From the Division of Hematology, Department of
Medicine, Jichi Medical School, Tochigi 329-0498, the
First
Department of Internal Medicine, Tokyo Medical University, Tokyo
160-8402, and the ** Department of Biochemistry, Jichi
Medical School, Tochigi 329-0498, Japan
Received for publication, November 13, 2002
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ABSTRACT |
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A member of the Forkhead
transcription factor family, FKHRL1, lies downstream of the
phosphatidylinositol 3-kinase-Akt activation pathway in cytokine
signaling. Because the phosphatidylinositol 3-kinase-Akt activation
pathway is required for BCR-ABL-mediated transformation and survival
signaling in chronic myelogenous leukemia (CML), in this study we
examined the involvement of FKHRL1 in the BCR-ABL-mediated signaling
pathway. FKHRL1 was constitutively phosphorylated in BCR-ABL-expressing
cell lines KCL22 and KU812, and its phosphorylation was inhibited by
treatment with STI571, a specific inhibitor of BCR-ABL tyrosine kinase.
Concomitantly, STI571 induced cell cycle arrest at the
G0/G1 phase, accompanied by up-regulation
of a cyclin-dependent kinase inhibitor p27/Kip1 in KCL22
cells. In addition, FKHRL1 was constitutively phosphorylated in the
TF-1/bcr-abl cell line ectopically expressing BCR-ABL but not in the
parent TF-1 cell line. Considering several lines of evidence that
phosphorylated FKHRL1 has lost transcriptional activity and that
p27/Kip1 expression is positively regulated by dephosphorylated "active" FKHRL1, BCR-ABL may down-regulate p27/Kip1 expression via
the loss of FKHRL1 function as a transcription factor. To demonstrate
this hypothesis, we generated a tamoxifen-inducible "active FKHRL1"
FKHRL1-TM (a triple mutant of FKHRL1, in which all three Akt
phosphorylation sites have been mutated), estrogen receptor system in
the KCL22 cell line. The addition of tamoxifen inhibited the cell
growth indicating that overexpression of FKHRL1 in the nucleus
antagonized deregulated proliferation of CML cells. Collectively,
FKHRL1 regulates the expression of p27/Kip1 as a downstream molecule of
BCR-ABL signaling in CML cells. BCR-ABL-induced loss of FKHRL1 function
may be involved in oncogenic transformation of CML partially via the
down-regulation of p27/Kip1 proteins.
The bcr-abl fusion gene originates from a reciprocal
translocation between the long arms of chromosomes 9 and 22, resulting in the formation of the Philadelphia chromosome (1). The resultant bcr-abl fusion gene encodes the chimeric BCR-ABL proteins of
p230, p210, and p185 (230, 210, and 185 kDa). These fusion proteins have constitutively active tyrosine kinase activity and are implicated in the pathogenesis of chronic myelogenous leukemia
(CML)1 and
Philadelphia-positive acute lymphoblastic leukemia (2). BCR-ABL exerts
diverse actions on hematopoietic cells as follows: transformation,
protection of apoptosis, cell cycle progression, altered cell
migration, and adhesion to the extracellular matrix (3-10). The
expression of BCR-ABL activates multiple signaling cascades, including
the JAK/STAT, Ras, and phosphatidylinositol 3-kinase (PI3K) pathways
(11). Among these, it was demonstrated that the activation of PI3K and
the downstream Akt signaling pathways is required for not only
BCR-ABL-mediated transformation but also cell survival of CML cells
(12).
Recently, it was demonstrated that members of human Forkhead
transcription factors FKHRL1, AFX, and FKHR are directly phosphorylated by activated Akt (13-17). We also reported that FKHRL1 is directly phosphorylated by activated Akt as one of the downstream molecules of
the PI3K/Akt activation pathway in erythropoietin and thrombopoietin signalings (18, 19). More than 100 Forkhead family transcription factors have been identified in diverse species ranging from yeasts to
humans (20). These transcription factors are related to embryogenesis, differentiation, and tumorigenesis. Interestingly, the three Forkhead transcription factors, FKHRL1, AFX, and FKHR, are human homologs of
DAF-16, which is involved in lifespan extension of Caenorhabditis elegans (21, 22). The genes encoding these three molecules were
originally identified in breakpoints of which chromosome translocations
are recognized in human tumors (23-25).
FKHRL1 has three potential Akt phosphorylation sites
(RXRXX(S/T)): Thr-32
(RPRSCT32), Ser-253
(RRRAVS253), and Ser-315
(RSRTNS315) (26). When cells are stimulated with
serum or growth factors, FKHRL1 is phosphorylated by activated Akt and
is exported from the nucleus to the cytoplasm, resulting in the
inhibition of target gene transcription (13-17). In contrast, when
cells are deprived of serum or growth factors, FKHRL1 becomes a
dephosphorylated form, translocates into the nucleus, and activates the
transcription of target genes. Thus, the transcriptional activity of
FKHRL1 is negatively regulated via Akt-induced phosphorylation.
Therefore, FKHRL1-TM, which is replaced at three amino acids, Thr-32,
Ser-253, and Ser-315 with alanine, is predicted to be an active form as a transcription factor (13). Medema et al. (27) and we (19) found that FKHRL1-TM induced cell cycle arrest at the
G0/G1 phase in a variety of cell lines.
Furthermore, Dijkers et al. (28) reported that "active"
FKHRL1 up-regulates the expression of cyclin-dependent kinase inhibitor p27/Kip1 at the transcriptional level, resulting in
blockage of cell cycle progression. These findings strongly suggested
that FKHRL1 controls cell cycling via regulating the p27/Kip1 expression. Taken together with several lines of evidence that
BCR-ABL suppressed p27/Kip1 expression via the PI3k/Akt activation pathway (29-31), FKHRL1 may lie downstream of the BCR-ABL signaling pathway as a phosphorylated "inactive" form, leading to the
down-regulation of p27/Kip1 expression. To address this notion, we
examined the phosphorylation of FKHRL1 in a CML-derived cell line. To
elucidate further the pathological role of FKHRL1 in CML, we generated
a tamoxifen-inducible FKHRL1-TM:estrogen receptor (ER) system in a
CML-derived cell line. We show here that FKHRL1 lies downstream of the
BCR-ABL signaling pathway, and that this molecule has a negative effect
on deregulated cell growth caused by BCR-ABL fusion protein. Loss of
function of FKHRL1 may be involved in the oncogenic transformation of
CML cells.
