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INTRODUCTION |
FKHRL1, a mammalian homolog of DAF-16, belongs to the Forkhead
transcription factor family (1). This family, characterized by the
presence of a highly conserved forkhead domain having a winged-helix
motif and DNA binding activity, is involved in embryogenesis, differentiation, and tumorigenesis (2). DAF-16 plays an important role
in the longevity of Caenorhabditis elegans (3). In C. elegans, the dauer lava ensures its survival in adverse conditions by lowering its metabolism and changing its shape and can live up to
ten times longer than a normal adult (4). DAF-16 induces dauer
formation and its activity is negatively regulated by DAF-2. Indeed, in
C. elegans, a loss-of-function mutation in the
DAF-2 gene causes developmental arrest at the dauer
stage via the enhancement of DAF-16 transcription activity, resulting
in an extension of the life span (5). These observations strongly
suggested that FKHRL1 plays an important role in mammalian biology.
This notion is supported by the evidence that there is a highly
conserved signaling pathway between C. elegans and human.
DAF-2 is an insulin receptor-like protein, AGE-1 is a catalytic subunit
of phosphatidylinositol 3-kinase
(PI3K)1-like protein, DAF-23
is a PI3K-like protein, AKT1/AKT2 is a serine/threonine kinase protein
kinase B (PKB) (also known as Akt)-like protein, and DAF-16 has high
homology to the mammalian Forkhead subfamily FKHRL1, AFX, and FKHR
(4).
Akt has been identified as a downstream target of PI3K necessary for
survival (6). Akt activated by growth factors phosphorylates apoptosis-associated molecules including Bad, caspase-9, IKK
, and
GSK-3 ensuring cell survival (7-10). In addition, the constitutively active form of Akt blocks apoptosis induced by growth factor
deprivation (11, 12). Thus, the PI3K-Akt activation pathway appears to be a prerequisite for cell survival. Recently, FKHRL1, AFX, and FKHR
have been identified as substrates of Akt (13-19). Phosphorylation of
these proteins by Akt regulates their nuclear translocation and targets
gene transcription. There are two or three potential Akt
phosphorylation sites (RXRXX(S/T)) on these three
members. For example, FKHRL1 has three putative phosphorylation sites; Thr32 (RPRSCT32),
Ser253 (RRRAVS253), and
Ser315 (RSRTNS315). When cells are
stimulated with serum or growth factors, a phosphorylated form of
FKHRL1 is retained in the cytoplasm and interacts with 14-3-3 proteins,
resulting in inhibition of target gene transcription. By contrast, when
cells are deprived of serum or growth factors, a nonphosphorylated form
of FKHRL1 translocates into nucleus and activates the transcription of
target genes. These findings indicate that FKHRL1, when not
phosphorylated by Akt, is an activator of transcription. Thus,
phosphorylation by Akt is essential for suppressing the transcription
activity of FKHRL1. In other words, Akt negatively regulates the
transcription activity of FKHRL1 by phosphorylation (13). This is also
true for both AFX and FKHR (14-18).
Very recently we identified FKHRL1 as one of the downstream molecules
of the phosphatidylinositol 3-kinase-Akt activation pathway in
erythropoietin (EPO) signal transduction (12), although the functional
role of this molecule in erythropoiesis is still open to question.
Based on several lines of evidence that there is a close relationship
between erythropoiesis and megakaryopoiesis (20-22), we examined
whether or not TPO, a major regulator of megakaryopoiesis, induces
phosphorylation of Akt and FKHRL1 in a human TPO-dependent leukemia cell line UT-7/TPO (23) and highly purified normal human
megakaryocytes. Moreover, to elucidate the function of FKHRL1 in TPO
signaling, we established a tetracycline (Tet)-inducible expression
system in UT-7/TPO cells. Our results suggest that FKHRL1 is
phosphorylated by TPO stimulation via Akt activation and that
unphosphorylated FKHRL1 negatively regulates the cell cycle, presumably
via the activation or inactivation of cell cycle-associated gene(s).
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EXPERIMENTAL PROCEDURES |
Hematopoietic Growth Factors and Reagents--
Recombinant TPO
was kindly donated by Kirin Brewery Co. Ltd. (Tokyo, Japan).
