From the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870
Received for publication, January 6, 2001, and in revised form, February 2, 2001
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ABSTRACT |
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The Tax protein, encoded by the human T-cell
leukemia virus type I (HTLV-I), is required for high level viral
transcription and HTLV-I-associated malignant transformation. Although
the precise mechanism of malignant transformation by Tax is unclear, it
is well established that Tax represses the transcription function of
the tumor suppressor p53, possibly accelerating the accumulation of
genetic mutations that are critical in HTLV-I-mediated malignant transformation. Tax repression of p53 transcription function appears to
occur, at least in part, through competition for the cellular coactivator CBP/p300. In this study, we characterize the effect of Tax
on the p53 family member, p73. We demonstrate that Tax also represses
the transcription function of p73 Human T-cell leukemia virus type I
(HTLV-I)1 is the etiological
agent of adult T-cell leukemia (ATL) which is an aggressive and fatal
hematological malignancy (1, 2). Only a small percentage of people
infected with HTLV-I develop ATL, generally 20-40 years following
infection (3). The infrequency of ATL, coupled with the long latency
period, suggests that ATL occurs as a consequence of multiple genetic
mutations that accumulate during the prolonged period of asymptotic
HTLV-I infection. Although the molecular basis of HTLV-I pathogenesis
is not well understood, there is strong emerging evidence that
expression of the viral trans-activator protein Tax plays an essential
role in the oncogenic process. Indeed, the expression of Tax is able to
immortalize primary T-cells (4), transform rat fibroblasts in
vitro (5), and promote tumorigenesis and leukemogenesis in a mouse
model (6).
With the goal of understanding the role of Tax in leukemogenesis,
several studies have examined how Tax affects proteins involved in
regulation of the cell cycle (for review see Ref. 7). These proteins
include the cyclin-dependent kinase inhibitors
p16INKA4, p18INK4A, p21Waf1/CIP1,
and p27Kip1 (8-13), cyclin D (8, 14, 15), the
transcription factors E2F-1 and E2F-2 (16-19), and the tumor
suppressor protein p53 (12, 20-30). p53 is a transcription factor that
is activated in response to genotoxic stress. Once activated, p53
induces expression of genes critical for cell cycle arrest or
apoptosis, thus preventing the transmission of genetic mutations to
progeny cells (31). Loss of p53 activity has been found in 60% of
human malignancies examined (32, 33), consistent with a role for p53 in
genome surveillance and suppression of malignant transformation.
Most interesting, in HTLV-I-infected and Tax-expressing cells, p53 is
present at elevated levels, with a relatively low frequency of mutation
(~25%) (12, 20, 21, 30, 34, 35). Paradoxically, several studies have
demonstrated that although generally wild type, p53 is functionally
inactive. For example, HTLV-I-infected cells do not respond
appropriately to a variety of p53 stimuli, including gamma and ionizing
irradiation (12, 22, 36, 37). Furthermore, Tax expression alone
abrogates p53-induced G1 arrest and apoptosis following DNA
damage (24) and inhibits the activation of a panel of known
p53-responsive genes (22). Several recent studies provide evidence
showing that Tax inhibition of the tumor suppressor activities of p53
is directly due to Tax inhibition of p53 transcription function (21,
22, 24).
It is well established that Tax does not directly bind p53 (12, 22, 27,
36, 38); thus, Tax appears to compromise p53 function via an indirect
mechanism. Several reports have indicated that Tax repression of p53
may occur through alterations in the phosphorylation state of p53 (23,
39). Alternatively, other studies indicate that the repression occurs
as a consequence of competition between Tax and p53 for the cellular
coactivators CBP/p300 (25-27). Both Tax and p53 utilize the cellular
coactivators CBP/p300 as mediators of transcriptional activation
(40-44). The site of competition on CBP/p300 appears to be the KIX
domain (amino acids 588-683), as both Tax and p53 have previously been
shown to bind to this region, and their binding is mutually exclusive (25, 27). This evidence suggests that Tax may attenuate p53 function by
abrogating the p53-KIX interaction, thus reducing the capacity of p53
to recruit CBP/p300 to target promoters.
