometry Analysis--
The flow cytometry were performed
as described (30). Briefly, cells were transfected with 2 µg of green
fluorescence protein expression plasmid pEGFP-N
(CLONTECH) and 10.0 µg of either control DNA
(empty vector), or p53, or TAFII31 expressing plasmid.
Transfected cells were harvested 48-72 h later and washed twice in
PBS. The cells were fixed in paraformaldehyde in PBS for 10 min and
permeabilized in 70% ethanol for 16 h at 4 °C. Cells were
resuspended in PBS containing 5 µg of propidium iodide and RNase A (5 µg/ml). Flow cytometric analysis was carried out in a
fluorescence-activated cell sorting analyzer (Coulter). GFP positive
cells were gated and analyzed for DNA content.
Stabilization of p53 in Vivo by TAFII31--
As a
first step to address the possible role of TAFs in regulating p53
stability, mammalian expression vectors encoding human TAFII31 and p53 were transfected into 10.1 mouse Balb/c 3T3
fibroblast cells that lack endogenous p53 (31). Co-transfection of
mdm2 and p53 decreased expression levels of p53 as a result of its destabilization, as previously reported (15, 16) (Fig.
1A). Expression of
TAFII31 in 10.1 cells increased the levels of p53 protein
by 2-fold (Fig. 1A). Furthermore, forced expression of TAFII31 attenuated mdm2-mediated on p53 degradation in
a dose-dependent manner (Fig. 1A).
TAFII31 did not change mRNA levels of p53 (Fig. 1A), suggesting that the elevation of p53 levels by
TAFII31 was not due to increased transcription of the p53
gene. In a duplicate experiment, expression levels of
TAFII31 protein in the transfection experiment were
confirmed by Western analysis (Fig. 1A). Pulse-chase labeling with [35S]methionine revealed that the half-life
of p53 was extended in cells expressing TAFII31 (Fig.
1B), supporting the notion that induction of p53 levels by
TAFII31 is due to enhanced p53 stability. To determine
whether TAFII31 mediates its effect on p53 through mdm2 or through an independent mechanism, p53 and
TAFII31 were cotransfected into a p53/mdm2 double null cell
line. The results demonstrated that p53 levels are not affected by the
expression of TAFII31 in p53/mdm2 null cells,
indicating that stabilization of p53 by TAFII31 is
mdm2 dependent (Fig. 1C). Because binding of p53 by
mdm2 is required for mdm2-mediated p53 degradation (15, 16), these
results suggest that TAFII31 either out-competes mdm2
for binding to p53 or excludes mdm2 from the p53 complex, thus
preventing mdm2-mediated p53 degradation.
TAFII31 Blocks mdm2-mediated Ubiquitination of
p53--
It has been shown that mdm2 targets p53 for
ubiquitination, and we demonstrated that TAFII31 inhibits
mdm2-mediated degradation of p53. To extend our finding that the
effect of TAFII31 on p53 is at the level of ubiquitination,
an in vitro ubiquitination assay was used to determine the
degree of p53 ubiquitination after expression of TAFII31
(19). In this assay nickel bead-bound histidine-tagged p53 (his-p53)
was incubated with TAFII31 and the targeting protein
mdm2. Subsequently, the ubiquitination reaction was carried out
using reticulocyte lysates depleted of mdm2 and JNK (Fig.
2A). The addition of mdm2
to p53 in the in vitro ubiquitination reaction resulted in
ubiquitinated p53. Although TAFII31 alone did not appear to
affect p53 ubiquitination, it inhibited mdm2-mediated p53
ubiquitination in a dose-dependent manner (Fig.
2A). These findings suggest that TAFII31
increases p53 stability by inhibiting mdm2-mediated p53
ubiquitination.
To determine the effect of TAFII31 on p53
ubiquitination in vivo, mdm2, TAFII31, and
his-p53 expression plasmids were cotransfected into Balb/c 3T3
fibroblasts in various combinations together with the HA-tagged
ubiquitin expression vector. After treatment with proteasome inhibitor
MG132, the transfected p53 was purified on nickel beads and detected by
immunoblotting with pAb421 antibody to p53. The degree of
polyubiquitinated p53 was detected using antibody against HA.
Consistent with the in vitro data, expression of mdm2
increased p53 ubiquitination and coexpression of TAFII31 blocked mdm2-mediated ubiquitination of p53 in vivo
(Fig. 2B). These data indicate that TAFII31
attenuates the effect of mdm2 on p53 ubiquitination.