2-mercaptoethanol, sodium orthovanadate, bovine serum albumin (BSA),
DNase, Iscove's modified Dulbecco's medium (IMDM), saturated
transferrin, oleic acid, L-
-phosphatidylcholine, cholesterol, and propidium iodide were purchased from Sigma. Vitamin B12 and folic acid were purchased from Sankyo Pharmaceutical Co. (Tokyo, Japan) and Takeda Pharmaceutical Co. (Osaka, Japan),
respectively. Dynabeads M (tm) 450 coated with goat anti-mouse IgG was
from Dynal Inc. (Great Neck, NY). Fetal calf serum (FCS), penicillin, and streptomycin were from Flow Laboratories, Inc. (McLean, VA). Insulin (porcine sodium, activity 26.3 United States Pharmacopoeia units/mg) was purchased from CalBiochem and Behring Diagnostics (La
Jolla, CA). Polyclonal antibodies against AKT (C-20) were purchased
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies against
phosphothreonine 32 [phospho-Thr32]FKHRL1, phosphoserine
253 [phospho-Ser253]FKHRL1, native FKHRL1, GST·FKHRL1
fusion protein, and cDNAs for wild-type and triple mutant FKHRL1
were kindly provided by Dr. Anne Brunet (Children's Hospital, Harvard
Medical School, Boston) (13). Phosphoplus AKT (Ser473)
Antibody Kit and AKT Kinase Assay Kit were purchased from New England
BioLabs Inc. (Beverly, MA). MEBCYTO-Apoptosis Kit was purchased from
MBL (Nagoya, Japan).
Cell Culture of UT-7/TPO Cell Line--
UT-7/TPO was maintained
in liquid culture with IMDM containing 10% FCS and 10 ng/ml TPO (23).
Colorimetric MTT assay for Cell Proliferation--
Cell growth
was examined by a colorimetric assay according to Mosmann with some
modifications (24). Briefly, cells were incubated at a density of
1 × 104/100 µl2 in 96-well plates in
IMDM containing 10% FCS in the presence of TPO (10 ng/ml). After
72 h of culture at 37 °C, 20 µl of sterilized 5 mg/ml
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;
Sigma) was added to each well. Following a 2-h incubation at 37 °C,
100 ml of 10% SDS was added to each well to dissolve the dark-blue
crystal product. The absorbance was measured at a wavelength of
595 nm using a microplate reader (model 3550; Bio-Rad, Richmond, CA).
Preparation of Cell Lysates, Immunoprecipitation, and Western
Blotting--
UT-7/TPO cells were deprived of growth factor for
24 h. After stimulation with TPO at 37 °C for a given period,
cells were washed and suspended in lysis buffer composed of 20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml aprotinin, and 2 mM sodium orthovanadate. After 20 min of incubation on ice, insoluble materials were removed by
centrifugation at 15,000 × g for 10 min. The
supernatants were immunoprecipitated with anti-Akt antibody (C-20)
attached to protein G-Sepharose for 4 h at 4 °C in an Eppendorf
shaker. Immunoprecipitates were collected by a brief centrifugation and
washed four times with 1 ml of lysis buffer. The immunoprecipitated
proteins were boiled for 5 min in SDS-PAGE sample buffer. After a brief
centrifugation, the supernatants were resolved by SDS-PAGE and were
electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad). The
blots were blocked with 5% skim milk in Tris-buffered saline for
1 h and then incubated with the appropriate concentration of
primary antibodies including anti-phosphoAkt(Ser473)
polyclonal antibody overnight at 4 °C. After a wash with TBS containing Tween 20 (1:1,000), the blots were probed with a 1:2,000 dilution of anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 20 min at room temperature. After a second wash, the
blots were incubated with an enhanced chemiluminescence substrate according to the instruction manual (New England BioLabs Inc.). In some
experiments, the supernatants were boiled for 5 min in SDS-PAGE sample
buffer containing 20 mM Tris, pH 7.4, 150 mM
NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 15 µg/ml aprotinin, 20 µg/ml
leupeptin, 2 mM sodium orthovanadate, and 20 mM
sodium fluoride. After a brief centrifugation, the supernatants were
resolved by SDS-PAGE and then electroblotted onto a PVDF membrane. The
blots were blocked with 5% skim milk in Tris-buffered saline for
1 h at room temperature and then incubated with the appropriate
concentration of primary antibody against
[phospho-Thr32]FKHRL1,
[phospho-Ser253]FKHRL1, or native FKHRL1 overnight at
4 °C. After a wash with TBS containing Tween 20 (1:2,000), the blots
were probed with a 1:5,000 dilution of anti-rabbit or anti-mouse
horseradish peroxidase-conjugated second antibodies for 90 min at room
temperature. After a second wash, the blots were incubated with an
enhanced chemiluminescence substrate (ECL Western blot detection
system; Amersham Pharmacia Biotech, Buckinghamshire, England) and
exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) to visualize
immunoreactive bands. The blots were stripped with 62.5 mM
Tris-HCI, pH 6.8, 2% SDS, and 100 µM 2-mercaptoethanol
at 50 °C for 30 min and were washed, blocked, and reprobed.
In Vitro Kinase Assay--
Immunoprecipitates were washed three
times with lysis buffer and once with the Akt kinase buffer (20 mM HEPES-NaOH, pH7.4, 10 mM MgCl2,
10 mM MnCl2). In vitro kinase
experiments were performed with a commercial kit (AKT Kinase Assay Kit)
using GSK-3 or GST·FKHRL1 fusion protein (13) as another substrate of
Akt. Reaction products were resolved by SDS-PAGE and visualized by ECL.
