Journal of General Virology (2003), 84, 897–906
DOI 10.1099/vir.0.18753-0
p53 facilitates degradation of human T-cell
leukaemia virus type I Tax-binding protein through
a proteasome-dependent pathway
Iqbal H. Chowdhury,1 Michael Radonovich,1 Renaud Mahieux,1,2
Cynthia Pise-Masison,1 Sumitra Muralidhar1 and John N. Brady1
1
Virus Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, National
Institutes of Health, Building 41/B201, 9000 Rockville Pike, Bethesda, MD 20892, USA
Correspondence
John Brady
2
[email protected]
Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France
Received 5 August 2002
Accepted 27 November 2002
Human T-cell leukaemia virus type 1 (HTLV-I), the aetiological agent of adult T-cell leukaemia (ATL)
and tropical spastic paraparesis (TSP/HAM), transforms human T-cells in vivo and in vitro. The Tax
protein of HTLV-I is essential for cellular transformation as well as viral and cellular gene
transactivation. The interaction of Tax with cellular proteins is critical for these functions. We
previously isolated and characterized a novel Tax-binding protein, TRX (TAX1BP2), by screening a
Jurkat T-cell cDNA library. In the present study, we present evidence that the tumour suppressor
p53 targets the TRX protein for proteasome degradation. Pulse–chase experiments revealed that
p53 enhanced the degradation of TRX protein and reduced the half-life from 2?0 to 0?25 h. p53
mutants R248W and R273H enhance TRX degradation suggesting a transcriptionally independent
mechanism. Both HTLV-I Tax and the proteasome-specific inhibitor MG132 inhibited p53mediated TRX protein degradation. These results suggest that TRX degradation is mediated
through activation of the proteasome protein degradation pathway independent of transcriptional
function of p53. Our results provide the first experimental evidence that Tax inhibits transciptiondependent and independent functions of p53.
INTRODUCTION
Human T-cell leukaemia virus type I (HTLV-I) is the
aetiological agent of adult T-cell leukaemia (ATL) and the
neurological disease tropical spastic paraparesis/HTLV-Iassociated myelopathy (TSP/HAM) (Gessain et al., 1985;
Osame et al., 1986; Poiesz et al., 1980; Yoshida et al., 1984).
HTLV-I provirus encodes a 40 kDa oncoprotein, Tax,
which plays a key role in virus replication and cellular
transformation. Tax activates transcription of viral as well
as cellular genes, including IL-2, IL-2Ra, IL-1, IL-6, IL-8,
IL-10, IL-15, GMCSF, c-fos and c-jun, and negatively
regulates b-polymerase, a DNA-repair enzyme and c-myb
(Azimi et al., 1998; Brady, 1996; Fujii et al., 1991; Jeang
et al., 1990; Mori et al., 1998; Mori & Prager, 1998; Nicot
et al., 2000; Nimer, 1991; Sawada et al., 1992; Siekevitz et al.,
1987; Yamashita et al., 1994). The deregulated expression
of some of these genes likely plays an important role in
transformation.
to integrate cellular responses including growth arrest at
the G1 phase of the cell cycle or apoptosis (Amundson
et al., 1998; Bates & Vousden, 1999; el-Deiry et al., 1993;
Gottlieb & Oren, 1998b; Harper et al., 1993; Polyak et al.,
1997; Selivanova & Wiman, 1995; Shen & White, 2001; Wu
et al., 1999). The biochemical activity required for p53
tumour suppression and the responses to DNA damage
involve the ability of p53 to bind DNA in a sequence-specific
manner and function as a transcriptional activator (elDeiry et al., 1992; Fields & Jang, 1990; Liu & Kulesz-Martin,
2001; Raycroft et al., 1990). Expression of p53 in cells
activates, through consensus p53 binding sites, a number of
genes involved in p53-induced cell cycle arrest or apoptosis,
including GADD45, WAF1, MDM2, Bax and PIG3 (Flatt
et al., 2000; Levine, 1997; Yu et al., 1999). The p53 protein
is a key regulator of apoptosis under a variety of physiological and pathological conditions (Bates & Vousden, 1999;
Kastan et al., 1995; Polyak et al., 1997).