Recently, a new member of the p53 gene family has been identified,
known as p73 (45). p73 is also a transcriptional activator protein and
carries an amino-terminal activation domain with moderate homology
(29%) to p53 (45, 46). At least 6 isoforms of p73 ( p73 has been classified as a possible tumor suppressor based on
substantial sequence homology and functional similarity with p53. This
classification was strengthened by the observation that the
p73 gene maps to chromosome 1p36, a region frequently
deleted in several malignancies including neuroblastomas, colorectal
cancers, and breast cancers (45). p73 activity is induced by exposure of cells to DNA-damaging agents such cisplatin, taxol, and
In this report, we investigate the function of p73 in
HTLV-I-transformed, Tax-expressing T-cell lines. We demonstrate that in
the presence of Tax the stability of p73 Cell Culture, Transient Cotransfection Assays, and Mammalian
Expression Plasmids--
HTLV-I-negative T-cells (CEM, HUT-78, Jurkat,
and Molt-4) and HTLV-I-transformed T-cells (C8166/45, C91/PL, MT2,
SLB-1, and HUT-102) were cultured in Iscove's modified Dulbecco's
medium supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and penicillin/streptomycin. The HCT-116 cells
and 293 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and penicillin/streptomycin. For transient
cotransfection assays (69), cells were grown to a density of
106 cells/ml and transfected with LipofectAMINE (Life
Technologies, Inc.) and a constant amount of DNA. After 24 h, the
cells were harvested and lysed, and luciferase activity was measured
using the Dual-Luciferase reporter assay system (Promega) with a Turner Designs model TD 20-e luminometer. Luciferase activity was normalized to Renilla luciferase from herpes simplex virus
thymidine kinase promoter (pRL-TK, Promega). Expression plasmids for
p73 Expression and Purification of Recombinant Proteins, in Vitro
Translation--
GST, GST-C/H1-(302-451), GST-C/H1-KIX-(302-683),
GST-KIX-(588-683), GST-CBP-(1514-1894), GST-CBP-(1894-2221), and
GST-CBP-(2212-2441) were expressed and purified as described
previously (25). Briefly, the DNA was transformed in BL21(DE3) pLysS,
expanded, induced, and purified by glutathione-agarose affinity
chromatography. Tax protein was expressed from the pTaxHis6 (75) and
pTet-K88A (73) expression plasmids and purified as described previously
(40). Briefly, the DNA was transformed in BL21(DE3) pLysS, expanded, induced, and purified to greater than 90% homogeneity by
Ni2+-nitrilotriacetic acid-agarose chromatography (Qiagen).
Purified Tax protein was dialyzed against TM 0.1 M KCl
buffer containing 50 mM Tris (pH 7.9), 12.5 mM
MgCl2, 100 mM KCl, 1 mM EDTA (pH 8), 1 mM dithiothreitol, 0.025% (v/v) Tween 20, and 20%
(v/v) glycerol, aliquoted, and then stored at Western Blot Analysis--
For whole cell extracts, cultured
cells were washed with cold phosphate-buffered saline and then lysed in
RIPA buffer (50 mM Tris-HCl, pH 8, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cells were incubated for 15 min at 4 °C, the lysates centrifuged, and the supernatant aliquoted and stored at Half-life Determination--
Cycloheximide chase experiments
were performed as described by Maki and Howley (76). Briefly, T-cells
were seeded at 2 × 106 cells/ml and adherent cells at
confluence in 100-mm dishes in their respective culture medium.
Cycloheximide (Sigma) was dissolved in absolute methanol and added
directly to the culture media to a final concentration of 20 µg/ml.
Cells were harvested at different time points after cycloheximide
treatment, washed with phosphate-buffered saline, and then whole cell
extracts were made by using RIPA buffer. Lysates of selected cells were
normalized for protein content by the Bradford assay, and 100 µg of
proteins were Western-blotted with anti-p73 GST Pull-down Assays--
All GST pull-down experiments were
performed using 20 µl of glutathione-agarose beads equilibrated in
pull-down buffer (20 mM Hepes, pH 7.9, 0.5 mM
EDTA, pH 8, 10% glycerol, 0.05% Nonidet P-40, 5 µM
ZnSO4, 2.5 mM MgCl2, 25 mM KCl, 1 mM dithiothreitol). The purified GST
fusion protein was incubated with the beads for 1-2 h at 4 °C and
then washed twice with pull-down buffer. The second protein(s) was then
added to the washed beads and incubated overnight at 4 °C. The beads
were washed four times, and bound proteins were eluted with SDS sample
dyes and resolved by electrophoresis on a 10% SDS-polyacrylamide gel.
To detect the binding of radiolabeled p73 p73 p73
The viral protein Tax has previously been shown to increase directly
p53 protein stabilization (21, 22), although the mechanism for this
stabilization is unknown. To determine if Tax is directly involved in
p73 Tax Inhibits the Transcription Function of p73
The Tax protein utilizes the coactivator CBP to mediate transcriptional
activation of the HTLV-I promoter (40, 41). Recently, two groups have
reported that p73
As an alternate means to examine whether competition for a common
coactivator may participate in Tax repression of p73, we next examined
whether overexpression of p73 p73
To determine whether Tax might also bind to C/H1, we tested purified
recombinant Tax protein in a GST pull-down assay with the C/H1 domain.