TAFII31 and p53 Interaction Is Required for p53
Stabilization--
To test whether TAFII31-p53 interaction
is required for the ability of TAFII31 to stabilize p53, we
generated mutant TAFII31 truncated at either the amino or
carboxyl terminus. The TAF-C expresses amino acids 130-264 of
TAFII31 and TAF-N produces amino acids 1-141 of the
protein (Fig. 3A).
Co-transfecting mutant TAFII31 with p53 in mammalian cells
showed that TAF-C coimmunoprecipitated with p53, and no interaction was
detectable between TAF-N and p53 in the same assay (Fig.
3B). To explore the effect of TAFII31 mutants on
p53 levels and stability, we first examined the ability of these
mutants to inhibit mdm2-mediated p53 degradation. Expression of
mdm2 resulted in p53 degradation (Figs. 1A and
3C). Co-transfection of TAF-C significantly attenuated the
ability of mdm2 to degrade p53 (Fig. 3C), although it
is not as effective as the full-length protein (Fig. 3C). On
the other hand, coexpression of TAF-N has no substantial effect on p53
levels (Fig. 3C), suggesting that TAFII31-p53
interaction is required for the inhibitory function TAFII31
inhibitory function on mdm2-mediated p53 degradation. To further
confirm this observation, we investigated the effect of mutant
TAFII31 on mdm2-induced p53 ubiquitination (Fig.
3D). Consistent with p53 stability data (Fig.
3C), expression of TAF-C, but not TAF-N, resulted in a
dose-dependent inhibition of mdm2-mediated p53
ubiquitination. Similarly, the anti-ubiquitination activity of TAF-C
appears to be weaker than its full-length counterpart (Fig.
3D), suggesting that other mechanisms are also involved in
stabilizing p53 by TAFII31 besides direct competition
with mdm2 binding.
Activation of p53-mediated Transcriptional Activation by
TAFII31--
Stabilization of p53 in cells is expected to
result in activation of p53 downstream pathways, including
activation of p53-mediated transcription activity, inhibition of cell
cycle progression, and/or activation of p53-mediated apoptosis. To
elucidate the biological implications of p53 stabilization mediated by
TAFII31, we first analyzed p53 transcriptional activity
using a luciferase reporter containing p53 binding sites from the human
p21/WAF-1 gene (29). Transfection of TAFII31 did not alter
the basal transcriptional activity of the reporter containing p53
binding site (Fig. 4A), whereas cotransfection of p53 and TAFII31 super-activated
the reporter activities in a dose-dependent manner (Fig.
4A). The synergetic activation of the p53 reporter gene by
TAFII31 may be attributed to its dual effects on p53
stability and its function as a p53 coactivator. The effect of
TAFII31 mutants on p53-mediated transcription activation
was also tested in similar assays. In contrast to the wild type
protein, both mutants inhibited p53-activated transcription
(Fig. 4B). Furthermore, these mutants inhibit wild type
TAFII31-mediated p53 transcription activation (Fig.
4B). Because TAFII31 functions as a bridge to
link p53 to the transcriptional machinery (26, 27), our data suggest
that expression of TAF mutants disrupts the formation of p53
transcription complex and thus blocks p53-mediated transcriptional
activity.
Forced Expression of TAFII31 Induces
p53-dependent Growth Suppression--
To further explore
the consequences of activation of p53-mediated transcription by
TAFII31, we used colony formation assay to monitor whether
expression of TAFII31 could affect cell growth (Fig.
4C and Table I). Transfection
of p53 into either 12.1 cells (p53 wild type) or 10.1 cells (p53-null)
resulted in a 8-fold reduction in plating efficiency, as a result of
p53-mediated growth arrest in both cell types. Transfection of
TAFII31 into 12.1 cells resulted in a 7-fold decrease in
colony formation. This effect was not seen in 10.1 cells. These data
suggest that the ability of TAFII31 to reduce the number of
colonies formed is mediated by p53. fluorescence-activated cell sorter
analysis of cell transiently transfected with TAFII31
further confirmed this finding. Expression of TAFII31
causes accumulation of cells in G1 and reduction of cells
in S phase of the cell cycle in a p53-dependent manner
(Fig. 4D). These results demonstrate that expression of
TAFII31 leads to activation of p53-mediated growth
arrest.
Association of TAFII31 with p53 in Cells after UV
Treatment--
Because forced expression of TAFII31 leads
to stabilization of p53 and subsequent growth suppression, the
association of p53 and TAFII31 in cells was then explored.
Analysis of the p53-TAFII31 complex was carried out in
Swiss3T3 cells before and after DNA damage (UV irradiation).