Mutation and inactivation of p53 are common in human
cancers, occurring in over half of all human tumours
(Harris, 1993; Hollstein et al., 1991; Ko & Prives, 1996;
Levine, 1997; Nigro et al., 1989; Prives & Manley, 2001; Ryan
et al., 2001). In response to various types of DNA damage
and cell stress signals, the p53 tumour suppressor functions
In view of recent results, it appears that p53 may also
regulate cell cycle progression through targeted degradation
of cell cycle regulatory proteins. Gottlieb & Oren (1998a)
have reported that p53 facilitates pRb cleavage and degradation by activating the caspase protease pathway. Wild-type
p53 has also been shown to accelerate degradation of
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I. H. Chowdhury and others
cellular proteins including FLIP, which inhibits apoptosis
(Fukazawa et al., 2001); b-catenin, which is implicated
in tumour development (Sadot et al., 2001); delta-Np63,
which is linked to accelerated tumorigenesis (Ratovitski
et al., 2001); and BRCA1, the breast and ovarian cancer
susceptibility gene, which has been suggested to be
involved in gene transcription and DNA repair (IrmingerFinger et al., 1999; Choi, 2001). In addition, Ravi et al.
(2000) reported that p53 enhances degradation of the
hypoxia-inducible factor 1a, which is involved in tumour
angiogenesis.
Tax-binding proteins, including CREB, p16, CBP/p300,
NF-kB, cyclin D and Mad1, which play pivotal roles in
Tax transactivation and cell cycle regulation, have been
identified (Adya et al., 1994; Bex et al., 1998; Brady, 1996;
Kanno et al., 1994; Kashanchi et al., 1998; Kwok et al., 1996;
Neuveut et al., 1998; Suzuki et al., 1996; Van Orden et al.,
1999). We previously reported the isolation of a Taxbinding protein, TRX (TAX1BP2), following screening of
a Jurkat T-cell cDNA expression library. Direct interaction
between Tax and TRX was demonstrated using Western
blot and coimmunoprecipitation assays (Mireskandari
et al., 1996). We further demonstrated that TRX RNA
was expressed ubiquitously in a number of cell lines and
human tissues. In the present report, we demonstrate that
the tumour suppressor p53 inhibits TRX expression by
targeting and enhancing TRX degradation through a
ubiquitin proteasome-mediated pathway. Similar to its
ability to inhibit p53 transactivation, Tax inhibits the
transcription-independent p53-mediated TRX proteolysis.
METHODS
Cells. Cos-1, HeLa and p53-negative human osteosarcoma cells
(Saos-2) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10 % foetal bovine serum (FBS),
2 mM L-glutamine and antibiotics (100 U penicillin ml21 and
100 U streptomycin ml21). The p53-negative human T-cell line
Jurkat was cultured in RPMI supplemented with 10 % FBS, 2 mM
glutamine and 100 U penicillin ml21 and 100 U streptomycin ml21.
Plasmids. The TRX cDNA (Mireskandari et al., 1996) was cloned
into the PCRII vector (Invitrogen) and was amplified using Pfu
DNA polymerase. The forward primer contained the 59-coding
sequences of TRX, with an additional ATG codon and KpnI site.
The reverse primer contained 39-coding sequences of TRX cDNA
and a XhoI site. The PCR product was gel-purified and digested
with KpnI and XhoI. The eukaryotic expression vector pcDNA3.1
His A or B (Invitrogen), containing the human cytomegalovirus
(CMV) promoter and tag sequence for antibody detection, was
digested with KpnI and XhoI and the TRX cDNA fragment inserted
in-frame. TRX protein expression was tested by in vitro transcription–translation using a rabbit reticulocyte lysate system (Promega),
immunoprecipitation with a tag antibody (anti-Xpress, Invitrogen),
or by Western blotting with anti-TRX antibody (rabbit polyclonal).
TRX protein expression was also confirmed by transfecting Jurkat,
Cos-1, HeLa and Saos-2 cells, followed by Western blotting and
immunostaining with anti-tag or anti-TRX antibody. Tax expression
vectors pc-Tax (wild-type) and Tax mutant M47 (L319R-L320S)
were kindly provided by W. C. Greene (Smith & Greene, 1990). The
expression vector for p53 mutants p53R248W and p53R273H, and
898
the p53 responsive element-containing reporter plasmids pG13-Luc
and MDM2-Luc, were gifts from V. Vogelstein (Johns Hopkins
University, Maryland, MD), and pCMVp53, which expresses wildtype (wt) human p53 from the HCMV immediate-early promoter,
was a gift from S. J. Kim (National Cancer Institute, Bethesda, MD).
