Published Ahead of Print 18 August 2006.
2006, 80(21):10497. DOI: 10.1128/JVI.00739-06.
Tanaka and Naoki Mori
Mariko Tomita, Akira Kikuchi, Tetsu Akiyama, Yuetsu
Human T-Cell Leukemia Virus Type 1 Tax
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JOURNAL OF VIROLOGY, Nov. 2006, p. 10497–10505
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 21
Human T-Cell Leukemia Virus Type 1 Tax Dysregulates
Mariko Tomita,1Akira Kikuchi,3Tetsu Akiyama,4Yuetsu Tanaka,2and Naoki Mori1*
Division of Molecular Virology and Oncology, Graduate School of Medicine,1and Division of Immunology, Faculty of Medicine,2
University of the Ryukyus, Nishihara, Okinawa, Japan; Department of Biochemistry, Graduate School of Biomedical Science,
Hiroshima University, Hiroshima, Japan3; and Laboratory of Molecular and Genetic Information, Institute for
Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan4
Received 11 April 2006/Accepted 8 August 2006
Dysregulation of ?-catenin signaling has been implicated in the malignant transformation of cells. However, the
Here we found that ?-catenin protein was overexpressed in the nucleus and that ?-catenin-dependent transcription
was significantly enhanced in Tax-positive HTLV-1-infected T-cell lines compared to that in Tax-negative HTLV-
1-infected T-cell lines. Transfection with ?-catenin-specific small interfering RNA inhibited the growth of the
Tax-positive HTLV-1-infected T-cell line HUT-102. Transient transfection of Tax appeared to enhance ?-catenin-
dependent transcription by stabilizing the ?-catenin protein via activation of the cyclic AMP (cAMP) response
element-binding protein. HTLV-1-infected T-cell lines overexpressing ?-catenin also showed increased Akt activity
via Tax activation of the cAMP response element-binding protein, resulting in the phosphorylation and inactivation
of glycogen synthase kinase 3?, which phosphorylates ?-catenin for ubiquitination. The phosphatidylinositol
3-kinase inhibitor LY294002 reduced ?-catenin expression in Tax-positive T-cell lines, and inactivation of glycogen
synthase kinase 3? by lithium chloride restored ?-catenin expression in Tax-negative T-cell lines. Finally, we
showed that dominant-negative Akt inhibited Tax-induced ?-catenin-dependent transcription. These results indi-
cate that Tax activates ?-catenin through the Akt signaling pathway. Our findings suggest that activation of
?-catenin by Tax may be important in the transformation of T cells by HTLV-1 infection.
Adult T-cell leukemia (ATL) is an aggressive and usually
fatal hematological malignancy that is etiologically linked to
infection with human T-cell leukemia virus type 1 (HTLV-1)
(14, 33, 43). Conventional chemotherapeutic drugs used for
the treatment of patients with ATL have yielded only a limited
improvement in prognosis (41). Currently, the molecular mecha-
Expression of the virally encoded Tax protein appears to be a
critical event during the leukemogenesis of ATL. Tax is a
transcriptional activator that modulates the expression of
HTLV-1 long terminal repeats (LTRs) and the transcription of
many cellular genes. Tax can immortalize primary human T
cells derived from peripheral blood or cord blood (11, 12) and
induce tumors in transgenic mice (29), probably by activating a
variety of proteins, including transcription factors such as cyclic
AMP response element-binding protein (CREB), serum-re-
sponsive factor, and NF-?B (10).
?-Catenin is a multifunctional protein that participates in
both cell-cell adhesion and the transcription of T-cell tran-
scription factor (Tcf)/lymphoid enhancer binding factor (Lef)
target genes. ?-Catenin levels are regulated posttranslationally
by the Wnt signaling pathway. In the absence of secreted Wnt
glycoprotein ligands, the modular axin protein provides a scaf-
fold for the binding of glycogen synthase kinase 3? (GSK-3?),
adenomatous polyposis coli (APC) protein, and ?-catenin.
This facilitates the phosphorylation of ?-catenin by GSK-3?,
allowing phosphorylated ?-catenin to be ubiquitinated for
rapid degradation by the proteasome (34). Mutations in APC,
?-catenin, or axin increase the steady-state level of ?-catenin,
and many cancers, including colorectal cancer, result from
hyperactivity of the Wnt/?-catenin signaling pathway due to
the constitutive ?-catenin-mediated transactivation of Tcf-de-
pendent genes (34). Thus, aberrant activation of ?-catenin has
major oncogenic effects (9, 31). Accordingly, disruption of this
signaling pathway holds promise for the development of new
?-Catenin is highly expressed in several leukemic cell lines
(4). In B-cell chronic lymphocytic leukemia, Wnt3, Wnt5b,
Wnt6, Wnt10a, Wnt14, and Wnt16, as well as the Wnt receptor
Frizzled 3, are highly expressed, resulting in the activation of
?-catenin-mediated transcription and an enhanced survival of
chronic lymphocytic leukemia lymphocytes (22). These results
suggested that ?-catenin is associated not only with epithelial
cancer but also with leukemia and lymphoma. However, nei-
ther the expression of ?-catenin in HTLV-1-infected T cells
nor the function of ?-catenin in leukemogenesis induced by
HTLV-1 has been elucidated. The goals of this study were to
determine the status of ?-catenin signaling in HTLV-1-in-
fected T cells and to elucidate the molecular activation of this
MATERIALS AND METHODS
Reagents. The proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL)
and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were pur-
* Corresponding author. Mailing address: Division of Molecular
Virology and Oncology, Graduate School of Medicine, University of
the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan.
Phone: 81 (98) 895-1130. Fax: 81 (98) 895-1410. E-mail: n-mori@med
?Published ahead of print on 18 August 2006.
on June 10, 2014 by guest
chased from Calbiochem (San Diego, CA). Cycloheximide and lithium chloride
were obtained from Sigma-Aldrich (St. Louis, MO).