Reagents and Antibodies--
Fetal calf serum (FCS) and
4-hydroxytamoxifen (4-OHT) were purchased from Sigma. Polyclonal
antibody against BCR was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Antibodies against phospho-FKHRL1 (Thr-32) and phospho-c-Abl
(Tyr-245) were purchased from Cell Signaling Technology Inc. (Beverly,
MA). Antibody against native FKHRL1 was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). An antibody against p27/Kip1 was
purchased from BD Biosciences. The 2-phenylamino-pyrimidine derivative
STI571 (molecular mass, 590 daltons) was developed and kindly
provided by Novartis (Basel, Switzerland). The stock solutions of this
compound were prepared at 1 mM with Me2SO and
stored at Cell Culture of CML-derived Cell Lines and Generation of
Transfectants--
KCL22, KU812 cell lines, and TF-1/bcr-abl cell
lines were maintained in liquid culture with IMDM containing 10% FCS
(32-34). TF-1 cells were maintained in liquid culture with IMDM
containing 10% FCS with granulocyte-macrophage
colony-stimulating factor (1 ng/ml). KCL22 cells were transfected with
mammalian expression vector (pcDNA3; Invitrogen) containing human
FKHRL1-TM-ER (27) or FKHRL1-DN cDNA by the Lipofectin method
according to the manufacturer's instructions (Promega, Madison, WI).
We selected three independent clones resistant to neomycin (1.0 mg/ml).
Colorimetric MTT Assay for Cell Proliferation--
Cell growth
was examined by a colorimetric assay according to Mosmann (35) with
some modifications. Briefly, cells were incubated at a density of
1 × 104/100 µl in 96-well plates in IMDM
containing 10% FCS. 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 2 h of incubation at 37 °C, 100 µl 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).
Preparation of Cell Lysates and Western Blotting--
The cells
were washed and suspended in lysis buffer containing 20 mM
Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
1.7 ng/ml aprotinin, 50 µg/ml leupeptin, 2 mM sodium
orthovanadate, and 20 mM sodium fluoride. After 20 min of
incubation on ice, insoluble materials were removed by centrifugation
at 15,000 × g for 20 min. The supernatants were boiled
for 5 min in SDS-PAGE sample buffer, resolved by SDS-PAGE, and
electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad). 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 overnight at 4 °C or for 1-2 h at
room temperature. After washing with Tris-buffered saline containing
Tween 20 (1:2,000), the blots were probed with a 1:10,000 dilution of
anti-rabbit or anti-mouse horseradish peroxidase-conjugated second
antibodies for 90 min at room temperature. After a second washing, the
blots were incubated with an enhanced chemiluminescence substrate (ECL
Western blot detection system; Amersham Biosciences) and exposed to
Hyperfilm ECL (Amersham Biosciences) to visualize the immunoreactive
bands. The blots were stripped with 62.5 mM Tris-HCl, pH
6.8, 2% SDS, and 100 µM 2-mercaptoethanol at 50 °C for 30 min, washed, blocked, and reprobed.
Cell Cycle Analysis--
Cell cycle analysis was performed by
staining DNA with propidium iodide in preparation for flow cytometry
with the FACScan/CellFIT system (BD Biosciences).
Luciferase Assay--
Oligonucleotides containing an array of
seven insulin-response elements (IREs) (three IRSA (CAAAACAA) and four
IRSB (TTATTTTG)) were cloned into the
KpnI-HindIII site of pGL3 basic vector to generate pGL3-7xIREs. The pGL3-7xIREs and pECE vector containing FKHRL1-TM cDNA with or without pCDNA3.1 containing FKHRL1-DN
cDNA were introduced into 293 cells by lipofection with the
internal control plasmid pRL-TKLUC (Promega, Madison, WI). After
transfection, the cells were cultured for 36 h and then harvested
for dual luciferase assay according to the manual (Promega). In some
experiments, a luciferase reporter containing the p27/Kip1 promoter was used.
STI571 Blocks Cell Cycle Progression at
G0/G1 Phase in KCL22 Cells--
In
the following experiments, we mainly used the KCL22 cell line. This
cell line was established from the bone marrow cells of a patient with
blastic crisis of BCR-ABL-positive CML (32). Therefore, KCL22 would be
a good model of CML cells for elucidating the biological function of
FKHRL1. We initially examined whether or not STI571 inhibits the
proliferation of KCL22 cells. KCL22 cells were exposed to various
concentrations of STI571 (0.03-3 µM) for 3 days. MTT
reduction assay revealed that STI571 inhibited the proliferation of
KCL22 cells in a dose-dependent manner (Fig. 1A). To clarify the mechanism
by which STI517 inhibited the cell growth of KCL22 cells, we examined
the effect of STI571 on cell cycling of the KCL22 cells. 1 µM STI571 induced the G0/G1
arrest in KCL22 cells in a time-dependent manner. The
G0/G1 ratio began to increase at 9 h and
reached a plateau at 24 h upon STI571 treatment, inversely to S
and G2/M populations (Fig. 1B). The ratio of
apoptotic cells did not increase during the observation periods up to
72 h (data not shown). As shown in Fig. 1C, a CDK
inhibitor, p27/Kip1 protein was detected after 3 h of exposure to
STI571, and the expression level was gradually increased up to 48 h.