As shown in Fig. 5C, both wild type Tax and KIX binding
defective K88A Tax bound well to the C/H1 domain (lanes 5 and 6). Together, these observations indicate that both Tax and p73 p73 In this report, we characterize the effect of the HTLV-I-encoded
oncoprotein Tax on the functionality of the p53 family member p73.
These studies were undertaken as several previous reports have shown
that Tax has dramatic effects on both the stability and transcription
function of p53. We show that, like p53, Tax represses the
transcription function of p73 while paradoxically enhancing the
stability of the protein. We demonstrate that the transcriptional
repression is reciprocal, as p73 represses the transcription function
of Tax. The molecular basis of the reciprocal repression appears to be
competition for the cellular coactivators CBP/p300, as both p73 We also show that p73 is overexpressed in HTLV-1-transformed T-cell
lines, due to an apparent increase in the half-life of the p73 protein.
This stability is directly due to the viral oncoprotein Tax. This
observation is supported by a prolonged p73 half-life in the
HTLV-I-transformed cell line C8166/45, which expresses the viral
protein Tax but not Rex (81). Furthermore, transient transfection
studies show that cotransfection of Tax promotes elevated p73 protein
levels, relative to that observed in the absence of Tax. The mechanism
of Tax stabilization of p73 remains unknown. In previous studies
examining the p53 degradation pathway, it has been suggested that the
interaction of p53 with the C/H1 domain CBP/p300 promotes degradation
(82). It is plausible that p53 and p73 competition with Tax for CBP
binding could potentially repress the degradation pathway, resulting in
an increase in the half-life of both proteins. The fact that CBP is
limiting in cells provides further support for a mechanism that
involves competition (83). Alternatively, Tax may contribute to higher
levels of p73 protein levels through transcriptional activation of the
p73 gene. Although this would not account for the observed
increase in the p73 half-life, precedence for this idea comes from the observation that the transcription factor E2F-1 has been shown to
stimulate p73 gene expression (68, 84, 85). Since Tax increases the level and activity of E2F-1 in HTLV-I-transformed T-cells
(16, 18), enhanced E2F levels may directly increase transcription of
the p73 gene.
Although p73 is present at elevated levels in Tax-expressing
HTLV-I-infected T-cells, it is functionally compromised for
transcription activation. Several possibilities might account for this
loss of p73 transcription function in the presence of Tax. For example, some viruses, such as adenovirus or hepatitis B, encode proteins that
bind and sequester p53 in the cytoplasm (86, 87). This is not the case
for Tax repression of p73, as we observe generally high levels of p73
in nuclear extracts (data not shown). This is consistent with a recent
study indicating that Tax and p73 Currently, Tax is known to bind the KIX domain and the
carboxyl-terminal SRC-interacting domain of CBP, with both interactions apparently contributing to Tax transactivation of HTLV-I transcription (40, 41).2 Interestingly, the
data presented herein indicate that Tax additionally interacts with the
C/H1 domain of CBP, perhaps further contributing to Tax transcription
function. The C/H1 domain, named for its cysteine/histidine-rich
domain, is composed of two zinc finger modules (88) that have recently
been shown to be involved in interactions with several proteins,
including two viral proteins (89, 90). C/H1 is located immediately
amino-terminal to the KIX domain, and this contiguous C/H1-KIX region
(aa 302-683) may form a strong binding platform for Tax. This idea is
supported by our observation that C/H1-KIX bound with high affinity
(relative to either domain alone) to the ternary complex containing
Tax, CREB, and the viral CRE
DNA.3 These data suggest that
multiple, distinct Tax-CBP interactions occur simultaneously and
perhaps cooperate to enhance coactivator-mediated transcriptional activation.
Several recent studies (45, 48, 52, 56-59) have shown that p73
promotes apoptosis in p53-deficient cells. Our observation that Tax
inhibits p73 transcription functions would suggest that Tax also
represses p73-mediated apoptosis. Thus, the inactivation of both p73
and p53 by Tax may together contribute to HTLV-I-dependent leukemogenesis. Although Tax levels are generally low in
HTLV-I-infected cells (91), intermittent high levels of Tax may
saturate multiple sites on CBP/p300, derailing both p53 and p73
transcription function. This event would likely promote an environment
in the infected T-cell that is tolerant of mutations and chromosomal
instability and resistant to apoptosis. This environment would promote
the survival of genetically compromised cells poised for further
genetic damage and malignant transformation.
and that the repression is
reciprocal in vivo, consistent with the idea that both
transcription factors may compete for CBP/p300 in vivo. We provide evidence showing that both Tax and p73 interact strongly with
the C/H1 domain of CBP and that their binding to this region is
mutually exclusive in vitro. This finding provides evidence supporting the idea that reciprocal transcriptional repression between
Tax and p73 is mediated through coactivator competition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
,
,
,
, and
) may be generated through alternative splicing of the
primary p73 transcript (47-49). The p73
and p73
proteins are
ubiquitously expressed at low levels in many tissues. An exception is
T-lymphocytes, which lack p73
, but express p73
(48).