Transfection. Transient transfection experiments with Cos-1, HeLa,
Saos-2 and Jurkat cells were performed using either electroporation
(Ausubel et al., 1989) or FuGene methods as described in the manufacturer’s instructions (Roche Diagnostics). Briefly, cells were
washed with RPMI without FBS and resuspended in 300 ml of the
same media at a concentration of 56106 per sample. The cells were
electroporated (Bio-Rad Gene pulser) using 230 to 250 V at a capacitance of 975 mF. The amount of DNA transfected was normalized
by the addition of vector control plasmid. TRX, Tax, p53, and p53
mutants R248W and R273H protein expression was assayed by
Western blot as described below.
Western blotting. Cells were washed with cold PBS, treated with
PBS containing 2 mM EDTA, scraped and lysed in RIPA buffer
(50 mM Tris, pH 8?0, 150 mM NaCl, 1 % Nonidet P-40, 0?5 % deoxycholate, 0?1 % SDS, containing 1 mM PMSF, aprotinin (1 mg ml21),
leupeptin (1 mg ml21) and 5 mM sodium fluoride at 4 ˚C for 1 h.
The lysate was cleared by centrifugation in a microcentrifuge at
14 000 r.p.m. for 15 min and the protein concentration was determined accordingly (Bio-Rad). Cell lysates (15 to 30 mg) were separated
on 12 % or 4 to 20 % SDS-polyacrylamide gradient gels, transferred
to an Immobilon membrane (Millipore) and assayed for TRX
expression using anti-Xpress Tag antibody (Invitrogen). Tax, p53,
mutant p53 (R248W and R273H) and the CMV immediate early 2
86 kDa gene product (IE-2) were detected using anti-Tax (Tab 172),
anti-p53 (Do-1, pab421) and anti-IE-2 (rabbit polyclonal) antibodies,
respectively. Antigen–antibody complexes were detected using the
enhanced chemiluminescence system (Amersham).
RNA isolation and Northern blotting. RNA was isolated from
transfected cells using TRIzol (Gibco-BRL). Fifteen mg of total RNA
was used for each Northern blot analysis. RNA was vacuum-dried
for 10 min, resuspended in denaturation buffer (10 mM phosphate
buffer pH 7?0, 16 MOPS buffer, 50 % formamide, 2?1 M formaldehyde) and incubated at 65 ˚C for 5 min followed by quenching on
ice. RNA loading buffer (2 ml) was added to each sample. Samples
were run on a 1 % agarose gel containing 16 MOPS and 0?66 M
formaldehyde and electrophoresis was performed at 50 V in 16
MOPS buffer. The RNA was blotted onto a nylon membrane
(Micron Separation) using capillary action, and then fixed onto the
membrane using a UV-Stratalinker 2400 (Stratagene). A 59 endlabelled TRX cDNA probe was used to detect the amount of TRX
RNA in each sample. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA (Clontech) was used as a control for RNA loading.
Immunoprecipitation. Cos-1 cells were co-transfected with TRX
alone or in the presence of p53. Cells were pulsed with [35S]
methionine and [35S]cysteine (200 mCi ml21 or 500 mCi per 6 cm
plate) for 30 min after 24 h of transfection, washed and chased for
3 h in complete medium. Samples were collected at 0, 0?5, 1?0, 2?0
and 3?0 h post-pulse. Cells were lysed in RIPA buffer, the protein
concentration was determined and 500 mg of protein used for
immunoprecipitation with an anti-Xpress (anti-Tag) antibody for
the detection of TRX. The amount of labelled protein was quantified
using the ImageQuant program (Molecular Dynamics).