STI571 Inhibits Phosphorylation of FKHRL1 Proteins in a Time- and
Dose-dependent Manner in KCL22 Cells--
We performed
Western blotting with anti-phospho-c-Abl (Tyr-245) or
anti-phospho-FKHRL1 (Thr-32) antibody in KCL22 cells. FKHRL1 and
BCR-ABL fusion protein were constitutively phosphorylated in this cell
line (Fig. 2A, lane
1). Next we examined whether or not STI571 inhibits the
phosphorylation of these two proteins. KCL22 cells were exposed to
STI571 (1 µM) for the indicated periods of up to 8 h
and then harvested. The phosphorylation of BCR-ABL fusion protein began
to diminish after a 1-h exposure to STI571 (Fig. 2A).
Moreover, KCL22 cells were exposed to increasing concentrations of
STI571 (0.05-5 µM) for 6 h. The phosphorylation of
BCR-ABL significantly diminished at 1 µM and completely
disappeared at 5 µM STI571 (Fig. 2B).
Consistent with the kinetics of BCR-ABL phosphorylation, STI571
inhibited the phosphorylation of FKHRL1 in a dose- and
time-dependent manner (Fig. 2, A and
B). However, STI571 did not affect the expression level of
BCR-ABL and FKHRL1 proteins (Fig. 2, A and
B).
FKHRL1 Is Commonly Phosphorylated in BCR-ABL-positive Cell
Lines--
To exclude the possibility that phosphorylation of FKHRL1
is limited to KCL22, we examined the phosphorylation of FKHRL1 in another Bcr-Abl-positive cell line, KU812. BCR-ABL was constitutively phosphorylated, and its phosphorylation was inhibited by STI571 in this
cell line (Fig. 3A).
Concomitantly, the constitutive phosphorylation of FKHRL1 was inhibited
by STI571 treatment. Collectively, these results suggested that FKHRL1
lies downstream of BCR-ABL signaling. To confirm this hypothesis, we
used the TF-1/bcr-abl cell line ectopically expressing BCR-ABL (34).
BCR-ABL was constitutively phosphorylated in TF-1/bcr-abl but not in
the parent TF-1 cells (Fig. 3B, 1st and 2nd
lanes), and its phosphorylation was inhibited after treatment with
1 µM STI571 (Fig. 3B, 3rd lane),
indicating that 1 µM STI571 indeed inhibited the BCR-ABL
activity in TF-1/bcr-abl cells. As expected, constitutive
phosphorylation of FKHRL1 was observed in TF-1/bcr-abl but not in the
parental TF-1 cells (Fig. 3B, 1st and 2nd
lanes), and its constitutive phosphorylation was inhibited after
STI571 treatment (Fig. 3B, 3rd lane). These
results strongly supported our hypothesis that FKHRL1 lies downstream of BCR-ABL signaling. p27/Kip1 protein was down-regulated in
TF-1/bcr-abl cells, compared with that in TF-1 cells (Fig.
3B, 1st and 2nd lanes). In addition,
STI571 induced inhibition of the BCR-ABL activity, accompanied by
up-regulation of p27/Kip1 protein in TF-1/bcr-abl cells (Fig.
3B, 2nd and 3rd lanes). These results strongly suggested that p27/Kip1 lies downstream of BCR-ABL signaling, as reported previously by other groups (29-31).
PI3K Activity Is Involved in BCR-ABL-dependent
Phosphorylation of FKHRL1--
We examined whether PI3K activity
is involved in BCR-ABL-dependent phosphorylation of FKHRL1
using a specific PI3K inhibitor LY294002. As shown in Fig.
3C, 20 µM LY294002 completely suppressed the
phosphorylation of FKHRL1, indicating that the phosphorylation of
FKHLR1 is dependent on PI3K activity in BCR-ABL signaling.
Active FKHRL1 (FKHRL1-TM-ER) Inhibits the Proliferation of KCL22
Cells--
Considering that FKHRL1 is constitutively phosphorylated in
BCR-ABL-expressing cells, FKHRL1 may be in an "inactive state" in
CML, leading to oncogenic transformation. To clarify this hypothesis, we generated transfectants expressing FKHRL1-TM-ER which became the
active form after the addition of 4-OHT. We selected three independent
positive clones highly expressing ectopic FKHRL1-TM-ER (Fig.
4A). The transfectant cells or
the parent KCL22 cells were treated with STI571 (1 µM) or
4-OHT (0.5 µM) for the indicated periods and then
harvested for MTT incorporation assay or cell cycle analysis. As shown
in Fig. 4B, STI571 inhibited the proliferation of the
transfectant cells to the same degree as the parent KCL22 cells. These
results suggested that the transfectant cells have still retained the
sensitivity to STI571 to the same degree as the parent KCL22 cells.
Induced activation of FKHRL1-TM by 4-OHT treatment led to the
suppression of MTT incorporation into the cells, whereas 4-OHT
treatment had no effect on MTT incorporation into the parental control
cells (Fig. 4C). These results suggest that FKHRL1-TM-ER is
actually functional in our system. As shown in Fig. 4D, the
percentage of cells in the G0/G1 phase was
significantly elevated 24 h after 4-OHT treatment, suggesting that
FKHRL1-TM-ER induced cell cycle arrest at the
G0/G1 phase. Concomitantly, the expression
level of p27/Kip1 protein was significantly elevated after 24 h of
exposure to 4-OHT (Fig. 4E).
As shown in Fig. 4D, the ratio of sub-G1
population representing apoptosis was increased after 4-OHT treatment
in these three clones, especially in clones 10-9 and 24-3. To elucidate
the mechanism by which the transfectant cells underwent rapid apoptosis
after the addition of 4-OHT, we focused our attention on the expression of Bim, a member of the BH3-only subfamily of cell death activators, because it was recently reported that Bim is transcriptionally regulated by FKHRL1 (36). As shown in Fig. 4E, the
expressions of BimEL, BimL, and BimS proteins were slightly
up-regulated 24 and 48 h after the addition of 4-OHT. A high dose
of STI571 up to 20 µM induced cell cycle arrest at the
G0/G1 phase but not apoptosis during the
observation periods up to 72 h (Fig.