Tet-inducible Expression System--
To express FKHRL1-TM
cDNA, we used a T-RExTM system (Invitrogen, Carlsbad,
CA), in which transcription of a target cDNA is initiated by Tet
treatment. In brief, UT-7/TPO cells initially were transfected with
expression vectors for the Tet repressor, pcDNA6/TR, by lipofection. After culture with 0.5 µg/ml of blasticidin (Invitrogen), one clone
(designated UT-7/TPO/pcDNA6/TR-2) was transfected further with a
Tet-inducible expression vector, pcDNA4/TO, for FKHRL1-TM. After
selection with Zeocin (Invitrogen) at a concentration of 200 µg/ml,
the induction levels of the FKHRL1-TM protein were examined before and
after 1 µg/ml Tet treatment by Western blotting analyses. The
transfectants designated UT-7/TPO/FKHRL1-TM clone 1 and clone 2 were
subjected to further analyses because the target proteins were induced
most efficiently by Tet treatment in these clones.
Confocal Microscopy--
Cytospin preparations were fixed and
permeabilized in PBS containing 4.0% paraformaldehyde and 0.4% Triton
X-100 for 20 min at room temperature. After a wash with PBS, the cells
were incubated with anti-FKHRL1 antibody diluted 1:20 in PBS containing
2% FCS and 0.05% Tween 20 at 4 °C at overnight. After a second
wash with PBS, the cells were incubated for 60 min with Alexa Fluor TM
488 goat anti-rabbit IgG (H+L) conjugate (Molecular Probes, Eugenes, OR) diluted 1:1000. Then, the cells were washed with PBS and mounted in
Perma Fluor Aqueous Mountant (Shandon/Lipshaw/Immunon, Pittusburgh, PA). Laser confocal scanning images were obtained using a TCS 4D
confocal system (Leica Instruments, Wetzlar, Germany).
Ex Vivo Generation of Megakaryocytic Cells--
Human
megakaryocytic cells were generated ex vivo, as described,
with minor modifications (25). In brief, recombinant human granulocyte
colony-stimulating factor (G-CSF; Chugai Pharmaceutical Co., and Kyowa
Hakko Pharmaceutical Co., Tokyo, Japan) was administered to healthy
volunteers (who had previously signed consent forms approved by the
Hokkaido University School of Medicine and the Hokkaido Red Cross Blood
Center committee for the Protection of Human Subjects), as described
(26). The mobilized peripheral blood CD34+ cells were isolated using
immunomagnetic beads (27, 28). The cells were then cryopreserved and
stored until use in a tank containing liquid nitrogen. The frozen
peripheral blood CD34+ cells were thawed and suspended in IMDM
containing 30% FCS and 100 units/ml DNase and then were centrifuged at
400 × g for 5 min at 4 °C. The cells were washed
twice with IMDM containing 0.3% deionized BSA and then resuspended in
IMDM containing 0.3% deionized BSA. The cells were next cultured in
liquid phase, as described elsewhere, with minor modifications (25). In
brief, cells ranging from 2.0 × 104 to 4.0 × 104 cells/ml were suspended in a mixture containing 5%
pooled human AB plasma, 1% BSA, 30 mg/ml of iron-saturated
transferrin, 10 µg/ml of insulin, lipid suspension (2.8 µg/ml oleic
acid, 4.0 µg/ml L-
-phosphatidylcholine, and 3.9 µg/ml cholesterol; Sigma) (29), vitamin B12 at 10 µg/ml, and folic
acid at 15 µg/ml, with TPO at 100 ng/ml, in the presence of 5 × 10
5 mole/ml 2-mercaptoethanol, penicillin at 50 units/ml,
streptomycin at 50 units/ml, and IMDM in a 50 ml polystyrene flask
(Corning Coster Corp., Cambridge, MA). After incubation for 10 days at 37 °C in a 95% N2, 5% O2 incubator, the
cells were collected, and washed twice with IMDM containing 0.3% BSA
(Day 10 cells).
Flow Cytometry--
Phenotyping of Day 10 cells was done by flow
cytometry using FACS (Vantage Becton Dickinson, Franklin Lakes, NJ), as
described (25). The following reagents were used: CD41 (TP80,
fluorescein isothiocyanate conjugate (FITC), Nichirei, Tokyo, Japan),
CD42b (phycoerythrin conjugate (PE), PharMingen, San Diego, CA), CD61 (FITC, PharMingen), and glycophorin A (FITC, DAKO Japan Co., Kyoto, Japan).
Cell Cycle Analysis--
Cell cycle analysis was performed by
staining DNA with propidium iodide in preparation for flow cytometry
with the FACScan/CellFIT system (Becton-Dickinson, San Jose, CA).