Immunoprecipitation using monoclonal antibodies against p53 and
immunoblot with antibodies to TAFII31 identified
TAFII31 bound to p53 after UV exposure. The amount of
TAFII31 protein associated with p53 was proportional to the levels of p53, whereas the levels of mdm2 associated with p53 were
inversely correlated with those of TAFII31 (Fig.
5). TAFII31 was in a complex
with p53 in untreated cells, because TAFII31 was
immunoprecipitated by anti-p53 antibody at time 0 where low levels of
p53 is expressed. This band is specific, because it is not present in
10.1 cells which lack p53 (data not shown). The increasing
TAFII31 associated with p53 after UV was not due to an
increase in TAFII31 levels as a result of UV exposure,
because the total TAFII31 protein did not change before and
after UV treatment. These results suggest that TAFII31
contributes to the stabilization of p53 after DNA damage.
We have demonstrated in this study that expression of the
coactivator protein TAFII31 contributes to the regulation
of p53 stability through mdm2-mediated p53 ubiquitination pathway.
Stabilization of p53 by TAFII31 leads to functional
activation of p53-mediated transcriptional activation and induction of
cell cycle inhibition, suggesting that TAFII31 contributes
positively to p53 functions in cells. Although most of the experiments
in this study are performed in transient transfection assays which
result in overexpression of proteins, the fact that there is a positive
correlation between endogenous p53 stabilization and
p53-TAFII31 association after DNA damage (Fig. 5) suggests
that TAFII31 plays an important role in the regulation of
p53 activity in vivo. This is consistent with the
observation that expression of TAFII31 induces growth arrest mediated by endogenous p53 (Fig. 4, C and
D, and Table I).
The mdm2 protein regulates both the activity and levels of p53
through a negative feedback loop. The TAFII31-p53
interaction, on the other hand, seems to form a positive loop to
activate p53 and its downstream pathways. It appears that
TAFII31 can directly inhibit mdm2-p53 interaction and
stabilize p53 as suggested in the in vitro ubiquitination
assay (Fig. 2A). It is further supported by the fact that
p53-TAFII31 interaction is required for
TAFII31-mediated p53 stabilization (Fig. 3). As a
transcription coactivator, TAFII31 may recruit p53 to the
transcription complex (26, 27), which leads to transcriptional
activation of p53 as seen in Fig. 4A. The recruitment of
p53 to the transcription complex by
TAFII31 may also protect p53 from the mdm2-mediated
degradation. These properties of TAFII31 reinforce its
function as coactivator of p53. Although the mechanistic basis of
regulation of p53 function by endogenous TAFII31 requires
additional investigation, the finding that the effect of
TAFII31 on p53 requires protein-protein interactions suggests that TAFII31 may directly compete with mdm2
for p53 binding, and a delicate balance between mdm2 and
TAFII31 determines the levels and stability of p53.
TAFII31 is part of the TFIID complex, containing TBP and
many other TAFs. It has been shown that TAF-TAF interactions are highly
conserved to allow interchange between homologues of TAFs from
different species in transcription reactions in vitro (26, 32). Sequence comparison of Drosophila dTAFII40
and human TAFII31 as well as dTAFII60 and
TAFII70 showed that the amino-terminal region is highly
conserved (data not shown), suggesting that this region may be used for
TAF-TAF interactions. Consistent with this notion, it has been shown
that the conserved amino-terminal domain of TAFII70
interacts with TBP, TAFII250, and TAFII31 (33).
The fact that the carboxyl half of TAFII31 contains the p53
binding domain further supports this possibility (Fig. 3). Thus,
TAFII31 functions as a bridge between p53 and the general
transcription machinery using its carboxyl terminus to interact with
p53 while binding to TFIID using its amino-terminal domain.
Although TAFII31 enhances p53-mediated transcription
activation, the mutant TAFII31 lacking either amino or
carboxyl terminus cannot form proper transcription complex and inhibits
p53 transcription activity (Fig. 4). The dominant negative effects of
TAFII31 mutants on p53 transactivation suggest that
TAFII31 plays an important role in mediating p53
transcriptional activity in vivo. Recent studies showed that
various TAF complexes (with or without TBP) mediate distinct functions,
such as transcription activation (34-36) and histone acetylation (37,
38). The findings presented in this report that TAFII31 can
stabilize p53 further expand the growing list of functions mediated by
the coactivator proteins.