Antibodies. Anti-?-catenin and anti-GSK-3? antibodies were purchased from
BD Transduction Laboratories (San Jose, CA). An anti-actin antibody was ob-
tained from NeoMarkers (Fremont, CA). An antibody to Tax (Lt-4) was de-
scribed previously (40). Anti-Akt, anti-phosphorylated Akt (Ser473), and anti-
phospho-GSK-3? (Ser9) antibodies were purchased from Cell Signaling Technology
(Beverly, MA). Anti-nucleolin and anti-I?B? antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated
anti-mouse and anti-rabbit immunoglobulin G antibodies for Western blotting
were purchased from Amersham Biosciences (Piscataway, NJ).
Cell culture. The HTLV-1-infected T-cell lines MT-2 (27), SLB-1 (20), HUT-
102 (33), MT-1 (26), TL-OmI (39), and ED-40515(?) (23) were maintained in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine se-
rum, 50 U/ml penicillin, and 50 ?g/ml streptomycin (Sigma-Aldrich) at 37°C in
5% CO2. MT-2 and SLB-1 are HTLV-1-transformed T-cell lines which were
established by an in vitro coculture protocol. MT-1, TL-OmI, and ED-40515(?)
are leukemic T-cell lines derived from patients with ATL. HUT-102 was estab-
lished from a patient with ATL, but its clonal origin is unclear. HeLa (human
cervix adenocarcinoma cell line) cells were maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 50
U/ml penicillin, and 50 ?g/ml streptomycin at 37°C in 5% CO2.
Plasmids. A ?-catenin expression plasmid (pCGN/?-catenin) and a human
Tcf-4 expression plasmid (pEF-BOS HA/Tcf-4E) were described previously (17,
18). The pGL3-OT and pGL3-OF reporter plasmids (37) were kindly provided
by B. Vogelstein (The Sidney Kimmel Comprehensive Cancer Center, Johns
Hopkins University School of Medicine, Baltimore, MD). pGL3-OT and
pGL3-OF contain three copies of the Tcf site (5?-AGATCAAAGG-3?) and a
mutant sequence (5?-AGGCCAAAGG-3?), respectively, upstream of the c-fos
promoter and the luciferase open reading frame. An HTLV-1 LTR luciferase
reporter plasmid (LTR-Luc), which contains the HindIII fragment from the
HTLV-1 LTR, was kindly supplied by I. Futsuki (Nagasaki University School of
Medicine, Nagasaki, Japan). An NF-?B reporter plasmid (?B-Luc) containing
five tandem repeats of an NF-?B binding site from the interleukin-2 receptor ?
chain gene was kindly provided by J. Fujisawa (Kansai Medical University,
Osaka, Japan). A series of expression vectors for Tax (Tax WT) and mutants
thereof (Tax M22, Tax 703, and Tax K88A) was described previously (13, 24). An
expression plasmid for dominant-negative CREB (pCMV-KCREB) was pur-
chased from BD Biosciences Clontech (Mountain View, CA). A dominant-
negative Akt expression plasmid (pCMV5-K169A, T308A, S473A-Akt) has Lys-
169-, Thr-308-, and Ser-473-to-Ala mutations and was kindly provided by D.
Alessi (University of Dundee, Dundee, United Kingdom).
Western blot analysis. Cells were lysed in sodium dodecyl sulfate (SDS)
sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10%
glycerol, 6% 2-mercaptoethanol, and 0.01% bromophenol blue. Nuclear and
cytoplasmic extracts were prepared by using a nuclear extraction kit (Active
Motif, Carlsbad, CA) according to the manufacturer’s instructions. The lysates
were resolved by electrophoresis on polyacrylamide gels and then electroblotted
onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Mem-
branes were incubated with the appropriate primary antibody, as indicated,
overnight at 4°C. After being washed, the blots were exposed to the appropriate
secondary antibody conjugated with horseradish peroxidase for 1 h at room
temperature. The reaction products were visualized using enhanced chemilumi-
nescence reagent (Amersham Biosciences) according to the manufacturer’s in-
Transfection and reporter assay. HeLa cells were transfected using Lipo-
fectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s
protocols. HTLV-1-infected T-cell lines were transfected using a previously
described DEAE-dextran method (30). In brief, 1 ? 107cells were incubated for
30 min at room temperature with 2.2 ml of transfection solution containing
plasmid DNA and 100 ?g of DEAE-dextran (Amersham Biosciences) in RPMI
1640 serum-free medium. Cells were then rinsed with 5 U/ml heparin (Wako,
Osaka, Japan) in RPMI 1640 and incubated in complete medium for 48 h. Cells
were transiently transfected with the indicated effecter plasmids and luciferase
reporter constructs. In all cases, the reference plasmid phRL-TK, which contains
the Renilla luciferase gene under the control of the herpes simplex virus thymi-
dine kinase promoter, was cotransfected to correct for transfection efficiency.
Luciferase assays were performed by using a dual-luciferase reporter system
(Promega, Madison, WI) in which relative luciferase activities are calculated by
normalizing transfection efficiencies according to the Renilla luciferase activity.
siRNA. To repress ?-catenin, a predesigned double-stranded small interfering
RNA (siRNA) (siGENOME SMARTpool CTNNB1; Dharmacon, Inc., Lafay-
ette, CO) was used. A siCONTROL non-targeting siRNA pool (Dharmacon,
Inc.) was used as a negative control. siRNAs were transfected into HUT-102 cells
with a Nucleofector device (program T-20) and a Cell Line Nucleofector kit V
(Amaxa, Inc., Cologne, Germany). Transfected cells were incubated for 12 h,
seeded into 24-well plates at 5 ? 104viable cells per well, and incubated for the
indicated times. The number of viable cells was determined every 24 h by
counting trypan blue-excluding cells in a hemocytometer.
Reverse transcriptase PCR (RT-PCR). Total cellular RNA was extracted from
cells by the use of TRIzol reagent (Invitrogen) as described by the supplier.