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RESULTS |
TPO Induced Phosphorylation of Akt Kinase in a Dose- and
Time-dependent Manner--
In the following experiments we
mainly used the UT-7/TPO cell line (23). This cell line absolutely
depends on TPO for growth and survival, and it has mature
megakaryocytic properties such as a developed demarcation membrane in
the cytoplasm, high expression of the megakaryocyte-specific markers,
platelet factor-4 and glycoprotein IIb mRNAs, and high DNA content
(23). Therefore, UT-7/TPO is a good model of megakaryocytic cells for
elucidating the biological role of the PI3K-Akt-FKHRL1 activation
pathway in TPO singaling. Based on the recent reports that Akt is
activated by several cytokines including interleukin-3, granulocyte
colony-stimulating factor and EPO (12, 30, 31), we initially examined
whether or not TPO activates Akt in UT-7/TPO cells. Growth
factor-deprived UT-7/TPO cells were exposed to TPO (100 ng/ml) for
given periods of up to 60 min and then harvested for
immunoprecipitation with anti-Akt antibody. Western blotting analysis
was performed using anti-[phospho-Ser473]Akt antibody,
which recognizes a phosphorylated serine473, one of two sites on Akt
phosphorylated in its active form. As shown in Fig.
1A, phosphorylated Akt
appeared within 1 min, and its level reached a maximum at 5 to 10 min,
and had diminished by 60 min. Moreover, growth factor-deprived UT-7/TPO
cells were exposed to increasing concentrations of TPO (0.1-100
ng/ml). As shown in Fig. 1B, phosphorylated Akt was detected
at 10 ng/ml of TPO, and the level reached a plateau at 100 ng/ml of
TPO. These findings indicate that TPO induced phosphorylation of Akt in
a dose- and time-dependent manner.

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Fig. 1.
TPO induces phosphorylation of Akt and FKHRL1
proteins in time- and dose-dependent fashions in UT-7/TPO
cells. TPO was removed from UT-7/TPO cells for 24 h. The
cells were then stimulated with TPO (100 ng/ml) for the periods
indicated (A and C), or with increasing
concentrations of TPO (0.1-100 ng/ml) for 10 min (B and
D). After solubilization, cell lysates were
immunoprecipitated with protein G-conjugated anti-Akt antibody
(A and B). Immunoprecipitates were eluted with
buffer containing SDS and were resolved by 10% SDS-PAGE. Proteins were
transferred onto a polyvinylidene difluoride membrane. Upper
panel, immunoblotting with anti-phosphoAkt antibody. Lower
panel, the blot was reprobed with anti-Akt serum to confirm equal
loading of protein. In some experiments, after solubilization, cell
extracts were resolved by 7.5% SDS-PAGE and immunoblotted with the
antibodies directed against phospho-Thr32 (C and
D, top panel, anti-phosphoT32) or
phospho-Ser253 (C and D, middle
panel, anti-phosphoS253). The blot was reprobed with anti-FKHRL1
antibody to confirm equal loading of protein (C and
D, bottom panel). Anti-FKHRL1 antibody recognizes
two bands; the upper band (*) is the phosphorylated form,
the lower band (**) is the unphosphorylated form.
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TPO-induced Phosphorylation of Akt Is Dependent on PI3K
Activity--
The growth factor-deprived UT-7/TPO cells were
pretreated with increasing concentrations of the PI3K-specific
inhibitor LY294002 (1 µM-100 µM) for 45 min
and then stimulated with TPO (100 ng/ml). Ten min later, the cells were
harvested for cell extraction. Western blotting analysis was performed
using anti-[phospho-Ser473]Akt antibody. As shown in Fig.
2A, the phosphorylation
density of Akt was markedly diminished at 10 µM LY294002
and equal to the basal level at 50 µM LY294002. This
result suggested that TPO-induced phosphorylation of Akt is mediated
via PI3K activity.

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Fig. 2.
Phosphorylation of Akt and FKHRL1 by TPO is
dependent on PI3K activity. TPO was removed from UT-7/TPO cells
for 24 h. The cells were pretreated with increasing concentrations
of LY294002 (1-100 µM) and then stimulated with TPO (100 ng/ml) for 10 min. After solubilization, cell lysates were
immunoprecipitated with protein G-conjugated anti-Akt antibody
(A). Immunoprecipitates were eluted with buffer containing
SDS and resolved by 10% SDS-PAGE. Proteins were transferred onto a
polyvinylidene difluoride membrane. Upper panel,
immunoblotting with anti-phosphoAkt antibody. Lower panel,
the blot was reprobed with anti-Akt serum to confirm equal loading of
protein. In some experiments, after solubilization, cell extracts were
resolved by 7.5% SDS-PAGE and immunoblotted with the antibodies
directed against phospho-Thr32 (B, top
panel, anti-phosphoT32) or phospho-Ser253
(B, middle panel, anti-phosphoS253). The blot was
reprobed with anti-FKHRL1 antibody to confirm equal loading of protein
(B, bottom panel). Anti-FKHRL1 antibody
recognizes two bands; the upper band (*) is the
phosphorylated form, the lower band (**) is the
unphosphorylated form.