5A). However, up-regulation of
Bim proteins was observed in the presence of STI571 in the range of
1-20 µM (Fig. 5B).
A Dominant-negative FKHRL1 (FKHRL1-DN) Enhances the Sensitivity of
KCL22 Cells to STI571--
To further elucidate the functional role of
FKHRL1 in STI571-induced cell cycle arrest of KCL22 cells, we generated
transfectants expressing FKHRL1-DN but lacking the transactivation
domain. Initially, we examined whether FKHRL1-DN functions as a
dominant-negative inhibitor. For this purpose, we used a pGL3-7xIREs
construct containing seven consecutive FKHRL1-binding sites, and we
introduced this construct into 293 cells with FKHRL1-TM cDNA and
then assayed the luciferase activity. As illustrated in Fig.
6A, FKHRL1-TM induced promoter
activity, which was completely inhibited by FKHRL1-DN. These results
indicate that FKHRL1-DN functions as a dominant-negative inhibitor on
the transactivation activity of FKHRL1-TM. Based on this result, we
transfected KCL22 cells with pcDNA3.1 containing FKHRL1-DN and
selected three independent positive clones expressing FKHRL1-DN protein
(Fig. 6B). Cell cycle analysis demonstrated that STI571
treatment significantly enhanced the ratio of the sub-G1
population in the transfectants expressing FKHRL1-DN but not vector
alone (Fig. 6C). The ratio of sub-G1 population
was increased in proportion to the expression level of FKHRL1-DN
protein (Fig. 6, A and C). Consistent with this
finding, cell viability of the transfectants was much lower than that
of the parent cells (Fig. 6D). This suggested that the
inhibition of endogenous FKHRL1 by FKHRL1-DN enhanced sensitivity to
STI571 in KCL22 cells.
In this study, we demonstrated that FKHRL1 is located downstream
to the BCR-ABL signaling pathway as an inactive phosphorylated form and functions as an effector when FKHRL1 is converted to the
dephosphorylated form by STI571 treatment in BCR-ABL-expressing cells.
We found that FKHRL1 is constitutively phosphorylated in KCL22, KU812,
and TF-1/bcr-abl cells and that STI571 induces down-regulation of the
BCR-ABL tyrosine kinase activity, dephosphorylation of FKHRL1, and cell
cycle arrest at the G0/G1 phase in KCL22 cells. This was accompanied by up-regulation of p27/Kip1 expression. Moreover,
4-OHT-inducible FKHRL1-TM induced the cell cycle
arrest and induced up-regulation of p27/Kip1 proteins in the
KCL22-derived transfectant cells. Taken together, our results suggested
that STI571 inhibited the cell growth of BCR-ABL-expressing cells, at
least in part via dephosphorylated FKHRL1-mediated up-regulation of
p27/Kip1 proteins. Therefore, constitutive phosphorylation of FKHRL1
induced by BCR-ABL fusion protein may be an important mechanism in
BCR-ABL-mediated cell cycle signaling (29-31).
STI571 (1 µM) inhibited the proliferation of cells but
did not induce apoptosis in KCL22 cells during the observation periods up to 72 h. Taken together with the previous reports that 1 µM STI571 was sufficient to induce cell death in
BCR-ABL-transformed cell lines and primary leukemia cells from CML
patient samples in chronic phase, KCL22 cells appear to be less
sensitive to STI571. This may be explained by several lines of evidence
that KCL22 was established from a patient with CML blastic crisis and
that survival pathways other than BCR-ABL tyrosine kinase possibly operate in the blastic crisis cells. However, because phosphorylated FKHRL1 was detected in TF-1/bcr-abl but not the parental TF-1 cells, we
concluded that FKHRL1 lies downstream of BCR-ABL tyrosine kinase.
The ectopic expression of active FKHRL1 did not induce cell cycle
arrest as robustly as ST571. In fact, in clones 10-9 and 24-4, the
changes in the ratio of G0/G1 population was
about 10%, whereas treatment with STI571 routinely elicited 20-40%
changes. Therefore, besides FKHRL1, other cell cycle-associated
molecules such as FKHR and AFX may be also involved in STI571-induced
cell cycle arrest, because these Forkhead family members commonly
induce p27/Kip1 (27, 37).
p27/Kip1 is generally regulated at the post-translational level via a
proteasome-dependent degradation pathway (38-40).
Consistent with this, it was previously shown that BCR-ABL fusion
inhibited the p27/Kip1 expression through the PI3K-Akt pathway and its
down-regulation mainly occurred through a
proteasome-dependent degradation pathway (31). However, it
was reported recently (28) that FKHRL1 elevated the p27/Kip1 promoter
activity in Ba/F3 cells expressing a 4-OHT-inducible FKHRL1-TM-ER
construct, suggesting that the transcriptional activity of FKHRL1
directly induced the p27/Kip1 gene expression. Thus, the
mechanism of how FKHRL1 regulates p27/Kip1 expression was still
controversial. To address this unresolved problem, we examined the
mechanism by which FKHRL1 regulates the p27/Kip1 expression in KCL22
cells. In this study, we found that both STI571 treatment and ectopic
expression of FKHRL1-TM induced the up-regulation of p27/Kip1
expression at the protein level but not at the mRNA level (Figs.
1D and 4E and data not shown). In addition, a
luciferase assay revealed that 4-OHT treatment did not enhance the
promoter activity of the p27/Kip1 gene in the KCL22
transfectants having 4-OHT-inducible FKHRL1-TM (data not shown).
Although we cannot completely exclude the possibility that the
discrepancy among these results was dependent on the different cell
lines used, our results suggested that the regulation of p27/Kip1
expression by FKHRL1 does not occur at the transcriptional level, at
least in KCL22-derived transfectant cells. If so, FKHRL1 may indirectly up-regulate the p27/Kip1 expression via an unidentified target molecule
of FKHRL1. Therefore, it would be of interest to identify the target
molecules of FKHRL1. Previously, it was reported that the von
Hippel-Lindau (VHL) tumor suppressor molecule controls cell cycle
progression by regulation of p27/Kip1 at both the mRNA and protein
levels (41). However, our preliminary data showed that 4-OHT did not
induce VHL proteins in the KCL22 transfectants expressing FKHRL1-TM-ER
(data not shown). Therefore, it is unlikely that VHL is a target
molecule for FKHRL1.