First-strand cDNAs were synthesized using an RNA-PCR kit (Takara Bio, Shiga,
Japan) with random primers. Thereafter, cDNAs were amplified for ?-catenin,
Tax, and ?-actin. The oligonucleotide primers used were as follows: for ?-cate-
nin, 5?-TGATGGAGTTGGACATGGCCATGG-3? (sense) and 5?-CAGACAC
CATCTGAGGAGAACGCA-3? (antisense); for Tax, 5?-CCCACTTCCCAGG
GTTTGGACAGA-3? (sense) and 5?-CTGTAGAGCTGAGCCGATAACGC
G-3? (antisense); and for ?-actin, 5?-GTGGGGCGCCCCAGGCACCA-3? (sense)
and 5?-CTCCTTAATGTCACGCACGATTTC-3? (antisense). Product sizes
were 570 bp for ?-catenin, 203 bp for Tax, and 548 bp for ?-actin. The ampli-
fication programs were as follows: for ?-catenin, 30 cycles of denaturing at 94°C
for 1 min, an annealing step at 60°C for 40 s, and an extension step at 72°C for
50 s; for Tax, 30 cycles of denaturing at 94°C for 30 s, an annealing step at 60°C
for 30 s, and an extension step at 72°C for 90 s; and for ?-actin, 28 cycles of
denaturing at 94°C for 30 s, an annealing step at 60°C for 30 s, and an extension
step at 72°C for 90 s. The PCR products were fractionated in 2% agarose gels and
visualized by ethidium bromide staining.
In vitro Akt kinase assay. The Akt kinase assay was performed using an Akt
kinase assay kit (Cell Signaling Technology) according to the protocol recom-
mended by the manufacturer. Briefly, the cells were washed with phosphate-
buffered saline, and 200 ?l of lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM
?-glycerol phosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and
1 mM leupeptin) was added to the cells for 10 min. Lysates were immunopre-
cipitated for 2 h at 4°C with anti-Akt antibody. The immunoprecipitates were
washed with lysis buffer and kinase buffer (25 mM Tris-HCl [pH 7.5], 5 mM
?-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2).
Kinase reactions were performed for 30 min at 30°C in kinase buffer supple-
mented with 200 ?M ATP and 1 ?g GSK-3?/? fusion protein. The samples were
loaded into a 12% acrylamide gel. Phosphorylation of GSK-3?/? was measured
by Western blotting with anti-phospho-GSK-3?/? (Ser21/9) antibody.
Overexpression of ?-catenin in Tax-positive HTLV-1-in-
fected T-cell lines. We first analyzed ?-catenin expression in six
HTLV-1-infected T-cell lines. The ?-catenin protein was
highly expressed in three of the lines, namely, MT-2, SLB-1,
and HUT-102, whereas only weak expression was detected in
the three ATL-derived T-cell lines, i.e., MT-1, TL-OmI, and
ED-40515(?) (Fig. 1A, top panel). Although the MT-2 cell
lysates showed two Tax-immunoreactive bands, consistent with
the 40-kDa Tax protein and a 69-kDa fusion between the
envelope and the Tax coding sequence reported previously
(15), Tax protein levels were similar in the three HTLV-1-
infected T-cell lines expressing high levels of ?-catenin protein.
In contrast, Tax was hardly detectable in the three ATL-de-
rived T-cell lines, which expressed low levels of ?-catenin (Fig.
1A, middle panel). No ?-catenin protein was detected in a
sample of normal peripheral blood mononuclear cells (data
not shown). To determine the cellular distribution of ?-cate-
nin, nuclear and cytoplasmic cell fractions from all cell lines
were analyzed by Western blotting. ?-Catenin was most abun-
dant in the nuclear fractions of the Tax-positive HTLV-1-
infected T-cell lines, while the Tax-negative cells showed rel-
atively smaller amounts of nuclear ?-catenin protein than of
the cytoplasmic pool (Fig. 1B).
Enhanced transcriptional activity of ?-catenin in Tax-pos-
itive HTLV-1-infected T-cell lines. To investigate whether the
nuclear accumulation of ?-catenin in Tax-positive HTLV-1-
10498TOMITA ET AL.J. VIROL.
on June 10, 2014 by guest
infected T-cell lines resulted in transcriptional activation of the
?-catenin/Tcf complex, both Tax-positive (MT-2, SLB-1, and
HUT-102) and Tax-negative [MT-1 and ED-40515(?)] T-cell
lines were transfected with Tcf reporter plasmids containing
three copies of the wild-type (pGL3-OT) or mutant (pGL3-
OF) Tcf site. The Tax-positive cells showing increased
?-catenin protein in the nucleus showed a higher level of
transcriptional activation of pGL3-OT than did the Tax-
negative HTLV-1-infected T-cell lines (Fig. 1C). Activation
of pGL3-OF was not observed in any cell lines. These results
suggested that the accumulation of ?-catenin in the nucleus
enhances the transcriptional activity of the ?-catenin/Tcf
Proteasome inhibition causes ?-catenin accumulation in
Tax-negative but not in Tax-positive HTLV-1-infected T-cell
lines. To determine whether the low level of ?-catenin protein
in the Tax-negative HTLV-1-infected T-cell lines was due to
proteasomal degradation, we treated the cells with LLnL, a
potent proteasome inhibitor, and then again analyzed the ex-
pression levels of ?-catenin protein by Western blot analysis
(Fig. 1D). In a Tax-negative T-cell line, ED-40515(?), signif-
icant accumulation of the ?-catenin protein was detected in the
presence of LLnL, in a time-dependent manner. A similar
result was obtained for another Tax-negative cell line, MT-1
(data not shown). In contrast, ?-catenin levels were not further
increased by LLnL treatment in the Tax-positive HUT-102 or
MT-2 cells (data not shown). LLnL treatment did not change
the expression level of Tax protein. Therefore, the difference in
?-catenin levels between Tax-positive and Tax-negative HTLV-
1-infected T-cell lines is attributable to differential degradation
but not to differential expression. These results tempted us to
investigate the role of the Tax protein in the activation of ?-cate-
Suppression of ?-catenin expression inhibited cell growth of
Tax-positive HTLV-1-infected T-cell line. To examine the role
of ?-catenin in HTLV-1-infected T-cell growth, HUT-102 cells
were transfected with ?-catenin siRNA. Cell growth was inhibited
in HUT-102 cells transfected with ?-catenin siRNA compared
with cells transfected with nontargeting siRNA (Fig. 2A). The
suppression of ?-catenin mRNA expression by ?-catenin siRNA
was confirmed by RT-PCR (Fig. 2B). These results indicated
FIG. 1. ?-Catenin is activated in Tax-positive HTLV-1-infected T
cells. (A) Overexpression of ?-catenin in a Tax-positive HTLV-1-
infected T-cell line. Total cell lysates were resolved by SDS-polyacryl-
amide gel electrophoresis and transferred to polyvinylidene difluoride
membranes for probing with anti-?-catenin and anti-Tax antibodies.