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TPO Indeed Induces Activation of Akt Kinase--
To confirm the
activation of Akt by TPO, we performed in vitro kinase assay
using GSK-3 as substrate (Fig.
3A). After exposure to TPO
(100 ng/ml) for given periods of up to 60 min, the cells were
immunoprecipitated with anti-Akt antibody and in vitro
kinase assay was performed according to the instructions of the AKT
Kinase Assay Kit. The density of phosphorylated GSK-3 was enhanced
after a 5-min exposure to TPO, and its enhancement continued until 60 min. This observation indicates that TPO indeed induced Akt kinase activation in UT-7/TPO cells.

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Fig. 3.
In vitro kinase assay revealed
that AKT kinase is activated by TPO stimulation and indeed
phosphorylates FKHRL1 proteins. TPO was removed from UT-7/TPO
cells for 24 h. Then the cells were stimulated with TPO (100 ng/ml) for the indicated periods. After solubilization, cell lysates
were immunoprecipitated with protein G-conjugated anti-Akt antibody.
Then immunoprecipitates were subjected to an in vitro kinase
assay. The kinase assays were carried out in the presence of 200 µM ATP using GSK-3 or GST·FKHRL1 as substrate. The
reactions were incubated at 30 °C for 30 min. Reaction products were
resolved by 15% SDS-PAGE and immunoblotted with anti-phosphoGSK-3
antibody (A) or the antibodies directed against
phospho-Thr32 (B, top panel,
anti-phosphoT32) or phospho-Ser253 (B,
middle panel, anti-phosphoS253). The blot was reprobed with
anti-FKHRL1 antibody to confirm equal loading of protein (B,
bottom panel). *, full-length GST·FKHRL1 protein; **,
smaller fragments of the GST·FKHRL1 protein are possible degradation
products (see Ref. 13).
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TPO Induces Phosphorylation of FKHRL1 in a Dose- and
Time-dependent Manner--
Recently it was reported that
FKHRL1 is a substrate of Akt kinase in vitro (13). We also
found that Akt kinase activated by EPO directly phosphorylated the
FKHRL1 protein (12). To confirm that FKHRL1 lies downstream of Akt
kinase in the TPO signaling pathway, we examined whether or not FKHRL1
is phosphorylated by TPO treatment. Growth factor-deprived UT-7/TPO
cells were exposed to TPO (100 ng/ml) for given periods of up to 60 min
or exposed to increasing concentrations of TPO (0.1-100 ng/ml) for 10 min, and then the cells were harvested for preparation of total cell lysates. Western blotting analysis was performed using
anti-phospho-Thr32 antibody and
anti-phospho-Ser253 that recognize a phosphorylated
threonine 32 and a phosphorylated serine 253, respectively. FKHRL1 was
phosphorylated at Thr32 and Ser253 at 1 min,
the level reaching a plateau at 5-20 min and declining thereafter
(Fig. 1C). Phosphorylated FKHRL1 was detectable at 0.1 ng/ml
of TPO, and the level reached a plateau at 1 ng/ml of TPO (Fig.
1D). These findings indicate that TPO induced
phosphorylation of FKHRL1 in a dose- and time-dependent manner.
TPO-induced Phosphorylation of FKHRL1 Is Dependent on PI3K
Activity--
The growth factor-deprived UT-7/TPO cells were
pretreated with increasing concentrations of the PI3K-specific
inhibitor LY294002 (1-100 µM) for 45 min and then were
stimulated with TPO (100 ng/ml). Ten min later, the cells were
harvested for cell extraction. Western blotting analysis with
anti-phospho-Thr32 antibody and
anti-phospho-Ser253 revealed that the phosphorylation
density of FKHRL1 at Thr32 and Ser253 was
markedly diminished at 10 µM LY294002 and completely
disappeared at 50 µM of LY294002 (Fig. 2B).
This result suggested that TPO-induced phosphorylation of FKHRL1 is
mediated via PI3K activity.
FKHRL1 Is One of the Target Molecules of AKT Kinase Activated by
TPO--
To demonstrate that FKHRL1 is directly targeted by AKT kinase
activated by TPO in vivo, we performed an in
vitro kinase assay using GST·FKHRL1 fusion protein as a
substrate. TPO-deprived UT-7/TPO cells were exposed to TPO for 10 or 20 min and then harvested for immunoprecipitation with anti-Akt antibody.
Immunoprecipitates were incubated with GST·FKHRL1 fusion protein
according to the instructions of the AKT Kinase Assay Kit with some
modifications. As shown in Fig. 3B, a phosphorylated FKHRL1
band was obtained with antibody, which recognizes phosphorylated
Thr32 and phosphorylated Ser253, respectively,
indicating that Akt activated by TPO directly phosphorylated FKHRL1 at
Thr32 and Ser253.