Bim is a member of the BH3-only subfamily that inhibits the function of
anti-apoptotic Bcl-2 family members by binding to them, resulting in
inhibition of release of cytochrome c from the mitochondria
to the cytosol (42). Recently, Shinjyo et al. (43) reported
that Bim lies downstream of cytokine signaling, and its down-regulation
is a prerequisite for the survival of hematopoietic cells. In addition,
Kuribara et al. (44) reported that BCR-ABL induced
deregulated hematopoiesis via the down-regulation of Bim expression in
CML. These findings suggested that Bim expression is critical for the
regulation of hematopoiesis, and its deregulated expression may
contribute to leukemogenesis such as CML. In this study, we found that
4-OHT induced apoptosis, accompanied by up-regulation of Bim proteins
(BimEL, BimL, and BimS) in BCR-ABL-positive KCL22 cells ectopically
expressing FKHRL1-TM-ER. However, even a high dosage of STI571 (20 µM) did not induce apoptosis, irrespective of the
up-regulation of Bim proteins (Fig. 5B). These results suggested that the expression of Bim proteins is required but not
sufficient for the induction of apoptosis, at least in KCL22 cells.
This notion is supported by the evidence that STI571 induced G0/G1 arrest and up-regulation of p27/Kip1 and
Bim proteins but not apoptosis in clone 24-4 (Fig. 4, D and
E).
It is noteworthy that although a dominant-negative form of FKHRL1
(FKHRL1-DN), though not completely, did significantly block the
STI571-induced cell cycle arrest at the G0/G1
phase, this mutant drastically enhanced STI571-induced apoptosis
in KCL22 cells. These results strongly suggest that active FKHRL1 could protect the KCL22 cells from apoptosis via the induction of cell cycle
arrest at the G0/G1 phase. Considering the
several lines of evidence that the C. elegans DAF2 pathway
regulates a state of diapause called dauer, which is an arrested
juvenile form triggered by food limitation, high temperature, and
crowding, and that the DAF16 is the main target of the DAF2 pathway, it
would not be surprising that active FKHRL1 protects cells from
apoptosis via cell cycle arrest at the G0/G1
phase in the presence of STI571. If so, FKHRL1 may play an important
role in the acquisition of "dormancy" in leukemia cells after
exposure to anti-leukemic agents.
However, it should be stressed that 4-OHT-induced expression of
FKHRL1-TM-ER also induced apoptosis in KCL22 cells. Previously, we
established a tetracycline-inducible system in a human
TPO-dependent cell line UT-7/TPO and showed that induced
expression of FKHRL1-TM lacking ER led to cell cycle arrest at
G0/G1 phase but not to apoptosis in this cell
line (19). Although we cannot exclude the possibility that this
discrepancy was due to the different cell lines used in the
experiments, overexpression of FKHRL1-TM-ER fusion protein may in part
act as a dominant-negative inhibitor of Akt activity. This notion can
be supported by several lines of evidence that overexpressed FKHRL1
binds to Akt (45) and that overexpression of the FKHR mutant replacing
Ser-256 with alanine strongly suppressed the phosphorylation of the
endogenous Akt substrates FKHRL1 and GSK3 The data presented here demonstrate that FKHRL1 is important in the
oncogenic transformation of CML. PTEN acts as a tumor suppressor, at
least in part, by antagonizing PI3K-Akt signaling (49). Recently, it
was reported (37) that Forkhead transcription factors including FKHRL1
are critical effectors of PTEN-mediated tumor suppression, suggesting
that FKHRL1 also functions as a tumor suppressor. Taken together with
our results that FKHRL1 lies downstream of BCR-ABL signaling as an
inactive phosphorylated form, and that FKHRL1-TM suppressed the cell
growth of CML-derived cell line KCL22, active FKHRL1 may be a potent
anti-proliferative molecule against BCR-ABL-induced tumorigenesis. In
addition, our finding that FKHRL1-DN caused apoptosis in STI571-treated
KCL22 cells suggests that FKHRL1 represents an attractive target for therapeutic manipulation in CML.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. pcDNA3 containing human FKHRL1-TM-ER cDNA
was kindly provided by Paul Coffer from the University Medical Center
(Heidelberglaan, The Netherlands). To prepare the dominant-negative
form of FKHRL1 (FKHRL1-DN), we restricted pcDNA3.1-containing human
FKHRL1-TM cDNA with Bst1107 and EcoRV
restriction enzymes, and we deleted the transactivation domain.
Anti-Bim antibody was a gift from Toshiya Inaba (Hiroshima, Japan).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
STI571 blocks cell cycle progression
at the G0/G1 phase in KCL22 cells.
A, effect of STI571 on proliferation of KCL22 cells. KCL22
cells were plated at a density of 10,000 cells/well in IMDM
supplemented with 5% FCS and cultured with various concentrations of
STI571 (0.03-3 µM). MTT reduction assay was performed
after 3 days of culture. The values represent the mean ± S.D.
from triplicate cultures and are expressed as a percentage of untreated
KCL22 cells. B, effect of STI571 on cell cycle. KCL22 cells
were treated with STI571 (1 µM), sequentially cultured
for the periods indicated, and harvested for cell cycle analysis.
C, up-regulation of p27/Kip1 protein after STI571 treatment.
KCL22 cells were treated with STI571 (1 µM), sequentially
cultured for the periods indicated, and harvested for Western blotting
analysis.
View larger version (63K):
[in a new window]
Fig. 2.