The arrow indicates the Tax protein, and the arrowhead indicates a
fusion protein between the envelope and the Tax coding sequence.
Actin was included as a loading control. (B) Western blot of nuclear
(N) and cytoplasmic (C) extracts from HTLV-1-infected T-cell lines
with anti-?-catenin antibody. ?-Catenin accumulated in the nuclei of
Tax-positive HTLV-1-infected T cells. Nucleolin and I?B? were used
as markers of nuclear and cytoplasmic integrity, respectively. (C)
Enhanced ?-catenin/Tcf transcriptional activity in Tax-positive HTLV-
1-infected T-cell lines. Cells were transfected with 0.1 ?g Tcf-4 expres-
sion plasmid and 2 ?g luciferase reporter plasmid containing the wild-
type (pGL3-OT) or mutant (pGL3-OF) Tcf site. After 48 h, cells were
collected, and transcriptional activity was determined by a luciferase
assay. Relative luciferase activities were measured in cell extracts and
normalized to the Renilla luciferase activity. Luciferase activity is pre-
sented as x-fold induction relative to the luciferase activity measured in
MT-1 cells. Data represent the means ? standard deviations (SD) for
three separate experiments. (D) Proteasome inhibition causes ?-cate-
nin accumulation in Tax-negative HTLV-1-infected T-cell lines. Tax-
negative [ED-40515(?)] and Tax-positive (HUT-102) HTLV-1-in-
fected T-cell lines were treated with 20 ?M LLnL for the indicated
times before collection. Western blots of total protein extracts from
the cells were probed with ?-catenin, Tax, and actin antibodies.
VOL. 80, 2006TAX DYSREGULATES ?-CATENIN 10499
on June 10, 2014 by guest
the specific role of ?-catenin in the growth of Tax-positive
HTLV-1-infected T cells.
Tax induces the accumulation of ?-catenin protein. To
determine whether Tax affects endogenous ?-catenin levels,
HeLa cells were transfected with increasing amounts of a wild-
type Tax (Tax WT) expression plasmid. Total cell extracts and
total RNA from transfected HeLa cells were analyzed for
?-catenin expression by Western blot and RT-PCR analyses,
respectively. Increased levels of ?-catenin protein were de-
tected in the presence of Tax, in a dose-dependent manner
(Fig. 3A). In contrast, ?-catenin mRNA levels were not
changed by Tax (Fig. 3B). Next, to determine the signaling
pathway responsible for the Tax-induced ?-catenin protein
accumulation, we used three Tax mutant expression plasmids,
which have been described previously (13, 24). Tax M22, which
has amino acid substitutions at residues 130 and 131, from
Thr-Leu to Ser-Ala, effectively activates the cyclic AMP re-
sponse element (CRE), which mediates the Tax-dependent
activation of the HTLV-1 LTR but not of the NF-?B element.
Tax 703 has amino acid substitutions at residues 319 and 320,
from Leu-Leu to Arg-Ser, which make it equivalent to mutant
M47, and Tax K88A carries a single amino acid substitution at
position 88, from Lys to Ala. Tax 703 and Tax K88A activate
the NF-?B element but do not affect the CRE. In the current
experiments, Tax M22, but not Tax 703 or Tax K88A, in-
creased the ?-catenin protein level (Fig. 3A). In contrast,
?-catenin mRNA levels were not altered by any of the Tax
mutants (Fig. 3B). The expression levels of Tax protein and
mRNA were increased independently of the amount of plas-
mid used (Fig. 3A and B). These results suggested that Tax
induces ?-catenin expression at the posttranscriptional level by
activation of the CREB signaling pathway.
Tax stabilizes ?-catenin protein. To elucidate the mecha-
nisms by which Tax induces ?-catenin protein accumulation,
we examined the effects of Tax on ?-catenin turnover. HeLa
cells were transfected with either empty vector [Tax (?)], wild-
type Tax (Tax WT), or mutant Tax (M22, 703, or K88A)
expression plasmids. To evaluate the degradation of ?-catenin
proteins, we also transfected ?-catenin expression plasmids
together with Tax. Cycloheximide (12.5 ?g/ml) was added to
the cell culture medium 24 h after transfection to block new
protein synthesis, and protein extracts were prepared 0, 2, 4,
and 6 h after the addition of cycloheximide. Western blotting
showed that ?-catenin was rapidly degraded in HeLa cells
transfected with empty vector [Tax (?)], whereas ?-catenin
levels remained stable in the presence of Tax WT throughout
FIG. 2. Repression of ?-catenin expression suppresses cell growth
of HTLV-1-infected T-cell line. (A) HUT-102 cells were transfected
with siRNA to repress ?-catenin (?) or with a nontarget siRNA (?).
The effect of siRNA on cell growth was examined by counting the
viable cell number in triplicate by the trypan blue dye exclusion
method. Data are expressed as means ? SD. (B) RT-PCR analysis
showing repression of ?-catenin mRNA in HUT-102 cells transfected
with ?-catenin siRNA (?) compared to that in cells transfected with a
nontargeting siRNA (?) 24, 48, and 72 h after transfection. ?-Actin
expression was used as the cDNA loading control.