Luciferase assay. To assay for luciferase activity, Saos-2 cells were
harvested 24 h after transfection by pG13-Luc or MDM2-Luc with
wt p53 or mutants p53R248W or p53R273H. The cells were washed
twice in cold PBS, and then lysed in 400 ml of Promega passive lysis
buffer (Promega). Cells were then gently vortexed and kept on
ice for 30 min. The supernatant was clarified by centrifugation at
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Tax-binding protein degradation
14 000 r.p.m. for 5 min at 4 ˚C. The extract was kept at 280 ˚C until
further use. The protein concentration was determined by the Bradford
protein assay (Bio-Rad). Equal amounts of protein (5 to 20 mg) were
added to 100 ml of luciferase substrate buffer according to the manufacturer’s protocol (Promega); these samples were then analysed in
a Berthold LB9500C Luminometer. Luminescence was measured in
relative light units (RLU). Luciferase activity was expressed as average light units produced per mg of protein present in the specific cell
lysate for each experiment. Each sample was assayed in triplicate and
the average value was determined±standard deviation.
RESULTS
Inhibition of TRX expression by p53
We have previously reported that HTLV-I Tax inhibits the
transactivation function of p53 (Pise-Masison et al., 1998,
2000, 2001). In addition we have demonstrated that Tax
interacts with a novel cellular protein, TRX (Mireskandari
et al., 1996). In a series of experiments designed to test the
biological function of TRX on Tax inhibition of p53
function, we observed a significant effect of p53 on TRX
expression. First, cells were transfected with the TRX/
pcDNA3.1 expression plasmid and TRX protein was
analysed 24 h post-transfection by Western blot using
anti-Xpress (Tag) antibody (Fig. 1). While the antibody
failed to react with proteins in the control extract (lane 1), a
protein migrating at 25 kDa was detected in cells transfected
with the TRX expression plasmid (lanes 2 to 4). Increasing
amounts of the TRX protein was observed as the amount
of TRX plasmid was increased in the transfection mix.
The identity of the protein was confirmed by Western
blot analysis with a peptide antibody raised against TRX
protein (data not shown).
Interestingly, we observed that cotransfection of p53
decreased the level of TRX protein in Cos-1 cells (Fig. 2A,
lanes 2 and 8). The ability of p53 to inhibit TRX expression
was dose-dependent (data not shown).
We next tested whether p53 inhibited TRX expression in
other cell lines including the p53-negative osteosarcoma
cell line, Saos-2. Consistent with the results presented
above, these experiments showed that p53 inhibited TRX
protein expression. Complete inhibition of TRX expression
was observed at 1 mg plasmid DNA in the transfected cells
(Fig. 2B, lane 5). The extracts were also used for Western
blot analysis with a p53-specific antibody. The results of
this experiment demonstrate that the level of p53 expression is slightly increased in samples containing TRX
(Fig. 2C; compare lanes 2 and 3 with 5 and 6).
Subsequently, TRX was transfected into Cos-1 cells in the
presence or absence of Tax and p53. Cotransfection of
Tax with TRX did not significantly alter the level of TRX
protein expression (Fig. 2A; compare lanes 2 and 7).
Fig. 1. Expression of TRX protein in Cos-1 cells. Cos-1 cells
were transfected by electroporation as described with TRX/
pcDNA3.1 expression plasmid and cells were harvested 24 h
post-transfection. TRX expression was determined by Western
blot and immunostaining using anti-Xpress (Tag) antibody, which
detects TRX protein, followed by chemiluminescence detection.
pCMV was used to normalize the amount of transfected DNA.
These figures represent one of five separate experiments.
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Fig. 2. Inhibition of the steady state level of TRX protein by
the tumour suppressor protein p53. (A) Cos-1 cells were transfected with TRX expression plasmid alone or in the presence of
either p53 or Tax expression plasmids. TRX expression was
determined by Western blot followed by immunostaining. (B
and C) Dose-dependent inhibition of TRX expression by p53 in
Saos-2 cells (p53 null). Cells were transfected with TRX
expression plasmid (10 mg per sample) in the presence of p53
expression plasmid at 1?0 or 0?3 mg per sample. Cells were
harvested 24 h post-transfection and TRX (B) and p53 (C)
expression was determined by Western blot analysis. pCMV
was used to normalize the amount of transfected DNA. These
figures represent one of five separate experiments.