Interestingly, one form of p73, which lacks the transactivation domain,
has been shown to be pro-apoptotic in neurons (50, 51). Specific p53 mutant proteins have been shown to associate with p73 (
,
,
, and
), resulting in interference with p73 transcriptional activity and the ability to induce apoptosis (52-54). As predicted from local
regions of strong homology in the DNA binding domains of both proteins,
p73 binds the canonical p53-binding sites (55). Furthermore, when
ectopically expressed, p73 can trans-activate a variety of p53
promoters (p21, bax, mdm2, and
GADD45) and induce apoptosis in p53-deficient cell lines
(45, 48, 52, 56-59). Similar to p53, the activation domain of p73
interacts with the cellular coactivators CBP/p300 (60); however, the
site of interaction appears to differ. Whereas p53 interacts with a
carboxyl-terminal region and the KIX domain (25, 43), p73 has recently
been shown to bind the C/H1 domain of CBP, located near the amino
terminus of the coactivator (60).
-irradiation (61-64). Furthermore, in lymphoid malignancies there
is evidence that hypermethylation, and thus silencing, of the
p73 gene may play a role in the development and/or
progression of the neoplasm (65, 66). Loss of a single p73 allele has
been linked to
-radiation-induced T-cell lymphomas (67). A recent
study suggests that deregulated E2F-1 induction of p73 might promote a
p53-independent, tumor suppressor pathway and that perhaps many of the
previously attributed "p53-independent" anti-tumorigenic activities
may be due directly to p73 (68). Despite these observations, other
studies have failed to show mutations in the p73 gene in a
wide variety of tumor types. Normal expression of p73 is observed in
many malignant and non-malignant cells, and some cancers actually shown
increased levels of p73. In contrast to p53-deficient mice,
p73
/
mice show no increased susceptibility
to spontaneous malignancies. These conflicting results suggest that
altered expression of the p73 gene, rather than a loss of
p73 function, may play a role in malignant transformation.
is increased, yet, paradoxically, the transcription function of p73
is decreased. We
also show that in the presence of p73 the transcription function of Tax
is reduced, indicating that the transcriptional repression is
reciprocal. The reciprocal transcriptional repression appears to arise
from intracellular competition for CBP/p300, an observation very
similar to that reported for Tax repression of p53 (25-27). Although
these effects of Tax on p73 function are very analogous to the effects
of Tax on p53 function, we provide evidence indicating that the site of
competition on the surface of CBP/p300 differs. We demonstrate
biochemically that both Tax and p73
bind strongly to the C/H1 domain
of CBP and that the binding of the two transcription factors is
mutually exclusive in vitro. These data suggest that Tax may
inactivate p73
transcription function by specifically competing for
the p73-C/H1 interaction in vivo. Disruption of both the p53
and p73
interactions with CBP/p300 in HTLV-I-infected cells may
alter the transcription function of these key regulatory proteins.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(pcDNA3.p73
; Ref. 70), p53 (pC53-SN3; Ref. 71), Tax
(pHTLV-Tax; Ref. 72) and K88A (CMV-K88A; Ref. 73) have been previously
described. The luciferase reporter plasmids pG13-Luc (74), viral
CRE-Luc (40) have also been described.
70 °C. In
vitro translation of p73
protein was performed using the TNT
translation system (Promega) according to the manufacturer's protocol.
70 °C. For nuclear extract preparation, cultured cells were washed with cold
phosphate-buffered saline, resuspended in lysis buffer (20 mM Hepes-KOH, pH 7.6, 20% glycerol, 10 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.1% Igepal, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride), and
incubated on ice for 10 min. The lysate was centrifuged for 5 min at
800 × g, and the nuclei were resuspended in SDS sample
buffer. For Western blot analysis, the whole cell and nuclear extracts
were electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies against p73
(Ab-3, Santa
Cruz Biotechnology), p53 (DO-1; Santa Cruz Biotechnology), or Tax
(rabbit immune serum recognizing the carboxyl-terminal domain of Tax).
antibody.