FIG. 3. Tax induces increased protein levels but does not change
mRNA levels for endogenous ?-catenin. HeLa cells were transfected
with increasing amounts of wild-type Tax (Tax WT) or Tax mutant
(Tax M22, Tax 703, and Tax K88A) expression plasmids. (A) Total cell
extracts from transfected HeLa cells were analyzed for ?-catenin and
Tax protein expression by Western blot analysis. Actin was used as a
loading control. (B) ?-Catenin mRNA expression was evaluated by
RT-PCR analysis of transfected HeLa cells. One microgram of total
RNA extracted from each transfected HeLa cell sample was used for
reverse transcription. PCR was then performed with the primers for
?-catenin, Tax, and ?-actin.
10500 TOMITA ET AL.J. VIROL.
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the 6-h chase period (Fig. 4). The increased ?-catenin levels
induced by Tax are therefore a consequence of protein stabi-
lization. Expression of the mutant protein Tax M22 also sta-
bilized ?-catenin protein levels for 6 h, whereas cells trans-
fected with the 703 and K88A mutants showed reduced levels,
as in the controls. These results suggested that the Tax-induced
activation of the CREB signaling pathway stabilizes the cellu-
lar levels of ?-catenin.
Tax enhances ?-catenin/Tcf transcriptional activity. We
next asked whether the stabilization of ?-catenin protein by
Tax affected ?-catenin/Tcf transcriptional activity (Fig. 5A). In
a transient transfection assay using pGL3-OT as a reporter, the
expression of Tax WT or ?-catenin alone in HeLa cells acti-
vated the reporter. The combination of ?-catenin and Tax WT
had an even greater effect. No significant responses were seen
with the mutant reporter (pGL3-OF) (data not shown). These
results suggested that ?-catenin and Tax activate Tcf synergis-
tically. We also tested the effects of Tax mutants on ?-catenin/
Tcf transcriptional activity. As with ?-catenin protein stabili-
zation, Tax M22, but not the 703 or K88A mutant, activated
the pGL3-OT reporter and acted synergistically with ?-cate-
nin. None of the Tax mutants affected the pGL3-OF reporter
activity (data not shown). Together, these sets of experiments
suggest that the stabilization of ?-catenin protein by Tax en-
hances the transcriptional activity of ?-catenin/Tcf and that
this effect of Tax is mediated via the CREB signaling pathway.
Dominant-negative CREB restores the Tax-induced activa-
tion of ?-catenin/Tcf transcriptional activity. To further ascer-
tain the biological role of CREB in transcriptional activation of
the ?-catenin/Tcf complex by Tax, HeLa cells were transfected
with KCREB, a dominant-negative version of CREB. The
pCMV-KCREB construct encodes a CREB protein with Arg
287 mutated to Leu in the DNA-binding domain. KCREB acts
as a dominant-negative repressor of wild-type CREB, blocking
its binding to CRE. HeLa cells were transfected with ?-cate-
nin, Tcf-4, and the pGL3-OT or pGL3-OF reporter plasmid in
the presence or absence of Tax WT and increasing amounts of
KCREB (Fig. 5B). KCREB inhibited the Tax-induced acti-
vation of pGL3-OT luciferase activity in a dose-dependent
manner, whereas no significant responses were seen with the
pGL3-OF reporter plasmid. An HTLV-1 LTR luciferase re-
porter (LTR-Luc) containing three unique CRE-containing
FIG. 4. The half-life of ?-catenin is extended in HeLa cells trans-
fected with Tax. HeLa cells were transfected with 0.5 ?g ?-catenin and
1 ?g empty vector (?) or a wild-type Tax (Tax WT) or Tax mutant
(M22, 703, or K88A) expression plasmid. Transfected HeLa cells were
incubated with 12.5 ?g/ml cycloheximide (CHX) to block new protein
synthesis. Extracts were prepared at the indicated times after the
cycloheximide block and then were analyzed for ?-catenin and Tax
protein expression by Western blotting. Actin was included as a load-
FIG. 5. Tax enhances transcriptional activity of ?-catenin/Tcf
through activation of the CREB signaling pathway. (A) HeLa cells
were transfected with 0.1 ?g Tcf-4 expression plasmid and 2 ?g lucif-
erase reporter plasmid containing the wild-type (pGL3-OT) Tcf site in
the presence (?) or absence (?) of 1 ?g Tax WT or a Tax mutant
(M22, 703, or K88A) and 0.5 ?g ?-catenin, as indicated. (B) HeLa cells
were transfected with 0.5 ?g ?-catenin, 0.1 ?g Tcf-4 expression plas-
mid, and 2 ?g luciferase reporter plasmid containing a wild-type
(pGL3-OT) or mutant (pGL3-OF) Tcf site in the presence (?) or
absence (?) of 1 ?g Tax WT and increasing amounts of dominant-
negative CREB (KCREB) expression plasmid (0, 0.1, 0.5, and 1 ?g), as
indicated. (C) HeLa cells were transfected with 0.1 ?g LTR-Luc or
?B-Luc reporter plasmid, together with 0.5 ?g Tax WT and increasing
amounts of KCREB (0, 0.1, 0.5, and 1 ?g), as indicated. After 48 h, the
cells were collected, and transcriptional activity was determined by a
luciferase assay. Relative luciferase activities were measured in cell
extracts, normalized to the Renilla luciferase activity, and presented as
x-fold induction relative to the basal level measured in cells transfected
with Tcf-4 and reporter plasmids without Tax or ?-catenin (A), with
?-catenin, Tcf-4, and reporter plasmids (B), or with reporter plasmids
alone without Tax or KCREB (C). Data represent means ? SD for
three separate experiments.