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I. H. Chowdhury and others
Effect of p53 on TRX RNA expression
It was of interest to determine if the p53 effect on TRX
expression was at the transcriptional or post-transcriptional
level. Cos-1 cells were cotransfected with TRX/pcDNA3.1
in the presence or absence of p53. Total RNA was prepared
and assayed for the level of TRX RNA with a TRX-specific
probe. TRX mRNA was detected in cells transfected with
TRX plasmid, but not the control cells (Fig. 3A, lanes 1 to 3).
The level of TRX mRNA expression was similar in the
presence and absence of p53 (lanes 2 and 4), indicating
that p53 did not have any significant effect on TRX RNA
expression. A control hybridization with the GAPDH
probe demonstrated that equal amounts of RNA were
present in all of the samples. The slight decrease in GAPDH
RNA in lane 4 is not significant. These results suggest that
the p53 effect is at the post-transcriptional level. Due to the
high level of TRX mRNA produced from the replicating
plasmid transfected into the cells, the endogenous 2?3 kb
TRX mRNA is not detected in the exposure shown in Fig. 3.
Fig. 3. p53 does not inhibit TRX RNA expression. Cos-1 cells
were transfected with TRX/pcDNA3.1 (4 mg per sample) alone
or in the presence of p53 (2 mg per sample). Cells were harvested at 48 h post-transfection. Total RNA was isolated as
described. (A) 15 mg total RNA from each sample was resolved
in a 1 % agarose/formaldehyde gel, transferred onto nylon membrane and hybridized with a TRX cDNA probe labelled with
[32P]dCTP. (B) The same blot was reprobed with a cDNA
probe for the internal housekeeping gene GAPDH.
p53 decreases the half-life of TRX protein
To determine if p53 affected the turnover or half-life of
TRX protein in the cell, a pulse–chase experiment was
performed. Cos-1 cells were transfected with TRX or TRX
plus p53. At 24 h post-transfection, cells were pulsed for
30 min with 500 mCi ml21 [35S]methionine and [35S]cysteine
and chased for 0 to 3 h. Cell extracts were immunoprecipitated with an anti-Xpress Tag antibody. The labelled
Fig. 4. Comparison of TRX protein half-life in the presence and absence of p53 by pulse–chase analysis. The half-life of TRX
protein was determined by pulse–chase experiments. Cells transfected with either TRX alone or in the presence of p53 were
metabolically labelled with [35S]methionine and [35S]cysteine. The cells were pulsed for 30 min with 500 mCi per plate in
2?5 ml medium, washed and chased for 0 to 3 h. 500 mg total protein (cell lysate) from each sample was used for
immunoprecipitation by anti-Xpress (Tag) antibody which detects transfected TRX. Proteins were separated by
electrophoresis in 12 % SDS-PAGE gels. Gels were analysed using the Molecular Dynamics PhosphorImager and
ImageQuant program. These figures represent one of two separate experiments.
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Tax-binding protein degradation
immunoprecipitates were then analysed by SDS-PAGE and
the radioactivity was quantified using the PhosphorImager
ImageQuant program. The results demonstrated that equal
amounts of TRX protein were present in the TRX- or TRX
plus p53-transfected cells at the 0 h time point (Fig. 4A,
lanes 1 and 6), indicating that p53 did not affect the
translation of TRX mRNA. Remarkably, the TRX protein
was much less stable in the p53-expressing cells. In the
absence of p53, TRX protein was degraded gradually with
a half-life of approximately 2?0 h (Fig. 4A, lanes 1 to 5;
Fig. 4B). In the presence of p53, the half-life of TRX was
0?25 h (Fig. 4A, lanes 6 to 10; Fig. 4B). These results suggest
that p53 decreases the steady state level of TRX protein by
enhancing degradation of the protein. In these studies, we
noted that the anti-Tag antibody immunoprecipitated
several lower molecular mass proteins. The fact that these
bands did not react with the anti-Tag antibody in Western
blot analysis (Fig. 1) suggests that the proteins may be
proteolytic products or TRX-associated proteins that
coimmunoprecipitate with TRX.