STI571 inhibits phosphorylation of FKHRL1
proteins in a time- and dose-dependent manner in KCL22
cells. The cells were then stimulated with increasing
concentrations of STI571 (0.05-5 µM) for 6 h
(A) or with STI571 (1 µM) for the periods
indicated (B). After solubilization, cell extracts were
resolved by 7.5% SDS-PAGE and immunoblotted with the antibodies
directed against phospho-Thr-32. The blot was reprobed with anti-FKHRL1
antibody to confirm equal loading of protein. Anti-phospho-FKHRL1
antibody recognizes two bands; the upper band (*) is the phosphorylated
form, the lower band (**) is nonspecific.
View larger version (16K):
[in a new window]
Fig. 3.
FKHRL1 is commonly phosphorylated in
BCR-ABL-positive cell lines. A, constitutive phosphorylation
of FKHLR1 in KU812 cells. KU812 cells were stimulated with STI571 (1 µM) for 6 h. After solubilization, cell extracts
were resolved by 7.5% SDS-PAGE and immunoblotted with the antibodies
directed against phospho-FKHRL1 (Thr-32) antibody. The blot was
reprobed with anti-FKHRL1 antibody to confirm equal loading of protein.
B, BCR-ABL-induced phosphorylation of FKHRL1.
Granulocyte-macrophage colony-stimulating factor-deprived TF-1
cells or TF-1/bcr-abl cells were treated with STI571 (1 µM) for 6 h. After solubilization, cell extracts
were resolved by 7.5 or 15% SDS-PAGE and immunoblotted with the
antibodies directed against phospho-abl, phospho-FKHRL1 (Thr-32), or
p27/Kip1. The blot was reprobed with anti-BCR or FKHRL1 antibody to
confirm equal loading of protein. C,
BCR-ABL-PI3K-dependent phosphorylation of FKHRL1.
TF-1/bcr-abl cells were treated with LY294002 (20 and 100 µM) for 1 h. After solubilization, cell extracts
were resolved by 7.5% SDS-PAGE and immunoblotted with the antibody
directed against phospho-FKHRL1 (Thr-32). The blot was reprobed with
FKHRL1 antibody to confirm equal loading of protein.
View larger version (79K):
[in a new window]
Fig. 4.
Active FKHRL1 (FKHRL1-TM-ER) inhibits the
proliferation of KCL22 cells via up-regulation of p27/Kip1.
A, generation of transfectants expressing FKHRL1-TM-ER.
After solubilization, cell extracts were resolved by 7.5% SDS-PAGE and
immunoblotted with the antibodies directed against anti-FKHRL1
antibody. The parent KCL22 cells were used as a control
(lane C). B, effect of STI571 on the
proliferation of transfectants. The cells were plated at a density of
10,000 cells/well in IMDM supplemented with 10% FCS and cultured with
various concentrations of STI571 (0.1-10 µM). MTT
reduction assay was performed after 3 days of culture. The values
represent the mean ± S.D. from triplicate cultures and are
expressed as a percentage of untreated transfectant cells. The parental
KCL22 cells were used as a control. C, effect of 4-OHT on
proliferation of transfectants. The cells were plated at a density of
10,000 cells/well in IMDM supplemented with 10% FCS and cultured with
various concentrations of 4-OHT (0.01-1 µM). MTT
reduction assay was performed after 3 days of culture. The values
represent the mean ± S.D. from triplicate cultures and are
expressed as a percentage of untreated transfectant cells. The parental
KCL22 cells were used as a control. D, effect of FKHRL-1-TM
on cell cycle. The transfectant cells and the parent cells were treated
with STI571 (1 µM) or 4-OHT (0.5 µM),
sequentially cultured for the periods indicated, and harvested for cell
cycle analysis. E, induction of p27/Kip1 or Bim protein
after STI571 and 4-OHT treatment. The parent KCL22 and the transfectant
cells were treated with STI571 (1 µM) or 4-OHT (0.5 µM), sequentially cultured for the periods indicated, and
harvested for Western blotting analysis with antibody against p27/Kip1
or Bim protein. The blot was reprobed with -actin antibody to
confirm equal loading of protein.
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[in a new window]
Fig. 5.
High dose of STI571 induces cell cycle arrest
at the G0/G1 phase and up-regulation of Bim
protein but not apoptosis in KCL22 cells. KCL22 cells were
cultured with various concentrations of STI571 (1-20 µM)
for 48 h and then harvested for cell cycle analysis or Western
blotting analysis. A, effect of high dose of STI571 on cell
cycle. B, effect of high dose of STI571 on the expression of
p27/Kip1 and Bim proteins. Western blotting analysis was performed with
antibody against p27/Kip1 or Bim protein. The blot was reprobed with
-actin antibody to confirm equal loading of protein.
View larger version (30K):
[in a new window]
Fig. 6.
A dominant-negative (DN) FKHRL1
(FKHRL1-DN) enhanced sensitivity to STI571 in KCL22 cells.
A, a dominant-negative FKHRL1 suppresses the transcription
activity of FKHRL1. Two hundred ninety three cells were co-transfected
with pGL3-7xIREs vector and pcDNA3.1 or pcDNA3.1-FKHRL1-WT
with or without pcDNA3.1-FKHRL1-DN expression vector. PRL-TK vector
was used as an internal control. After a 36-h incubation, the cells
were harvested for dual luciferase assay. The results are expressed in
terms of the increased induction of luciferase activity from
pcDNA3.1 vector alone. The values represent the mean ± S.D.
from triplicate experiments. White column, minus
pcDNA3.1-FKHRL1-DN; black column, plus
pcDNA3.1-FKHRL1-DN. B, expression of FKHRL1-DN proteins
in the transfectants. After solubilization, cell extracts were resolved
by 7.5% SDS-PAGE and immunoblotted with antibodies directed against
anti-FKHRL1 antibody. C, effect of FKHRL-1-DN on cell cycle.