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21-bp repeats was used to confirm the effects of KCREB on the
action of Tax. HeLa cells were transfected with LTR-Luc or a
?B-Luc reporter plasmid in the presence or absence of Tax WT
and increasing amounts of KCREB. Tax could activate gene
expression from both the HTLV-1 LTR and NF-?B reporters.
However, KCREB inhibited only HTLV-1 LTR transcriptional
activity, not NF-?B transcriptional activity (Fig. 5C), indicating
that KCREB blocks Tax-induced activation of the CREB, but
not NF-?B, pathway. These results indicated that Tax activates
the ?-catenin/Tcf transcriptional activity through the activation
of CREB signaling.
Accumulation of ?-catenin protein correlates with activa-
tion of Akt signaling by Tax. The activation of Akt signaling
has been associated with an accumulation of ?-catenin (36).
Akt activation can be mediated by the activation of PI3K, and
Tax activates the PI3K pathway (21). In addition, Jeong and
colleagues recently showed that Akt is activated in HTLV-1-
transformed T-cell lines and that Tax activates Akt in these
cells (16). To determine whether Akt activation is associated
with the stabilization of ?-catenin in the HTLV-1-infected
T-cell lines, we examined the phosphorylation status of Akt
and the in vitro Akt kinase activity relative to ?-catenin protein
expression (Fig. 6A). Phosphorylated Akt was strongly de-
tected in the three Tax-positive HTLV-1-infected T-cell lines
(MT-2, SLB-1, and HUT-102), whereas only weak expression
FIG. 6. Tax enhances ?-catenin/Tcf transcriptional activity through
the Akt signaling pathway. Total lysates of HTLV-1-infected T-cell lines
(A), of HeLa cells transfected with 1 ?g Akt and 1 ?g Tax WT or a Tax
mutant (M22, 703, or K88A), as indicated (B), of MT-2 cells treated with
the PI3K inhibitor LY294002 (20 ?M) for the indicated periods (C), or
of MT-1 and MT-2 cells treated with 10 mM lithium chloride (LiCl)
(GSK-3? inhibitor) for 24 h (D) were resolved by SDS-polyacrylamide gel
electrophoresis, and Western blots were probed with anti-phosphorylated
Akt (Ser473 [p-Akt]), anti-Akt, anti-phosphorylated GSK-3? (Ser9 [p-
GSK-3?]), anti-GSK-3?, anti-Tax, and anti-actin antibodies, as indicated.
To assess Akt kinase activity, in vitro Akt kinase assays with GSK-3?/? as
a substrate were performed. Phosphorylated GSK-3?/? was detected with
B). (E) HeLa cells were transfected with 0.1 ?g Tcf-4 expression plasmid
and 2 ?g luciferase reporter plasmid containing the wild-type (pGL3-OT)
or mutant (pGL3-OF) Tcf site together with 1 ?g Tax and 1 ?g of either
wild-type Akt (Akt-WT) or dominant-negative Akt (Akt-DN) expression
plasmid, as indicated. After 48 h, the cells were collected, and transcrip-
tional activities were determined by a luciferase assay. Relative luciferase
activities were measured in cell extracts, normalized to the Renilla lucif-
erase activity, and presented as x-fold induction relative to the basal level
measured in cells transfected with Tcf-4 and reporter plasmids without
Tax or Akt. Data represent means ? SD for three separate experiments.
10502TOMITA ET AL. J. VIROL.
on June 10, 2014 by guest
of this phosphorylated protein was detected in the Tax-nega-
tive T-cell lines [MT-1, TL-OmI, and ED-40515(?)]. Total Akt
protein was similarly expressed in all cell lines. The in vitro Akt
kinase activity was also higher in Tax-positive T-cell lines than
in those that were Tax negative. Next, to examine how Tax
regulates Akt signaling activity, Tax WT or a Tax mutant (M22,
703, or K88A) was expressed in HeLa cells together with Akt.
The activation of Akt was assessed by Western blot analysis of
phosphorylated Akt and GSK-3?, which is a downstream tar-
get of Akt kinase. Tax WT and Tax M22 increased the phos-
phorylation of Akt and GSK-3?, while Tax 703 and Tax K88A
induced no phosphorylation of either protein. Total Akt and
GSK-3? protein levels were not affected by any of the ex-
pressed plasmids. These results indicated that Tax also acti-
vates Akt signaling via activation of the CREB pathway. The
expression of ?-catenin protein was also increased by Tax WT
and Tax M22, but not by the 703 and K88A mutants (Fig. 3),
suggesting that the accumulation of ?-catenin protein might be
mediated by activation of the Akt signaling pathway, resulting
in inactivation of GSK-3?.
Accumulation of ?-catenin protein is regulated by the PI3K/
Akt signaling pathway in HTLV-1-infected T-cell lines. To
examine the role of Akt signaling in the accumulation of
?-catenin in HTLV-1-infected T cells, Tax-positive MT-2 cells
were treated with the PI3K inhibitor LY294002 (Fig. 6C). In
the presence of 20 ?M LY294002, ?-catenin expression was
significantly reduced, in a time-dependent manner. Phosphor-
ylation of both Akt and GSK-3? was inhibited by LY294002,
whereas the total levels of both proteins were unaffected. The
expression of Tax was not changed by LY294002 treatment.
These data suggested that overexpression of ?-catenin in MT-2
cells is mediated by constitutive activation of PI3K/Akt signal-
ing. Next, to elucidate the role of GSK-3? activity in regulating
?-catenin expression, we used lithium chloride, which acts as a
noncompetitive inhibitor of GSK-3? (19) (Fig. 6D), and ob-
served an induction of ?-catenin protein expression in Tax-
negative MT-1 cells, whose GSK-3? proteins are active (de-
phosphorylated at Ser9). Similar results were obtained with
another Tax-negative cell line [ED-40515(?) (data not shown)].