FKHRL1 Protein Is Present in Normal Megakaryocytes--
We
examined whether or not FKHRL1 is expressed in normal megakaryocytes.
To obtain a large amount of megakaryocytes for Western blotting
analysis, human CD34-positive cells were cultured in the presence of
Interleukin-3, SCF, and TPO for 10 days. Isolated human megakaryocytic
cells expressed the specific megakaryocytic markers CD41 at 93.7%,
CD61 at 75.5%, and CD42b at 42.8%, but did not express the
erythroid-specific marker GPA (Fig. 4,
A-D). Virtually all CD42b-positive cells expressed CD41
antigens (Fig. 4C). Using these highly purified
megakaryocytes, we examined whether or not Akt and its downstream
molecule FKHRL1 are indeed phosphorylated in primary megakaryocytic
cells. After a 2-h deprivation of growth factors, the cells were
stimulated with TPO (100 ng/ml) for 20 min and then harvested for
Western blotting analysis. As shown in Fig. 4, E and
F, Akt and FKHRL1 proteins were indeed expressed in
megakaryocytes. As expected, Akt at Ser473 and FKHRL1 at
Thr32 and Ser253 were clearly phosphorylated by
TPO treatment.

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Fig. 4.
Expression and TPO-induced phosphorylation of
FKHRL1 in highly purified megakaryocytes. Purified human CD34+
cells were induced to undergo megakaryocytic differentiation with 100 ng/ml of TPO. After 10 days of culture, the cells were collected and
counterstained with CD41, CD61, and GPA. A dot plot, forward
scatter (vertical axis) versus side light scatter
(horizontal axis), by gating whole viable cells
(A). A dot plot for cells stained by anti-mouse
IgG1 conjugated with PE/FITC (B), CD42b/CD41
(C) and CD61/GPA (D). E and
F, phosphorylation of Akt and FKHRL1 in highly purified
normal megakaryocytes. Normal megakaryocytes were obtained as described
under "Experimental Procedures." After solubilization, cell
extracts (2 × 105 cells/lane) were resolved by 7.5%
SDS-PAGE and immunoblotted with anti-Akt or anti-FKHRL1
antibodies.
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FKHRL1 Blocks Cell Cycle Progression from
G0/G1 to S Phase in UT-7/TPO
Cells--
Considering that FKHRL1 lies downstream of the PI3K-Akt
activation pathway, these results raised the possibility that FKHRL1 is
involved in the TPO-induced cell survival as a target molecule of the
PI3K-Akt pathway. To test this possibility, we expressed the triple
mutant of FKHRL1 (FKHRL1-TM) in UT-7/TPO by using a Tet-inducible
system, in which expression of the target protein is induced by Tet
treatment. FKHRL1-TM in which Thr32, Ser253,
and Ser315 are all converted to alanines is localized to
the nucleus and activates the Fas ligand (FasL) gene
promoter in vitro, indicating that FKHRL1-TM is an
"active" form for activation of target gene(s). We transfected
UT-7/TPO/pcDNA6/TR-2 with pcDNA4/TO containing FKHRL1-TM cDNA and
isolated several stable clones. Among them, UT-7/TPO/FKHRL1-TM clone 1 and clone 2 were used for the subsequent experiments because Tet
treatment led to the strong induction of FKHRL1-TM protein in these
clones. Western blotting analysis of the whole cell lysates revealed
that the treatment with Tet resulted in rapid induction of FKHRL1-TM
proteins in UT-7/TPO/FKHRL1-TM cells (Fig.
5A). Next, we investigated the
nuclear localization of FKHRL1-TM in these transfectants. Confocal
microscopic study revealed that induced FKHRL1-TM was localized to the
nucleus after Tet treatment (Fig. 5B). Induced expression of
FKHRL1-TM by Tet treatment led to the suppression of TPO-induced MTT
incorporation into the cells (Fig.
6A), whereas Tet treatment had
no effect on MTT incorporation into a control cell line that was
transfected with an empty expression vector (data not shown). These
results suggest that FKHRL1-TM is actually functional in our systems. To further elucidate the biological role of FKHRL1, using clone 1 we
examined the effect of FKHRL1-TM on cell cycling of UT-7/TPO cells.
After a 12-h exposure to Tet in the absence of TPO, the cells were in
part harvested for cell cycle analysis (Fig. 6B, left
panel). The remaining cells was sequentially cultured with TPO (10 ng/ml) for 24 h, and then harvested for cell cycle analysis (Fig.