The transfectant cells and parent cells were treated with STI571 (1 µM), sequentially cultured for the periods indicated, and
harvested for cell cycle analysis. D, cell viability of
transfectants and the parent KCL22 cells after STI571 treatment. Cells
were treated with increasing concentrations of STI571 (0.01-1
µM) for 4 days and then the cell viability was assessed
by trypan dye exclusion (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(46).
Alternatively, overexpression of FKHRL1-TM may deprive endogenous
FKHRL1 of co-factors prerequisite for the transcription activity, and
as a result, FKHRL1-TM might function as a dominant-negative inhibitor
against anti-apoptotic effect of endogenous FKHRL1, although this
concept is speculative at present. Thus, as proposed by Woods and Rena
(47), caution may be needed when considering any results of
overexpression experiments with FKHRL1-TM. Very recently, Kops et
al. (48) reported that glucose deprivation-induced apoptosis is
significantly blocked by a PI3K inhibitor LY294002 in a human colon
carcinoma cell line. However, our preliminary experiments revealed that
LY294002 up to 20 µM did not prevent 4-OHT-induced
apoptosis in the KCL22 transfectant expressing FKHRL1-TM-ER. Therefore,
it is unlikely that constitutive activation of PI3K activity by BCR-ABL
tyrosine kinase enhanced 4-OHT-induced apoptosis in the
FKHRL1-TM-ER-expressing cells.
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ACKNOWLEDGEMENTS |
---|
We thank Paul Coffer (University Medical Center Utrecht, The Netherlands) for FKHRL1-TM-ER cDNA and Toshiya Inaba (Hiroshima, Japan) for anti-Bim antibody. We also thank Novartis Pharmaceuticals (Basel, Switzerland) for the generous gift of STI571.
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FOOTNOTES |
---|
* This work was supported by grants-in-aid for Cancer Research and Scientific Research from the Ministry of Education, Science, and Culture of Japan, by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, and the Mochida Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Division of Hematology, Dept. of Medicine, Jichi Medical School Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-0498, Japan. Tel.: 81-285-58-7353; Fax: 81-285-44-5258; E-mail: nkomatsu@jichi.ac.jp.
¶ Present address: Dept. of Hematology, Nanfang Hospital, Guangzhou city, Guangdong 510515, China.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M211562200
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ABBREVIATIONS |
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The abbreviations used are: CML, chronic myelogenous leukemia; PI3K, phosphatidylinositol 3-kinase; ER, estrogen receptor; 4-OHT, 4-hydroxytamoxifen; FCS, fetal calf serum; MTT, 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IMDM, Iscove's modified Dulbecco's medium; IREs, insulin-response elements; VHL, von Hippel-Lindau.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | McLaughlin, J., Chianese, E., and Witte, O. N. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6558-6562[Abstract] |
2. | Lugo, T. G., Pendergast, A. M., Muller, A. J., and Witte, O. N. (1990) Science 247, 1079-1082[Medline] [Order article via Infotrieve] |
3. | Tauchi, T., and Broxmeyer, H. E. (1995) Int. J. Hematol. 61, 105-112[CrossRef][Medline] [Order article via Infotrieve] |
4. | Daley, G. Q., and Baltimore, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9312-9316[Abstract] |
5. |
Bedi, A.,
Barber, J. P.,
Bedi, G. C., El-,
Deiry, W. S.,
Sidransky, D.,
Vala, M. S.,
Akhtar, A. J.,
Hilton, J.,
and Jones, R. J.
(1995)
Blood
86,
1148-1158 |
6. |
Bedi, A.,
Zehnbauer, B. A.,
Barber, J. P.,
Sharkis, S. J.,
and Jones, R. J.
(1994)
Blood
83,
2038-2044 |
7. |
Jonuleit, T.,
van der Kuip, H.,
Miething, C.,
Michels, H.,
Hallek, M.,
Duyster, J.,
and Aulitzky, W. E.
(2000)
Blood
96,
1933-1939 |
8. | Cortez, D., Reuther, G., and Pendergast, A. M. (1997) Oncogene 15, 2333-2342[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Bazzoni, G.,
Carlesso, N.,
Griffin, J. D.,
and Hemler, M. E.
(1996)
J. Clin. Invest.
98,
521-528 |
10. |
Salgia, R., Li, J. L.,
Ewaniuk, D. S.,
Pear, W.,
Pisick, E.,
Burky, S. A.,
Ernst, T.,
Sattler, M.,
Chen, L. B.,
and Griffin, J. D.
(1997)
J. Clin. Invest.
100,
46-57 |
11. | Sattler, M., and Salgia, R. (1997) Cytokine Growth Factor Rev. 8, 63-79[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Skorski, T.,
Bellacosa, A.,
and Nieborowska-Skorska, M.
(1997)
EMBO J.
16,
6151-6161 |
13. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |
14. |
Tang, E. D.,
Nunez, G.,
Barr, F. G.,
and Guan, K. L.
(1999)
J. Biol. Chem.
274,
16741-16746 |
15. |
Rena, G.,
Guo, S.,
Cichy, S. C.,
Unterman, T. G.,
and Cohen, P.
(1999)
J. Biol. Chem.
274,
17179-17183 |
16. | Kops, G. J., de Ruiter, N. D., De, Vries-Smits, A. M., Powell, D. R., Bos, J. L., and Burgering, B. M. (1999) Nature 398, 630-634[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Biggs, W. H., III,
Meisenhelder, J.,
Hunter, T.,
Cavenee, W. K.,
and Arden, K. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7421-7426 |
18. |
Kashii, Y.,
Uchida, M.,
Kirito, K.,
Tanaka, M.,
Nishijima, K.,
Toshima, M.,
Ando, T.,
Koizumi, K.,
Endoh, T.,
Sawada, K.,
Momoi, M.,
Miura, Y.,
Ozawa, K.,
and Komatsu, N.
(2000)
Blood
96,
941-949 |
19. |
Tanaka, M.,
Kirito, K.,
Kashii, Y.,
Uchida, M.,
Toshima, M.,
Endoh, T.,
Sawada, K.,
Ozawa, K.,
and Komatsu, N.