Transcription-independent function of p53
targets TRX degradation
Next, we examined the activity of transcriptionally inactive
p53 point mutants R248W and R273H on TRX degradation
in Cos-1 cells. The results indicated that similar to wt
p53 the p53 mutants also increased TRX degradation
(Fig. 5A). The transcriptional activity of wt p53 and
mutants R248W and R273H were confirmed by cotransfecting the p53 plasmids with pG13-Luc (which contains
136 p53-responsive elements) or MDM2-Luc into p53
Fig. 5. Transactivation defective p53 mutants retain the ability to target TRX degradation. (A) Cos-1 cells were transfected
with TRX expression plasmid alone or in the presence of either wt p53 or transcriptionally inactive mutants p53R248W or
p53R273H. TRX expression was determined by Western blot followed by immunostaining as described (B) and (C) Saos-2
cells were cotransfected with p53 responsive element-containing reporter plasmids pG13-Luc (B) or MDM2-Luc (C) in
absence or presence of wt p53 or mutant p53R248W or p53R273H. The relative luciferase activity was determined in
triplicate±standard deviation. The control luciferase activity for pG13-Luc and MDM2-Luc was 1818 and 8708 units,
respectively. (D) 35S-labelled TRX was synthesized in rabbit reticulocyte lysate and incubated with purified GST or GST–p53.
Following incubation, bound proteins were analysed by SDS-PAGE and autoradiography. The TRX protein band is indicated.
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I. H. Chowdhury and others
negative Saos-2 cells (Fig. 5B and C). The results showed
that wt p53 activated p53RE-containing pG13-Luc (153fold) (Fig. 5B) and MDM2-Luc (25-fold) (Fig. 5C). As
expected, p53 mutants R248W and p53R273H failed to
activate the p53RE-containing reporter plasmid pG13-Luc
or MDM2-Luc. These results suggest that p53-induced TRX
protein degradation is mediated through a p53 transcriptionindependent pathway.
To analyse the potential interaction between TRX and p53,
we performed in vitro protein binding assays. 35S-labelled
TRX was prepared using the rabbit reticulocyte lysate
system. TRX was incubated with purified GST or GST–p53
protein. After centrifugation and washing of the pellets to
remove unbound protein, SDS-gel analysis of the proteins
demonstrated that TRX bound to GST–p53 but not the
GST control (Fig. 5D).
TRX degradation is mediated through the
proteasome pathway
Next, we wanted to determine whether p53-mediated TRX
degradation was regulated through a specific proteolytic
pathway. The proteasome-specific inhibitor MG132 and the
cysteine protease inhibitor E64 were added to transfected
cells at concentrations of 3 and 10 mM for 16 h. Consistent
with previous reports, these conditions are not toxic to
cells as shown when trypan blue exclusion is used to measure cell viability (Magae et al., 1997; Meriin et al., 1998)
(data not shown). Interestingly, the results show that the
proteasome-specific inhibitor MG132 could block p53mediated TRX protein degradation (Fig. 6A, compare lanes
1, 3 and 7). Complete protection of TRX degradation was
achieved at an MG132 concentration of 10 mM. In contrast,
the cysteine protease inhibitor E64 (lanes 4 and 5) failed
to inhibit TRX degradation. In parallel experiments, the
calpain-specific inhibitor ALLN also failed to prevent
p53-mediated TRX degradation (data not shown). These
results suggest that p53 targets TRX proteolysis through the
proteasome degradation pathway.
A Western blot analysis of the extracts with a p53-specific
antibody is presented in Fig. 6(B). Consistent with the
results presented in Fig. 2(C), coexpression of TRX with
p53 leads to a slight increase in the steady state level of
p53 in the cells (lanes 2 and 3). No further increase in the
level of p53 expression was observed when E64 or MG132
was added to the cells.
Specificity of TRX protein degradation by p53
To determine if p53-mediated degradation of TRX was
specific, we used the CMV IE-2 86 kDa gene product. IE-2
transactivates the CMV immediate early gene and has
been shown to deregulate cell cycle and apoptosis pathways
(Castillo et al., 2000; Salvant et al., 1998; Wiebusch &
Hagemeier, 1999; Zhu et al., 1995). The IE-2 expression
vector was transfected into Saos-2 cells in the presence or
absence of p53. As an experimental control, cells were
transfected with TRX alone or in the presence of p53.