It appears that the p53 stability is regulated by multiple factors in
cells. Besides mdm2 and TAFII31, many other proteins have been shown to regulate p53 stability, such as WT-1 (7), JNK (11),
HIF (12), p19ARF (13), and p300 (14). How TAFII31 interacts
with these proteins in stabilizing p53 is unknown. It is possible that
the balance between these factors determines p53 levels. The emerging
model suggests that the contributions of different factors to p53
stability may depend on cell context, cell cycle, and cell stressing
signals. One example is the finding that JNK appears to regulate p53
ubiquitination in nonstressed cells at the G0/G1 phase of the cell
cycle, whereas mdm2 controls the stability of p53 at S and
G2/M (11).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
S (Sigma), and 33% RL (v/v) in a total volume of
30 µl at 30 °C for 5 min. The reaction was stopped by adding 0.5 ml of 8 M urea in sodium phosphate buffer (pH 6.3) with
0.1% of Nonidet P-40. The beads were washed three times with the stop
buffer and once with PBS supplemented with 0.5% Triton X-100, and the
protein moiety was eluted with Laemmli sample buffer at 100 °C and
subjected to Western blotting as described (11). For the in
vivo ubiquitination assay, the cells were transfected with his-p53
(1 µg), mdm2 (3 µg), TAFII31 (3 µg), and HA-ubiquitin
(1 µg). The cells were treated with MG132 (10 µM,
CalBiochem) for 12 h at 48 h after transfection and lysed in
kinase buffer. The p53-ubiquitin complex was precipitated by nickel-nitrilotriacetic acid beads and analyzed on SDS-polyacrylamide gel electrophoresis as described (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stabilization of p53 by
TAFII31. A, the p53-deficient murine Balb/c
3T3 fibroblast 10.1 cells were transiently transfected with DNA
encoding p53 (1 µg), mdm2 (2 µg), and TAFII31 (2, 5, and 10 µg). The transfected cells were harvested 48 h after
transfection. Half of the material was processed to determine the level
of p53 protein by immunoprecipitation with pAb421 and immunobloting
using polyclonal antisera to p53. The other half was used for RNA
preparation to assess p53 mRNA levels via Northern
blots. In a separate transfection, cells were analyzed for
TAFII31 expression by Western blots using monoclonal
antibody against HA epitope. B, increase of p53 half-life by
TAFII31. 10.1 cells were transfected with p53,
TAFII31, and mdm2. The cells were pulse-labeled with
[35S]methionine for 10 min and chased at the time
indicated. P53 was immunoprecipitated by pAb421. C,
stabilization of p53 by TAFII31 is
mdm2-dependent. Immortalized fibroblast cells from
p53/mdm2 double knockout mice were cotransfected with p53
expressing plasmid (1 µg) and TAFII31 (2-10
µg). The levels of p53 were detected by immunoprecipitation-Western
blotting.
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Fig. 2.
TAFII31 inhibits
mdm2-induced p53 ubiquitination. A,
TAFII31 blocks mdm2-mediated p53 ubiquitination
in vitro. Recombinant hisp53 proteins, purified
mdm2 (11), and the TAFII31 protein produced using an
in vitro transcription and translation kit (Promega) were
added to a ubiquitination reaction containing reticulocyte lysates
predepleted of mdm2 and supplemented with bacterially produced
HA-tagged ubiquitin, ATP and ATP S, as described (19, 39). The degree
of ubiquitination was measured using immunoblots probed with anti-HA
antibody. The p53-ubiquitin is represented as smears of more slowly
migrating proteins in the upper part of the membrane. B,
inhibition of p53 ubiquitination by TAFII31 in
vivo. 10.1 cells were transfected his-p53 (1 µg), HA-ubiquitin
(1 µg), mdm2 (3 µg), and TAFII31 (3 µg) in
various combinations. The p53 complex was captured on nickel beads, and
p53-ubiquitin was detected by Western blots against HA. Levels of p53
were determined using antibody against p53 on a Western blot.
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Fig. 3.
TAFII31-p53 interaction is
required for stabilization of p53. A, expression of
TAFII31 and mutants. Cells transfected with indicated
plasmids or vector alone (control) were lysed. TAFII31
proteins were detected using anti-HA antibody on a Western blot. The
TAF-C expresses amino acids 130-264 of TAFII31 and TAF-N
produces amino acids 1-141. B, association of
TAFII31with p53. Cells were transiently transfected with
indicated plasmids or vector alone (control). The p53 protein was
immunoprecipitated using monoclonal pAb421 antibodies from 1 mg of
protein lysates. A polyclonal p53 antibody was used for p53 detection.