(2001)
J. Biol. Chem.
276,
15082-15089 |
20. | Kaufmann, E., and Knochel, W. (1996) Mech. Dev. 57, 3-20[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Lin, K.,
Dorman, J. B.,
Rodan, A.,
and Kenyon, C.
(1997)
Science
278,
1319-1322 |
22. | Ogg, S., Paradis, S., and Gottlieb, S. (1997) Nature 389, 994-999[CrossRef][Medline] [Order article via Infotrieve] |
23. | Galili, N., Davis, R. J., and Fredericks, W. J. (1993) Nat. Genet. 5, 230-235[Medline] [Order article via Infotrieve] |
24. |
Hillion, J., Le,
Coniat, M.,
Jonveaux, P.,
Berger, R.,
and Bernard, O. A.
(1997)
Blood
90,
3714-3719 |
25. | Borkhardt, A., Repp, R., Haas, O. A., Leis, T., Harbott, J., Kreuder, J., Hammermann, J., Henn, T., and Lampert, F. (1997) Oncogene 14, 195-202[CrossRef][Medline] [Order article via Infotrieve] |
26. | Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A., and Cohen, P. (1996) FEBS Lett. 399, 333-338[CrossRef][Medline] [Order article via Infotrieve] |
27. | Medema, R. H., Kops, G. J., Bos, J. L., and Burgering, B. M. (2000) Nature 404, 782-787[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Dijkers, P. F.,
Medema, R. H.,
Pals, C.,
Banerji, L.,
Thomas, N. S.,
Lam, E. W.,
Burgering, B. M.,
Raaijmakers, J. A.,
Lammers, J. W.,
Koenderman, L.,
and Coffer, P. J.
(2000)
Mol. Cell. Biol.
20,
9138-9148 |
29. |
Jonuleit, T.,
van Der Kuip, H.,
Miething, C.,
Michels, H.,
Hallek, M.,
Duyster, J.,
and Aulitzky, W. E.
(2000)
Blood
96,
1933-1999 |
30. |
Parada, Y.,
Banerji, L.,
Glassford, J.,
Lea, N. C.,
Collado, M.,
Rivas, C.,
Lewis, J. L.,
Gordon, M. Y.,
Thomas, N. S.,
and Lam, E. W.
(2001)
J. Biol. Chem.
276,
23572-23580 |
31. |
Gesbert, F.,
Sellers, W. R.,
Signoretti, S.,
Loda, M.,
and Griffin, J. D.
(2000)
J. Biol. Chem.
275,
39223-39230 |
32. | Kubonishi, I., and Miyoshi, I. (1983) Int. J. Cell Cloning 1, 105-117[Abstract] |
33. | Kishi, K. (1985) Leuk. Res. 9, 381-390[Medline] [Order article via Infotrieve] |
34. | Nakajima, A., Tauchi, T., and Ohyashiki, K. (2001) Leukemia (Baltimore) 15, 989-990[CrossRef] |
35. | Mosmann, T. (1983) J. Immunol. Methods 65, 55-63[CrossRef][Medline] [Order article via Infotrieve] |
36. | Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (2000) Curr. Biol. 10, 1201-1204[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Nakamura, N.,
Ramaswamy, S.,
Vazquez, F.,
Signoretti, S.,
Loda, M.,
and Sellers, W. R.
(2000)
Mol. Cell. Biol.
20,
8969-8982 |
38. | Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995) Science 269, 682-685[Medline] [Order article via Infotrieve] |
39. | Hengst, L., and Reed, S. I. (1996) Science 271, 1861-1864[Abstract] |
40. |
Shirane, M.,
Harumiya, Y.,
Ishida, N.,
Hirai, A.,
Miyamoto, C.,
Hatakeyama, S.,
Nakayama, K.,
and Kitagawa, M.
(1999)
J. Biol. Chem.
274,
13886-13893 |
41. | Kim, M., Katayose, Y., Li, Q., Rakkar, A. N., Li, Z., Hwang, S. G., Katayose, D., Trepel, J., Cowan, K. H., and Seth, P. (1998) Biochem. Biophys. Res. Commun. 253, 672-677[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Strasser, A.,
Puthalakath, H.,
Bouillet, P.,
Huang, D. C.,
O'Connor, L.,
O'Reilly, L. A.,
Cullen, L.,
Cory, S.,
and Adams, J. M.
(2000)
Ann. N. Y. Acad. Sci.
917,
541-548 |
43. |
Shinjyo, T.,
Kuribara, R.,
Inukai, T.,
Hosoi, H.,
Kinoshita, T.,
Miyajima, A.,
Houghton, P. J.,
Look, A. T.,
Ozawa, K.,
and Inaba, T.
(2001)
Mol. Cell. Biol.
21,
854-864 |
44. | Kuribara, R., Honda, H., Shnjyo, T., Inukai, T., Sugita, K., Nakazawa, S., Hirai, H., and Ozawa, K. (2000) Blood 96, 347a |
45. |
Zheng, W. H.,
Kar, S.,
and Quirion, R.
(2000)
J. Biol. Chem.
275,
39152-39158 |
46. | Rena, G., Prescott, A. R., Guo, S., Cohen, P., and Unterman, T. G. (2001) Biochem. J. 354, 605-612[CrossRef][Medline] [Order article via Infotrieve] |
47. | Woods, Y. L., and Rena, G. (2002) Biochem. Soc. Trans. 30, 391-397[CrossRef][Medline] [Order article via Infotrieve] |
48. | Kops, G. J., Dansen, T. B., Polderman, P. E., Saarloos, I., Wirtz, K. W., Coffer, P. J., Huang, T. T., Bos, J. L., Medema, R. H., and Burgering, B. M. (2002) Nature 419, 316-321[CrossRef][Medline] [Order article via Infotrieve] |
49. | Stambolic, V., Suzuki, A., de La Pompa, J. L., Brothers, G. M., Mirtsos, C., Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. (1998) Cell 95, 1-20[Medline] [Order article via Infotrieve] |