Consistent with results presented above, the expression of
TRX protein was reduced in the presence of p53 (Fig. 7B,
lanes 2 and 3). In contrast, the level of IE-2 protein
expression was similar in the presence and absence of p53
(Fig. 7A, lanes 4 and 5). Two background protein bands
were routinely detected in the Western blots with the IE-2
antibody (Fig. 7A). The level of reactivity, however, did not
change significantly in the different experimental conditions
and thus does not interfere with the interpretation of results.
Fig. 6. TRX degradation is through the proteasome pathway.
Saos-2 (p53 null) cells were transfected with TRX alone or cotransfected with p53 and cultured for 24 h. Transfected cells
were treated with medium alone, the cysteine protease inhibitor
E64, or the 26S proteasome-specific inhibitor MG132, at concentrations of 3 and 10 mM. Cells were harvested and TRX (A)
and p53 (B) protein was analysed by Western blotting. These
figures represent one of five separate experiments.
902
Tax inhibits p53-mediated degradation of TRX
We have shown that Tax inhibits the transactivation
function of p53 (Pise-Masison et al., 1998, 2000, 2001).
To test whether Tax inhibited the p53-mediated degradation
of TRX, transfection experiments were repeated in the
presence and absence of a Tax expression vector. Similar
to the results presented above, we observed that the level
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Tax-binding protein degradation
DISCUSSION
Fig. 7. p53 fails to target CMV IE-2 protein for degradation.
Saos-2 cells (p53 null) were transfected with TRX or CMV IE-2
alone or in the presence of p53. Cells were harvested 24 h
post-transfection and expression of IE-2 (A) and TRX (B) was
determined by Western blot followed by immunostaining with
anti-IE-2 for IE-2 detection and anti-Xpress for TRX. These
figures represent one of two independent experiments.
The p53 tumour suppressor protein is critical for controlling cell proliferation, protection against oncogenic transformation and is an important regulator of apoptosis
under a variety of physiological and pathological conditions. Transcriptional activation of target genes is one of
the key mechanisms by which p53 induces cell cycle arrest
and apoptosis (Kastan et al., 1995; Levine, 1997). Many lines
of evidence support a role for the activation of the cyclindependent kinase inhibitor waf1/Cip1/p21 in p53-induced
cell cycle arrest (el-Deiry et al., 1993; Harper et al., 1993).
Transcriptional activation of Bax (Miyashita & Reed, 1995)
and IGF-BP3 (Buckbinder et al., 1995) play a significant
role in the apoptosis response pathway. p53 can also induce
cell cycle arrest by repressing transcription from a variety
of growth-promoting genes, including those of the c-fos,
c-jun, c-myc, Rb, MDR1, IL-6 and PCNA (Chin et al., 1992;
Ginsberg et al., 1991; Lechner et al., 1992; Ragimov et al.,
1993; Santhanam et al., 1991; Shiio et al., 1992; Subler et al.,
1992; Yamaguchi et al., 1994).
p53 may also regulate tumour development and cell cycle
progression through targeted degradation of cell cycle
of TRX protein expression was diminished in the presence
of p53 (Fig. 8A, compare lanes 1, 5 and 6). In contrast,
cotransfection with Tax resulted in the inhibition of TRX
degradation (lanes 7 and 8). This result suggests that Tax
is able to inhibit the p53-targeted degradation of TRX protein. Western blot analysis with control p53 and Tax-specific
antibodies is shown in Fig. 8(B) and (C), respectively.
To demonstrate the specificity of Tax inhibition of p53
function, we tested whether a Tax mutant was able to inhibit p53-mediated TRX degradation. As demonstrated above,
cotransfection of TRX with p53 led to a significant reduction in the level of TRX expression. Quantitative analysis
of the blot indicated there was approximately an 85 %
reduction in TRX expression in the presence of p53.