, the gel was dried and
analyzed by PhosphorImager analysis. To detect the binding of Tax,
proteins were transferred to nitrocellulose and probed with an
anti-His6 antibody (H-15; Santa Cruz Biotechnology).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Is Overexpressed in HTLV-I-transformed T-cell Lines--
It
is well established that the HTLV-I Tax protein promotes stabilization
of endogenous p53. To determine whether Tax might similarly stabilize
p73, we examined the steady-state levels of p73 in whole cell extracts
from the following Tax-expressing, HTLV-I-transformed cell lines:
C8166/45, C91/PL, MT2, SLB-1, and HUT-102 (Fig.
1, lanes 1-5). For
comparison, we examined p73 expression levels in the following
uninfected human leukemic T-cell lines: CEM, HUT-78, Jurkat, and Molt-4
(Fig. 1, lanes 7-10). Kidney 293 cells, which express high
levels of the p73
isoform (55), were used as a positive control
(Fig. 1, lane 6). Each of the HTLV-I-transformed cell lines,
with the exception of HUT-102, had detectable levels of p73
(Fig. 1,
lanes 1-5). Interestingly, we were able to detect p73
in
HUT-102 nuclear extracts (data not shown). In contrast, we did not
detect p73
expression in any of the uninfected T-cell lines (Fig. 1,
lanes 7-10). For comparison, we also measured p53 expression levels and found that, as previously reported, p53 was
detectable in all of the HTLV-I-transformed cell lines (12, 20, 22, 24,
30, 35, 37) as well as uninfected CEM and MOLT-4 (77) (Fig. 1,
lanes 11-20). We also examined Tax expression levels in the
HTLV-I-infected cell lines (Fig. 1, lanes 21-25). Most
interesting, we observed a direct correlation between higher p73
levels and the presence of the native 40-kDa form of Tax (Fig. 1,
lanes 21-25). The larger forms of Tax detected in MT-2 and
HUT-102 cells appear to be a fusion protein that carries both envelope
and Tax sequences (78). The correlation between expression of this Tax
fusion protein and reduced p73 levels suggest that the fusion protein
is defective for p73 stabilization. Together, these observations
suggest that Tax may play a role in p73
stabilization. We have also
examined whether the nuclear localization of p73 is affected in HTLV-I
cell lines. In all cell lines examined, we detected generally high
levels of p73
in the nucleus, suggesting that the localization of
p73
is unaffected in HTLV-I transformed T-cells (data not
shown).
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Fig. 1.
Overexpression of p73
in HTLV-I-transformed T-cells. Total cell extracts (100 µg) from five HTLV-I-infected T-cell lines were analyzed for p73
expression by Western blot analysis (lanes 1-5). For
comparison, four uninfected human T-cell lines were examined in
parallel (lanes 7-10). As a positive control, Western blot
analysis of p73
expression in 293 cells was performed (lane
6). The blots were reprobed with both anti-p53 (lanes
11-20) and anti-Tax antibodies (lanes 21-25). The
high molecular weight forms of Tax, produced by fusion of Tax with
Env-coding sequences (78), are indicated by an
asterisk.
Is Stabilized by Tax--
Previous studies have shown that
the half-life of p53 is significantly increased in HTLV-I-transformed
T-cells (20) and HTLV-I-immortalized T-cells (21). To determine if an
increase in the p73 half-life might account for the elevated protein
levels observed in HTLV-I-infected cell lines, we measured p73
stability in SLB-1 and C8166/95 cells, the two HTLV-I-transformed lines that expressed a high level of p73
protein (see Fig. 1). Cells were
treated with cycloheximide for various lengths of time, and p73
levels were measured by Western blot analysis. As a control, we
determined the half-life of p73
in uninfected HCT-116 cells, as they
have previously been used in p73
half-life studies (62). Representative Western blots of p73
levels following cycloheximide treatment in SLB-1 and C8166/45 are shown in Fig.
2A. Graphical representation
of the data is presented in Fig. 2B. In the control HCT-116
cells, we found that, like p73
(62), the p73
half-life in these
cells was 45 min (Fig. 2B, left panel). Most interesting, the p73
half-life in SLB-1 and C8166/45 was measured at 210 and 165 min, respectively (Fig. 2B, center and right
panels). These data correspond to a p73
half-life increase of
~4-fold in these HTLV-I-infected T-cells.
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Fig. 2.
p73 half-life
determination in HTLV-I-infected T-cells. A,
cycloheximide was added to a final concentration of 20 µg/ml to the
HTLV-I-transformed cell lines SLB-1 and C8166/45. At the indicated
times following cycloheximide addition, cells lysates were prepared and
examined by Western blot using an anti-p73
antibody. A
representative Western blot for both cell lines is shown. B,
graphical representation of the half-life data. p73
levels from the
SLB-1 and C8166/45 Westerns, shown in A (averaged with a
second independent experiment), were quantified by densitometry and
represented graphically (normalized to that of the zero time point).