6B, right panel). The ratio of cells at the
G0/G1 phase increased after Tet treatment,
compared with the control cells (Tet
versus Tet+; 0 h, 43.3% versus 44.9%; 24 h, 31.6% versus 48.0%). These results strongly suggested that FKHRL1 activates or
inactivates the transcription of the cell cycle-associated gene(s). The
subG1 population was not increased by Tet treatment compared with the control cells (Fig. 6B). Indeed, the ratio
of annexin V-positive cells was not increased in the cells treated with
Tet (data not shown). Similar results were obtained in clone 2 (data
not shown). Therefore, active FKHRL1 induced cell cycle arrest at
G0/G1 phase but did not induce apoptosis in
UT-7/TPO cells.

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Fig. 5.
Inducible expression of FKHRL1-TM after Tet
treatment. A, Western blotting analysis with
anti-FKHRL1 antibody. UT-7/TPO/FKHRL1 -TM clone 1 and clone 2 were
cultured with Tet (1 µg/ml) for 12 or 24 h. Then the cells were
harvested for Western blotting analysis with anti-FKHRL1
antibody. B, nuclear localization of FKHRL1-TM after
Tet treatment. UT-7/TPO/FKHRL1-TM clone 1 was cultured with Tet (1 µg/ml) for 12 h and the nuclear localization of FKHRL1-TM was
investigated by confocal microscopy as described in " Experimental
Procedures."
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Fig. 6.
Inducible FKHRL1 blocks cell-cycle
progression at G0/G1 phase in UT-7/TPO
cells. A, effect of Tet-induced FKHRL1-TM on
proliferation of UT-7/TPO transfectants. UT-7/TPO/FKHRL1-TM clone 1 (square) and clone 2 (circle) cells were plated
at a density of 10,000 cells/well in IMDM supplemented with 5% FCS and
cultured with (filled) or without (open) Tet (1 µg/ml) at various concentrations of TPO (0.01-100 ng/ml). MTT
reduction assay was performed after 3 days of culture. The values
represent the percentage of the result obtained at 100 ng/ml of TPO in
each clone. The data are shown as the mean ± S.D. from triplicate
cultures. B, effect of Tet-induced FKHRL1-TM on cell cycle.
After a 12-h exposure to Tet (1 µg/ml) in the absence of TPO,
UT-7/TPO/FKHRL1-TM (clone 1) cells were stimulated with TPO (10 ng/ml),
sequentially cultured for the periods indicated, and harvested for cell
cycle analysis.
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DISCUSSION |
In this study we showed that Akt and FKHRL1 were rapidly and
transiently phosphorylated by TPO in a human leukemia cell line UT-7/TPO. We found that PI3K inhibitor LY294002 completely blocked the
TPO-induced phosphorylation of Akt and FKHRL1 in UT-7/TPO cells,
indicating that the activation of Akt and FKHRL1 is completely regulated by PI3K activity. In addition, in vitro kinase
assay revealed that Akt kinase activated by TPO directly phosphorylated FKHRL1 protein at threonine 32 and serine 253 residues. These findings
indicate that FKHRL1 functions as a downstream molecule of the PI3K-Akt
activation pathway, as is the case for EPO (12). Finally, we
demonstrated that unphosphorylated active FKHRL1 induced cell cycle
arrest at the G0/G1 phase in UT-7/TPO cells.
Importantly, Akt and FKHRL1 were present and phosphorylated in highly
purified normal human megakaryocytes. This strongly suggested that
these molecules are involved in normal megakaryopoiesis and presumably in platelet production.
It is noteworthy that inducible expression of active FKHRL1 (FKHRL1-TM)
induced cell cycle arrest at G0/G1 phase in
UT-7/TPO. Very recently it was reported that AFX, a member of the
Forkhead subfamily, blocked cell cycle progression in phase
G1 via activation of the p27Kip1 gene at the
transcriptional level (32). This observation prompted us to examine
whether or not activation of p21WAF1/Cip1 and
p27Kip1 is involved in FKHRL1-induced cell cycle arrest at
the G0/G1 phase in UT-7/TPO cells. However,
luciferase assay revealed that FKHRL1-TM did not activate
p21WAF1/Cip1 or p27Kip1 gene promoter in the
Tet-inducible system (data not shown). In addition, neither
p21WAF1/Cip1 nor p27Kip1 were enhanced at the
protein level after Tet treatment (data not shown). These findings
indicate that some cell cycle-associated molecule(s) other than
p21WAF1/Cip1 and p27Kip1 influence the
FKHRL1-induced cell cycle arrest at G0/G1 phase in UT-7/TPO cells. There was a discrepancy in the dosage of LY294002 between the inhibition of phosphorylation of Akt-FKHRL1 and induction of apoptosis in UT-7/TPO cells. Although a high dose of PI3K inhibitor (50 and 100 µg/ml of LY294002) induced apoptosis in more than 50% of
UT-7/TPO cells (data not shown), phosphorylation of Akt and FKHRL1 by
TPO was significantly blocked at 10 µM LY294002 at which
dosage apoptosis did not occur in UT-7/TPO cells (data not shown).