Consistent with the results presented above, we observed
that wild-type Tax inhibited p53-mediated TRX degradation (Fig. 9A, lanes 1, 3 and 4). In contrast, Tax mutant
M47, which is defective in CREB activation and binding to
CBP and PCAF, was significantly less efficient at inhibiting
p53-mediated TRX degradation (lane 5). Quantitative
analysis of the Western blot indicated there was a 65 to
70 % reduction in TRX expression in the presence of p53
and the Tax mutant M47. Consistent with these results,
M47 does not inhibit p53 transactivation function in Saos-2
and HeLa cells (Pise-Masison et al., 2001; Mulloy et al.,
1998). The inability of M47 to inhibit p53 function was not
due to protein expression levels, since an equal or greater
level of M47 protein was observed in the cell extracts
(Fig. 9B, lanes 4 and 5).
http://vir.sgmjournals.org
Fig. 8. Tax inhibits p53-mediated TRX degradation. Saos-2 cells
were transfected with TRX (8 mg) alone or in the presence of p53
at 0?3 and 1?0 mg plasmid DNA. The Tax expression plasmid
pc-Tax (7 mg) was cotransfected with TRX and p53 as indicated.
Cells were harvested 24 h post-transfection and 30 mg of protein
was analysed by Western blot analysis using anti-Xpress antibody
for TRX (A), antibody DO-1 for p53 (B) and Tab-172 for Tax (C).
These figures represent one of five independent experiments.
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903
I. H. Chowdhury and others
delta Np63 degradation occurred via the caspase pathway. We used cysteine protease inhibitor E64, the 26Sproteasome inhibitor MG132 and the calpain 1 protease
inhibitor ALLN. The cysteine protease inhibitor E64 and
calpain 1 protease inhibitor ALLN failed to protect the p53mediated TRX degradation. In contrast, proteasome-specific
inhibitor MG132 blocked p53-mediated TRX degradation.
The analysis of TRX protein by the PeptideCutter program
revealed that there are no caspase (caspase1–caspase10)
cutting sites in the TRX protein. In contrast, chymotrypsin
(30 cutting sites) and trypsin (20 cutting sites) protease
sites are abundant.
Fig. 9. Tax mutant M47 is unable to block p53-mediated TRX
degradation. Saos-2 cells were cotransfected with TRX alone
or with p53. The TRX+p53 samples were cotransfected with
either wt Tax, or with Tax mutant M47. Cells were harvested
24 h post-transfection and TRX (A) and Tax (B) protein expression
were determined by Western blot followed by immunostaining.
The figure represents one of two independent experiments.
The emerging model of the functional interaction of Tax
and p53 interaction supports the ability of Tax to inhibit
several functions of p53. We and others have previously
shown that Tax inhibits the transactivation function of
p53 (Ariumi et al., 2000; Cereseto et al., 1996; Lemasson
& Nyborg, 2001; Mulloy et al., 1998; Pise-Masison et al.,
1998, 2000, 2001; Suzuki et al., 1999; Van Orden et al., 1999).
The data presented in this study demonstrate that Tax
also blocks transcription-independent functions of p53. It
will be of interest to further identify the functional consequences of TRX degradation, interaction with tumour
suppressor p53 and its involvement in HTLV-I-induced
leukaemogenesis.
ACKNOWLEDGEMENTS
We thank Dr Paul Lambert for helpful discussions and suggestions and
Janet Duvall for valuable editorial and technical support.
regulatory proteins (Gottlieb & Oren, 1998a; Ratovitski
et al., 2001; Choi, 2001; Ravi et al., 2000). In the present
study, we demonstrate that p53 promotes proteasomedependent degradation of the cellular protein TRX. In cell
cycle analysis, overexpression of TRX protein in a variety of
cells did not result in cell cycle arrest at either the G1/S or
G2/M border, suggesting that the function of TRX is not
inhibition of cell cycle progression. Conversely, expression
of TRX protein in cells blocked with hydroxyurea facilitated the progression of cells into S phase suggesting that
TRX may act as a positive factor in G1/S progression (data
not shown). Since expression of p53 in Saos-2 cells has been
reported to increase G1/S arrest, it is interesting to speculate
that p53 targets TRX for degradation in part to inhibit the
G1/S transition. We have recently found that TRX functions as a coactivator, facilitating Tax transactivation of
the HTLV-I LTR (data not shown). It will be of interest to
determine if the cell cycle regulation is linked to the TRX
transcription function.
To understand the mechanism of TRX degradation by
p53, we have analysed p53 mutants which are transcriptionally inactive. The results of these studies demonstrate
that TRX degradation is a transcription-independent
function of p53, since p53R248W and p53R273H stimulate
degradation. Ratovitski et al. (2001) showed similar results
in the degradation of delta Np63 protein by p53. Of interest,
904
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