The half-life of p73
in HCT-116 cells is also shown (left
graph). The half-life was determined at 50% remaining p73
protein.
stabilization, we cotransfected expression plasmids for p73
and Tax into human Jurkat T-lymphocytes, and we measured Tax and p73
protein levels by Western blot analysis. We selected the
HTLV-I-negative Jurkat cells, as they are negative for both p73
and
p53 (see Fig. 1, lane 9 and 19, respectively) (79, 80). Fig. 3A shows that
expression of Tax produced a significant increase in p73
protein
levels (lanes 2 and 3). In the absence of Tax, a
small amount of p73
was detected by Western blot, consistent with
the short half-life of the protein (Fig. 3A, lane 1). Both 1 and 2 µg of the transfected Tax expression plasmid produced a
comparable increase in p73 levels, suggesting that the lower amount of
Tax is saturating for p73
stabilization. As a control, Tax
stabilization of p53 is also shown (Fig. 3B).
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Fig. 3.
Stabilization of p73
by Tax. A, an expression vector for p73
(500 ng) (70) was cotransfected into Jurkat cells (lanes 1-3)
with increasing amounts (1 and 2 µg) of an expression vector for Tax
(72) (lanes 2 and 3). After 24 h, cells were
analyzed by Western blot with anti-p73
and anti-Tax antibodies, as
indicated. B, as a control, the same experiment was
performed with an expression vector for p53 (500 ng) (71) (lanes
1-3) and Tax (1 and 2 µg) (lanes 2 and 3)
and probed with anti-p53 and anti-Tax antibodies, as indicated.
--
Several
groups have previously shown that the viral oncoprotein Tax, in
addition to stabilizing p53, represses p53 transcription function (21,
22, 24, 25). Based on these observations, we were interested in
determining whether Tax similarly repressed the transcription function
of p73
. To examine this possibility, we utilized the p53-responsive
reported plasmid, pG13-Luc for these studies, as overexpression of
p73
has been shown to activate many p53-responsive genes (45,
48, 52, 56-59). (Genes that are specifically responsive to p73 have
not yet been identified.) We transfected the pG13-Luc reporter plasmid,
which carries 13 copies of the consensus p53-response element driving
expression of the luciferase gene, into HTLV-I-negative Jurkat T-cells
(Fig. 4A). Consistent with the
observation that Jurkat T-cells do not express p73 or p53 (Fig. 1,
lanes 9 and 19), we did not detect significant
luciferase activity produced from the pG13-Luc reporter plasmid in
these cells (Fig. 4A, lane 1). As expected, cotransfection of the p73
expression plasmid strongly activated transcription from
p53-responsive reporter plasmid (Fig. 4A, compare
lanes 1 and 2). Interestingly, cotransfection of
the Tax expression plasmid produced a dose-dependent
repression of p73
-mediated transcriptional activation, strongly
supporting a direct role for Tax in repression of p73
transcription
function (Fig. 4A, lanes 3-5).
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Fig. 4.
Reciprocal repression of transcription
function between Tax and p73 .
A, Tax represses p73
-activated transcription. The
p53-responsive pG13-luc reporter plasmid (100 ng) (74) was
cotransfected with a constant amount of the p73
expression plasmid
(200 ng) and increasing amounts (200, 400, and 800 ng) of the
expression plasmids for either wild type Tax (lanes 3-5) or
Tax K88A (73) (lanes 6-8), as indicated. Values shown are
the average luminescence ± S.E. from two independent experiments
performed in duplicate. B, Tax K88A is defective for
transactivation. The Tax-responsive viral CRE-luc reporter plasmid (40)
(100 ng) (lane 1) was cotransfected with 200 ng of the
expression plasmid for either wild type (wt) Tax (lane
2) or Tax K88A (lane 3). Values shown are the average
luminescence ± S.E. from two independent experiments performed in
duplicate. C, expression of wild type or mutant K88A Tax
protein in the nucleus. Nuclear extracts from Jurkat cells transfected
with 400 ng of the wild type or mutant K88A Tax expression plasmid
(lanes 2 and 3) were analyzed by Western blot
with an anti-Tax antibody. D, p73
represses Tax-activated
transcription. The Tax-responsive viral CRE-luc reporter plasmid (100 ng) was cotransfected with 200 ng of the expression plasmid for wild
type Tax (lane 2-5) and increasing amounts (200, 400 and
800 ng) of the p73
expression plasmid (lanes 3-5) as
indicated. Values shown are the average luminescence ± S.E. from
two independent experiments performed in duplicate.
also utilizes CBP to mediate transcriptional
activation (59, 60). Since there is significant emerging evidence for
coactivator competition as a mechanism of transcriptional repression
(25-27), we were interested in testing whether CBP competition may
account for the observed Tax repression of p73 transcription function.