Therefore, it is unlikely that phosphorylation of Akt and FKHRL1 is
closely involved in the anti-apoptotic effect of TPO. This notion is in
part supported by the evidence that overexpression of different levels
of FKHRL-TM did not induce apoptosis in UT-7/TPO cells (Fig.
6B and data not shown). Alternatively, our results may
suggest that FKHRL1 in a dephosphorylated state is not sufficient to
induce apoptosis in UT-7/TPO cells. If so, a third factor may be
required for FKHRL1-induced apoptosis in this cell line.
FKHRL1 was originally identified as a novel partner of the
MLL gene, in undifferentiated acute leukemia with
t(6;11)(q21;q23) translocation (33). Interestingly, it has been
reported that AFX·MLL and FKHR·PAX3 fusion genes are detectable in
acute leukemias with t(X;11)(q13;q23) and alveolar rhabdomyosarcoma,
respectively (34, 35). Therefore, aberrant expression or
loss-of-function of these three members appears to be involved in
tumorigenesis. Considering our results that inducible expression of
FKHRL1-TM inhibited TPO-induced proliferation of UT-7/TPO cells (see
Fig. 6A), FKHRL1 may function as a tumor suppresser in
normal cells, although this is speculation.
There is a close relationship between cell differentiation and cell
cycle arrest. To examine whether or not FKHRL1 affects TPO-induced
megakaryocytic differentiation of UT-7/TPO cells, we cultured
UT-7/TPO-FKHRL1-TM cells in the presence of TPO and Tet for 5 days.
However, inducible expression of FKHRL1-TM by Tet did not enhance the
expression of CD41 and CD61 antigens, which are specific for the
megakaryocytic lineage (data not shown). This finding suggests that
FKHRL1-TM-induced cell cycle arrest at the
G0/G1 phase is not sufficient for
megakaryocytic differentiation.
There was a discrepancy between Akt and FKHRL1 in the dosage of TPO
needed for their phosphorylation. Phosphorylation of Akt by TPO was
induced at 1 ng/ml of TPO, whereas FKHRL1 was phosphorylated by TPO at
0.1 ng/ml. In addition, the in vitro kinase assay revealed that the phosphorylation level of FKHRL1 by Akt was much weaker than
that in vivo. Although we cannot completely exclude the
possibility that the discrepancy in phosphorylation level between
in vitro and in vivo is because of technical
problems, the discrepancy may be because of the different sensitivity
of antibody used in this experiment, or that not only Akt but other
protein kinase(s) are involved in the phosphorylation of FKHRL1 protein.
FKHRL1 has three putative Akt consensus phosphorylation sites,
Thr32, Ser253, and Ser315. Among
them, Thr32 and Ser253 were phosphorylated by
TPO stimulation in UT-7/TPO cells. Indeed, an in vitro
kinase assay revealed that both sites were directly phosphorylated by
Akt activated by TPO. However, we could not demonstrate the
phosphorylation of Ser315 induced by TPO because adequate
anti-phospho-Ser315 antibody was not available. To overcome
this obstacle, we took advantage of the finding that phosphorylation of
FKHRL1 at Ser315 but not Thr32 or
Ser253 had a significant effect on the mobility of FKHRL1
on an SDS gel (13). As shown in Figs. 1C, 1D,
2B, and 4F, TPO induced a shift up in the
mobility of FKHRL1. This result strongly suggested that FKHRL1 at
Ser315 was phosphorylated by TPO stimulation, although
it is still unknown whether or not Akt directly phosphorylated
Ser315.
Because the FasL promoter contains three Forkhead-responsive
elements that bind FKHRL1 (13), it was predicted that FasL is one of the target molecules of FKHRL1 in UT-7/TPO cells. Indeed, the
nonphosphorylated active form of FKHRL1 can activate the
FasL promoter in vitro and induce apoptosis in
cerebellar neurons, fibroblasts, and Jurkat T lymphoma cells (13).
Based on these observations, we examined whether or not FKHRL1 can
activate the FasL gene using a Tet-inducible system in
UT-7/TPO cells. However, unexpectedly, reverse transcriptase-polymerase
chain reaction revealed that FasL mRNA was undetectable
after the addition of Tet (data not shown), suggesting that the
FasL gene is not a target molecule for FKHRL1 at least in
UT-7/TPO cells.
To our knowledge, this is the first report that shows that Akt and
FKHRL1 are present in normal megakaryocytes and that these molecules
are actually phosphorylated by TPO. The evidence that FKHRL1 is
ubiquitously expressed in all tissues strongly suggests that FKHRL1 is
a housekeeping molecule (13). In conclusion, considering our results
that FKHRL1 induced cell cycle arrest at G0/G1
phase but had no effect on differentiation and apoptosis, FKHRL1 may
play an important role in keeping the cells in the quiescent state via
the activation or inactivation of cell cycle-associated gene(s).