In contrast, ?-catenin levels were not increased after lithium chlo-
ride treatment in the Tax-positive MT-2 cells (Fig. 6D) or HUT-
102 cells (data not shown), whose GSK-3? proteins are already
inactive (phosphorylated at Ser9). The expression of Tax itself
was not changed by lithium chloride treatment. These results
indicated that LiCl increases ?-catenin levels in cell lines which
3?, suggesting that the difference in ?-catenin levels between
Tax-positive and Tax-negative HTLV-1-infected T-cell lines is
attributable to the differential activity of GSK-3?.
Dominant-negative Akt inhibits Tax-induced ?-catenin/Tcf
transcriptional activity. To further study the enhancive effect
of Tax and Akt activation on ?-catenin/Tcf-mediated transcrip-
tion, we used wild-type (Akt-WT) and dominant-negative mu-
tant (Akt-DN) Akt expression plasmids to directly examine the
involvement of Akt in Tax-induced ?-catenin/Tcf transcription
(Fig. 6E). Akt-WT enhanced pGL3-OT activity induced by
Tax, while Akt-DN suppressed the Tax-induced pGL3-OT ac-
tivity. Both the wild-type and dominant-negative mutant Akt
plasmids did not affect pGL3-OF activity. These data demon-
strated that ?-catenin/Tcf transcriptional activation by Tax is
mediated via the Akt signaling pathway.
In this study, we observed an accumulation of nuclear
?-catenin protein and an enhanced transcriptional activity of
?-catenin/Tcf in Tax-positive HTLV-1-infected T-cell lines but
not in those that were Tax negative. Proteasome inhibition
restored ?-catenin protein expression in Tax-negative, but not
Tax-positive, T-cell lines, suggesting that Tax might stabilize
the ?-catenin protein by inhibiting protein degradation. Trans-
fection with ?-catenin siRNA inhibited cell growth of HUT-
102 cells, a Tax-positive HTLV-1-infected T-cell line. We
further demonstrated that HTLV-1 Tax activates ?-catenin/
Tcf-dependent transcription by stabilizing the ?-catenin pro-
tein through the CREB signaling pathway in Tax-transfected
HeLa cells. Furthermore, transient expression of Tax in HeLa
cells led to the CREB-dependent phosphorylation and activa-
tion of Akt and the subsequent phosphorylation and inactiva-
tion of the Akt target, GSK-3?. In turn, dominant-negative Akt
inhibited the Tax-induced ?-catenin/Tcf transcriptional activ-
ity, and upon inactivation of the negative Wnt signaling regulator,
GSK-3?, ?-catenin was stabilized to therefore activate ?-catenin/
Tcf-dependent transcription (Fig. 7). Treatment of Tax-positive
T-cell lines with a PI3K inhibitor and inactivation of GSK-3? in
Tax-negative T-cell lines implicated GSK-3? inactivation in the
process of ?-catenin accumulation in HTLV-1-infected T cells.
The Akt signaling pathway is important for the survival and
growth of numerous types of cancer cells. Previous studies
showed that Tax induces PI3K signaling activation and that this
activation is associated with transformation of Rat-1 fibroblast
cells stably expressing Tax (21). More recently, Jeong and
FIG. 7. Schematic representation of the effects of Tax on the
?-catenin signaling pathway. Tax activates PI3K/Akt through activat-
ing the CREB signaling pathway, although the mechanism of this
activation remains to be elucidated. Akt subsequently phosphorylates
and inhibits GSK-3?, a negative regulator of ?-catenin. Inactivation of
GSK-3? prevents proteasomal degradation of ?-catenin, and in turn,
activated ?-catenin can translocate to the nucleus and bind to the
transcription factor Tcf.
VOL. 80, 2006TAX DYSREGULATES ?-CATENIN10503
on June 10, 2014 by guest
colleagues demonstrated that Akt signaling is activated in
HTLV-1-transformed cells and that Tax can activate this sig-
naling pathway by inducing Akt phosphorylation (16). These
observations indicated that Tax-induced Akt signaling activa-
tion plays an important role in the transformation of HTLV-
1-infected cells. Consistent with these findings, we found here
that phosphorylation and activation of Akt were associated
with Tax expression in HTLV-1-infected T-cell lines and that
Tax induced Akt activation in transfected HeLa cells. More-
over, we demonstrated, for the first time, an association be-
tween the activation of CREB signaling by Tax and the Tax-
induced activation of Akt. Recently, silencing of CREB gene
expression by RNA interference decreased the phosphoryla-
tion of Akt induced by forskolin stimulation (25). However, it
remains unclear exactly how Tax activates Akt through the
CREB signaling pathway, and future studies need to address
this issue. During preparation of this article, a study showing
Tax-mediated Akt activation was published by Kuan-Teh
Jeang’s lab (32). They concluded that Tax-mediated Akt acti-
vation depended on the ability of Tax to interact with the p85?
subunit of PI3K but not on the CREB-activating activity of
Tax. Moreover, they found that a Tax mutant, Tax M22, could
not activate Akt in mouse embryonic fibroblasts (MEFs). In
the present study, we demonstrated that Tax M22 could acti-
vate Akt in HeLa cells. However, they reported that a Tax
mutant with disrupted NF-?B activation, Tax S258A, could
activate Akt in MEFs. The precise reason for these differences
is not clear, but we cannot exclude the possibility that these
differences could be attributable to the differences in the cell
lines which were used for the Akt assay. We used HeLa cells,
which are highly transformed, but primary cells (such as
MEFs) were used in Jeang’s study. Further analysis is needed
to elucidate whether the differences between the two studies
are due to the differences in the cells which were used for the
Recently, Yang et al. demonstrated that the APC gene pro-
moter region was methylated in some cases of acute or chronic
ATL (42). Epigenetic modifications can affect gene expression
and contribute to the pathogenesis of tumor formation and
growth. Methylation of CpG islands within tumor suppressor
genes is an important oncogenic mechanism in certain cancers,
including hematological malignancies. Yang’s results suggested
that a loss of APC gene expression by methylation of its pro-
moter might lead to ?-catenin stabilization. However, we ob-
served normal expression of the APC protein in all HTLV-1-
infected T-cell lines tested here (data not shown). Therefore,
diminished APC function by hypermethylation may not be a
common mechanism for inducing ?-catenin activation in HTLV-
1-infected T cells.