To test this hypothesis, we utilized a point mutant of Tax (K88A) that
has previously been shown to be defective for interaction with the KIX
domain of CBP (73) and Tax transactivation in vivo (25, 73).
If Tax repression of p73
is occurring through competition
specifically for the KIX domain of CBP, then Tax K88A should be unable
to repress p73 function. Unexpectedly, cotransfection of mutant Tax
K88A strongly repressed the transactivation function of p73
to a
level greater than that observed with wild type Tax (Fig.
4A, compare lanes 2-8). We then tested whether
K88A was defective for Tax transactivation, as previously described
(73), by examining K88A function on a reporter plasmid carrying three
copies of the Tax-responsive viral CREs (viral CRE-Luc) driving
expression of the luciferase gene (40). Transfection of the wild type
Tax expression plasmid strongly activated transcription from the
Tax-responsive promoter, whereas Tax K88A was defective for activation
(Fig. 4B, lanes 2 and 3). Western blot analysis
of nuclear extracts showed that both the wild type and mutant Tax
proteins were expressed in the transfection assay (Fig. 4C, lanes
1-3). These data suggest that, if Tax repression of p73
is
mediated through coactivator competition, the site of competition
resides outside of the KIX domain of CBP.
similarly repressed Tax function. We
reasoned that if repression of p73
by Tax occurs as a consequence of
competition for CBP, then overexpression of p73
should similarly
repress Tax function. To test this possibility, we performed the
reciprocal experiment using the Tax-responsive viral CRE-Luc reporter
plasmid. As expected, cotransfection of the Tax expression plasmid
strongly activated transcription from the Tax-responsive reporter
plasmid (Fig. 4D, lanes 1 and 2). Consistent with the theory of coactivator competition, cotransfection of increasing amounts of the expression plasmid for p73
repressed Tax transactivation in a dose-dependent fashion (Fig.
4D, lanes 3-5). Together, these data support a mechanism of
reciprocal repression where Tax and p73 bind to a common coactivator,
ultimately recruiting the coactivator to their respective target genes.
and Tax Both Bind to the C/H1 Domain of CBP--
The
transient transfection data suggesting that Tax and p73
were
competing for CBP binding led us to determine whether there might be a
common site on CBP where both transcription factors interact. We
considered the amino-terminal C/H1 domain of CBP, as Zeng et
al. (60) reported, that p73
binds to this domain of CBP. To
confirm this observation, we analyzed the binding of p73
to several
regions of CBP using the GST pull-down assay. Purified GST fusion
proteins carrying various regions of CBP (see Fig.
5A) were bound to
glutathione-agarose beads and then incubated with labeled and in
vitro translated p73
protein. Fig. 5B shows that, as
expected, p73
bound well to the two GST constructs that carried the
C/H1 domain of CBP (lanes 3 and 4). We did not
detect significant binding of p73
to GST alone, KIX (aa 588-683),
or to three carboxyl-terminal regions of CBP, comprising amino acids 1514-1894, 1894-2212, and 2212-2441 (Fig. 5B, lanes 2 and
5-8). These results are consistent with the report by Zeng
et al. (60) who showed that the interaction of the
amino-terminal activation domain of p73
with C/H1 modulates p73
transcription function.
View larger version (44K):
[in a new window]
Fig. 5.
Tax and p73 bind to
the C/H1 domain of CBP. A, schematic illustration of
the CBP protein and positions of the C/H1 and KIX domains.
B, p73
binds to the C/H1 domain of CBP in
vitro. The p73
35S-labeled in vitro
translation product (0.5 µl) was incubated with GST alone or the
indicated GST fusion proteins (10 pmol). Bound p73
and protein
standards are indicated. p73
onput (40%) is shown (lane
1). C, wild type and K88A mutant Tax protein bind to
the C/H1 domain of CBP. Purified wild type and mutant recombinant Tax
proteins (10 pmol) were incubated with 10 pmol of GST alone
(lanes 3 and 4) and GST-C/H1-(aa 302-451)
(lanes 5 and 6). Bound Tax and proteins standards
are indicated. Wild type and mutant Tax onput (20% each) is shown
(lanes 1 and 2).
bind to the C/H1 domain of CBP, possibly to recruit
CBP to their target promoters. This common interaction site on CBP provides support for the hypothesis that Tax and p73
may compete for
CBP in vivo, accounting for the observed reciprocal
transcriptional repression.
and Tax Binding to C/H1 Is Mutually Exclusive--
To test
directly whether the binding of Tax and p73
to C/H1 is mutually
exclusive, we performed a GST pull-down competition assay.