Associated TAFII31proteins were detected using a HA
antibody. C, expression of carboxyl-terminal half of
TAFII31 inhibits mdm2-induced p53 degradation The DNA
expressing p53 (1 µg), mdm2 (3 µg), TAFII31 (3 µg), TAF-C (3 µg), TAF-N (3 µg), and vector control (3 µg) were
transfected in various combinations into 10.1 cells. The levels of p53
were detected by immunoprecipitation-Western blots and
mdm2, and TAFII31 and mutants were detected by Western
blots. D, effects of TAFII31 mutants on
mdm2-mediated p53 ubiquitination. 10.1 cells were transfected
hisp53 (1 µg), HA-ubiquitin (1 µg), mdm2 (3 µg),
TAFII31 (3 µg), TAF-N (3 µg), and TAF-C (1 and 3 µg)
in various combinations. The ubiquitinated p53 was detected by Western
blotting using an antibody against HA after precipitation with nickel
beads. Aliquots of the cell lysates were examined for mdm2 and p53
expression by Western blots.
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Fig. 4.
TAFII31 activates p53-mediated
transcriptional activation and growth arrest pathway. Saos-2 cells
were transfected with 2 µg of the p53 reporter vector, 1 µg of pCMV
or pCMV-p53wt, and increasing amounts of TAFII31 expressing
vector (0.1, 0.5, and 2 µg). A corresponding appropriate amount of
empty vector was included to maintain a total of 2 µg in all
transfections. Cells were maintained at 37 °C and assayed to
determine luciferase units/µg protein. The indicated values are the
average of two independent experiments that were performed in
duplicate. A, expression of TAFII31 has no
effect on basal transcription and enhances transcription via the p53
response element in the p21 promoter. Fold activation was
calculated as the fold increase in luciferase units/µg protein
relative to reporter alone for the effect on basal transcription. The
effect on p53-activated transcription was calculated as the fold
increase relative to reporter and TAFII31 plasmids in the
presence and absence of p53 expression plasmid. B, effect of
TAFII31 mutant on p53 activated transcription and
TAFII31-mediated p53 transcriptional activity. All activities were normalized to
reporter alone. C, TAFII31 suppresses colony
formation in a p53-dependent manner. 10.1 (no p53) and 12.1 (wild type p53) cells were transfected with neo vector, p53,
TAFII31 (2 µg each). The Geneticin-resistant colonies
were counted 2-3 weeks after transfection and stained with the Giemsa
stain. D, effect of p53 and TAFII31 expression
on cell cycle progression. 10.1 and 12.1 cells were transiently
transfected with control vector, p53, and TAFII31 DNA, as
indicated. A GFP expressing plasmid was cotransfected to mark the
transfected cells. The cells were subjected to fluorescence-activated
cell sorter analysis, and GFP positive cells were analyzed for DNA
content. Cell cycle distribution for each fluorescence-activated cell
sorter was presented. The experiments were repeated, and data represent
one set of the experiments.
p53-dependent suppression of cell plating efficiency by
TAFII31
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Fig. 5.
Association of TAFII31 with p53
in Swiss 3T3. Swiss 3T3 cells were treated with UV at
50J/m2. The cells were harvested at the indicated time
points. Lysates were immunoprecipitated by pAb421, and the membrane was
blotted with anti-p53, anti-mdm2, or anti-TAFII31
antibodies. Purified TAFII31 served as a positive control
(pos. cont.). Amount of TAFII31 in untreated and
UV-treated cells were analyzed using whole cell extracts
(WCE), and 100 µg quantities of lysates were subjected to
Western blotting analysis using the anti-TAFII31 antibody.
IP, immunoprecipitation; IB, immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Stuart Aaronson, David Sassoon, Z. Q. Pan, and J. Licht for helpful discussions, Dr. S. Jones at University of Massachusetts for the gift of p53/mdm2 double-null fibroblasts, and Dr. Z. Luo at Boston University for providing the pMTN plasmid.
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FOOTNOTES |
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* This work was supported by a NCI, National Institutes of Health Grants P01 CA80058 (to Z. R. and X. W.), CA78419 (to Z. R.), and CA 69161 (to J. J. M.).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.
Supported by National Institutes of Health and American Cancer
Society funds.
** To whom correspondence should be addressed: The Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030. E-mail: xiangwei@ bcm.tmc.edu.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M007955200
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ABBREVIATIONS |
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The abbreviations used are:
TBP, TATA-binding
polypeptide;
TAF, TATA-binding polypeptide-associated factor;
HA, hemagglutinin;
ATPS, adenosine
5'-O-(thiotriphosphate);
PBS, phosphate-buffered saline;
GFP, green fluorescent protein.
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REFERENCES |
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