In this study, we demonstrated that the CREB-activating ac-
tivity of Tax is important for Akt and ?-catenin activation, which
enhances cell growth and survival. Previous studies reported that
the CREB pathway is required for the clonal expansion of CD4?
and CD8?T cells (1), and a Tax mutant which is active for CREB
but deficient in NF-?B signaling can immortalize human primary
T lymphocytes (35). Consisting with our findings, these results of
previous studies indicated that the Tax-activated CREB pathway
plays an important role in the permanent growth and immortal-
ization of human T lymphocytes.
What is the specific role of ?-catenin in Tax-mediated biol-
ogy? To answer this question, we demonstrated that transfec-
tion with ?-catenin siRNA inhibited the growth of the HTLV-
1-infected T-cell line HUT-102, which expresses high levels of
?-catenin protein. Our results are consistent with a previous
study showing that the growth of HUT-102 cells was inhibited
by overexpression of a dominant-negative ?-catenin or domi-
nant-negative Tcf expression plasmid (4). These results impli-
cated ?-catenin in the cell growth of HTLV-1-infected T cells.
Enhanced expression of the ?-catenin-regulating genes, such
as cyclin D1 and c-myc, which regulate cell cycle progression
and apoptosis, has been observed in Tax-positive HTLV-1-
infected T-cell lines (data not shown). It could thus be pro-
posed that overexpression of these proteins might result in
malignant cell growth of HTLV-1-infected T cells. Impor-
tantly, ?-catenin expression was not increased in peripheral
blood mononuclear cells from ATL patients (data not shown).
Because the expression of Tax was not detected in these ATL
cells, overexpression of ?-catenin may depend on Tax expres-
sion and may not be necessary to maintain the malignant phe-
notype in the late (Tax-independent) stage of ATL.
The Wnt/?-catenin signaling pathway has been identified as a
common target for perturbation by viruses, as demonstrated by
the following examples from the literature. Similar to the effect of
Tax on ?-catenin signaling, Epstein-Barr virus encodes latent
membrane protein 2A, which activates PI3K and Akt, resulting
in GSK-3? inactivation and ?-catenin stabilization (28). The
latency-associated nuclear antigen of Kaposi’s sarcoma-associ-
ated herpesvirus binds to GSK-3? and sequesters it in the
nucleus, preventing ?-catenin phosphorylation (6, 7). The large T
antigen of JC virus, a human polyomavirus, interacts with ?-cate-
nin, stabilizing it and promoting its nuclear accumulation as well
as activating a c-myc promoter (5, 8). The Vpu protein of human
immunodeficiency virus type 1 binds to the ?-transducin repeat-
containing protein and blocks the ubiquitination and proteasomal
degradation of ?-catenin (2). Hepatitis B virus X protein achieves
?-catenin stabilization by suppressing GSK-3? activity in an Src
kinase-dependent manner (3). Finally, hepatitis C virus NS5A
activates PI3K, resulting in stabilization of ?-catenin (38). There-
fore, the Wnt pathway is targeted by many different oncogenic
viral proteins via distinct mechanisms, indicating the importance
of this pathway in the genesis of virus-associated tumors.
In summary, the data presented here show that ?-catenin
signaling is activated in Tax-positive HTLV-1-infected T-cell
lines. Activation of the Akt pathway by Tax induced the inac-
tivation of GSK-3?, leading to stabilization of the ?-catenin
protein. The increased ?-catenin expression induced by Tax
was followed by an upregulation of ?-catenin-induced tran-
scriptional activity, and the activation of ?-catenin through the
Akt signaling pathway was mediated by the activation of
CREB signaling via Tax activation. Together, our results im-
plicate an important role for Tax in the activation of the
?-catenin signaling pathway and thereby in the transformation
of T lymphocytes by HTLV-1 infection.
We thank M. Maeda for providing the ED-40515(?) cell line; the
Fujisaki Cell Center, Hayashibara Biomedical Laboratories (Okayama,
Japan), for providing the HUT-102 and MT-1 cell lines; B. Vogelstein
for providing pGL3-OT and pGL3-OF; J. Fujisawa for providing ?B-
Luc; I. Futsuki for providing LTR-Luc; K. Matsumoto for providing
Tax WT, Tax M22, and Tax 703; C.-Z. Giam for providing Tax K88A;
10504 TOMITA ET AL.J. VIROL.
on June 10, 2014 by guest
and D. Alessi for the wild-type and dominant-negative Akt expression
plasmids. We also acknowledge all members of our laboratories for
their helpful comments and collaborations.
This work was supported in part by grants-in-aid 16590951 and
17790654 from the Japan Society for the Promotion of Science and
grant-in-aid 16017289 from the Ministry of Education, Culture, Sports,
Science and Technology of Japan and by the Takeda Science Foun-
dation, the Uehara Memorial Foundation, and the Foundation for
Promotion of Cancer Research in Japan.
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on June 10, 2014 by guest
JOURNAL OF VIROLOGY, Feb. 2011, p. 1417
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 3
Human T-Cell Leukemia Virus Type 1 Tax Dysregulates ?-Catenin Signaling
Mariko Tomita, Akira Kikuchi, Tetsu Akiyama, Yuetsu Tanaka, and Naoki Mori
Division of Molecular Virology and Oncology, Graduate School of Medicine, and Division of Immunology, Faculty of Medicine,
University of the Ryukyus, Nishihara, Okinawa, Japan; Department of Biochemistry, Graduate School of
Biomedical Science, Hiroshima University, Hiroshima, Japan; and Laboratory of Molecular and
Genetic Information, Institute for Molecular and Cellular Biosciences,
University of Tokyo, Tokyo, Japan
Volume 80, no. 21, p. 10497–10505, 2006. The publisher hereby retracts the above article due to evidence of data manipulation,
a clear violation of ASM’s ethical standards.