Glutathione-agarose beads bound with GST-C/H1 (CBP aa 302-451) were
incubated with a constant amount of in vitro
transcribed/translated p73
and increasing amounts of purified Tax
(Fig. 6). If the two proteins bind to
C/H1 in a mutually exclusive manner, we reasoned that increasing
amounts of Tax, relative to p73
, would displace p73
from C/H1.
Fig. 6 shows that the coincubation of increasing amounts of purified
Tax protein in the binding reaction reduced p73 binding to C/H1
(compare lanes 3-6, upper panel). The competition was
dose-dependent and corresponded directly with a concomitant increase in Tax binding (Fig. 6A, lanes 3-6, lower panel).
We next tested whether the Tax point mutant K88A, which bound C/H1 similar to wild type Tax, could also compete with p73
for CH/1 binding. As expected, increasing amounts of the Tax point mutant also
dramatically reduced p73
binding to C/H1 (Fig. 6A, lanes 7 and 8). In the reciprocal competition experiment,
GST-C/H1 was incubated with a constant amount of purified Tax protein
and increasing amounts of in vitro transcribed/translated
p73
. Similar to the results obtained above, increasing amounts of
p73
reduced the binding of Tax, with a concomitant increase in
p73
binding to the C/H1 domain (Fig. 6B, lanes 3-6).
Together, these data indicate that the binding of p73
and Tax to
C/H1 is mutually exclusive in vitro, providing a possible
mechanism for the observed repression of p73 by Tax.
View larger version (47K):
[in a new window]
Fig. 6.
Tax and p73 binding
to C/H1 is mutually exclusive. A, Tax inhibits p73
binding to C/H1. The p73
35S-labeled in vitro
translation product (0.5 µl) was incubated with GST alone or
GST-C/H1-(aa 302-451) (10 pmol) in the presence of increasing amounts
of purified recombinant wild type (10, 20, and 40 pmol) or K88A mutant
Tax protein (20 and 40 pmol) (lanes 4-6 and 7 and 8, respectively). p73
and Tax were detected as
described under "Materials and Methods." Protein standards are
indicated. p73
onput (40%) is shown (lane 1).
B, p73
inhibits Tax binding to C/H1. Purified recombinant
Tax protein (10 pmol) was incubated with GST alone or GST-C/H1-(aa
302-451) (10 pmol) in the presence of increasing amount of p73
35S-labeled in vitro translation product (0.5, 2.5, and 10 µl) (lanes 4-6). Tax and p73
were detected
as described under "Materials and Methods." Protein standards are
indicated. Tax onput (20%) is shown (lane 1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
Tax bind to the amino-terminal C/H1 domain of CBP, and their binding is
mutually exclusive in vitro.
do not directly interact (28). We
have also determined that Tax does not compete with p73 binding to the
DNA (data not shown). The observation that Tax and p73 reciprocally
repress transcription function, in the absence of a direct interaction,
strongly suggests competition for limiting CBP/p300. There is
precedence for this scenario with Tax and p53, as we and others
(25-27, 38) have previously shown that the reciprocal transcriptional
repression between these two factors occurs, at least in part, through
competition for CBP. In support of this hypothesis, we show in this
report that both Tax and p73 bind the C/H1 domain of CBP in
vitro, and that this binding is mutually exclusive. Furthermore,
we show that a Tax mutant, K88A, which is defective for KIX binding
(73) but not C/H1 binding, can also repress p73 activation. This
observation strongly supports the idea that Tax and p73
compete for
CBP utilization in vivo, specifically through the C/H1
domain of the coactivator. Our observation that Tax K88A represses
p73
transcription function is in contrast to a recent report by
Kaida et al. (28) who showed that this Tax mutant failed to
repress the p73
activity. The nature of this discrepancy is not
known, however Kaida et al. (28) used Saos-2 cells, whereas
we used mature CD4+ Jurkat T-cells. This difference in cell
type may account for the observed discrepancy.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Dobbelstein for the p73
expression plasmid; H. Giebler and N. Polakowski for helpful
discussions; J. Mick for sharing reagents; and S. McBryant for critical
reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 970-491-5017;
E-mail: il@lamar.colostate.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M100131200
2 K. E. S. Scoggin, A. Ulloa, and J. K. Nyborg, submitted for publication.
3 J. Mick and J. K. Nyborg, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HTLV, human T-cell leukemia virus type I; GST, glutathione S-transferase; aa, amino acid; ATL, adult T-cell leukemia.
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REFERENCES |
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