From the George Whipple Lab for Cancer Research, Departments of Pathology, Urology, and Radiation Oncology and The Cancer Center, University of Rochester Medical Center, Rochester, New York 14642
Received for publication, December 26, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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APPL may function as an adapter protein to
modulate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Although
we have previously proven that the PI3K/Akt pathway can suppress
androgen receptor (AR) transactivation, the potential linkage from APPL to the AR remains unclear. Here we demonstrated that APPL could suppress AR-mediated transactivation in a dose-dependent
manner in LNCaP and PC-3 cells. This suppressive effect could be
blocked by either dominant-negative Akt or dominant-negative PI3K or
LY294002, suggesting that the APPL-mediated suppression of AR
transactivation is dependent on the PI3K/Akt pathway. We also observed
that APPL could further enhance the Akt-mediated suppression of AR
transactivation and AR target gene using the reporter gene and Northern
blot assay. APPL was able to enhance insulin-like growth factor
(IGF-1)-mediated Akt activation. The abrogation of IGF-1-mediated Akt
activation by the dominant-negative PI3K or LY294002 or antisense APPL
suggests that APPL may function as an important adapter protein in
controlling the IGF-1 The androgen receptor
(AR)1 is a member of the
nuclear receptor superfamily that cooperates with multiple proteins to
exert its biological function (1-4). Upon binding to ligand
testosterone/5 The phosphatidylinositol 3-kinase (PI3K) consists of regulatory (p85)
and catalytic (p110) subunits that participate in multiple cellular
processes, including cell survival, growth, transformation, and
differentiation (9). Akt, a serine/threonine kinase can prevent cell
apoptosis by phosphorylation and inactivation of several pro-apoptotic
proteins such as Bad, caspase-9, and forkhead transcription factors
(10-12) and is the key effector of the PI3K pathway (12, 13). The
binding of PI3K-generated phospholipids to Akt results in the
translocation of Akt from the cytoplasm to the inner surface of the
plasma membrane, where Akt is phosphorylated by the upstream kinases
phosphoinositide-dependent protein kinase (PDK)-1, PDK-2,
and integrin-linked kinase (14, 15). Three Akt homologs, Akt1,
Akt2, and Akt3, have been identified and characterized (16-18). Tissue
distribution studies suggest that these Akt homologs are expressed
ubiquitously in human tissue and may share similar mechanisms in
exerting their biological functions (19).
APPL (adapter protein containing PH domain,
PTB domain, and leucine zipper motif) was
identified as an Akt-interacting protein (20) and detected in many
human tissues including the prostate. The ability of APPL to interact
with Akt1 and P110 Materials--
pCDNA3-cAkt (a constitutively active Akt with
a deletion at amino acids 4-129 replaced with a consensus
myristoylation domain) and pCDNA3-dAkt (a kinase-deficient mutant,
K179A) were obtained from Dr. Robert Freeman (22). APPL Constructs--
APPL cDNA was PCR-amplified from the
human testis Marathon-Ready cDNA library
(Clontech) using primers APPL-5' (5' CTT CTC
GAG ATG CCG GGG ATC GAC AAG 3') and APPL-3' (5'-CGC GGG
CCC TTA TGC TTC TGA TTC TCT-3'). The underlined nucleotides
represent BamHI in APPL-5' and ApaI in APPL-3'.
The PCR products were cut as a BamHI to ApaI
fragment and inserted between the BamHI and ApaI sites in pCDNA3-FLAG and in-frame to the down-stream of the FLAG tag. APPL N- and C-terminal DNA fragments (1-1284 and 1329-2130 bp,
respectively) were produced by PCR from pCDNA3-FLAG-APPL and inserted between the BamHI and XbaI sites in
pCDNA3-FLAG. The APPL N-terminal DNA fragment was inversely
inserted into pFLAG-CMV vector (Sigma) with XbaI and
BamHI sites as antisense APPL.
Reverse Transcription (RT)-PCR Analysis--
Reverse
transcription was performed at 42 °C for 50 min in a total volume of
20 µl of first-strand buffer (Invitrogen) containing 0.5 µg of
oligo(dT)12-18 primers (Invitrogen), 0.5 mM
each dNTP (Invitrogen), 10 mM dithiothreitol, 200 units of
Moloney murine leukemia virus reverse transcriptase (U. S. Biochemical Corp.), 200 units of SuperScript II reverse transcriptase, and 1 µg
of total RNA. The absence of contaminating DNA from each RNA sample was
checked by omitting the reverse transcriptase from the RT reaction (RT
control). When the reaction was complete, 2 µl of the cDNA
solutions were amplified in a final volume of 50 µl of PCR buffer (20 mM Tris/HCl, 50 mM KCl, 1.5 mM
MgCl2) containing 0.2 µM each primer, 0.2 mM each dNTP (Invitrogen), and 1 unit of Taq DNA
polymerase (Invitrogen). The PCR primers used were: PTB-N 5', 5'-CGC
GGA TCC CAG TTA TTT ATT GTC CGA-3', and PTB-N 3', 5'-GCT CTA GAT TAT
GGA AAC GTG AGC CTT GT-3' (250-bp product).
Cell Culture and Transfections--
The human prostate cancer
PC-3 and African green monkey kidney COS-1 cells were maintained in
Dulbecco's minimum essential medium containing penicillin (25 units/ml), streptomycin (25 µg/ml), and 10% fetal calf serum. The
androgen-dependent human prostate cancer LNCaP cells were
purchased from ATCC, and the passages numbering between 22 and 30 were
used in these studies and maintained in ATCC-recommended RPMI 1640 with
10% fetal calf serum. Transfections were performed using
SuperFectTM (Qiagen). In brief, cells were plated in 10%
charcoal-dextran-treated FBS (CD-FBS)-containing medium in 24-well
plates at 8 × 104 cells/well. One day after plating,
the cells were transfected according to standard procedures.
Luciferase Reporter Assays--
The cells were transfected with
plasmids in the 10% CD-FBS media for 16 h and then treated with
ethanol or 10 nM DHT for 24 h. After the cells were
washed with phosphate-buffered saline and harvested, cell lysates were
prepared and used for luciferase assay according to the manufacturer's
instructions (Promega). The results were obtained from at least three
sets of transfection and are presented as the mean ± S.D.
Glutathione S-Transferase (GST) Pull-down Assay--
APPL was
subcloned into pGEX-KG vector (Amersham Biosciences). GST fusion
proteins were generated from BL21 (DE3) strain and purified as
described by the manufacturer (Amersham Biosciences). The purified GST
proteins were resuspended with 100 µl of interaction buffer (20 mM Tris-HCl/pH 8.0, 60 mM NaCl, 1 mM EDTA, 6 mM MgCl2, 1 mM dithiothreitol, 8% glycerol, 0.05% (v/v) Nonidet P-40,
1 mM phenylmethylsulfonyl fluoride, and proteinase
inhibitors) and incubated with 800 µg of LNCaP cell lysates or
35S-labeled TNT-expressed AR in the presence or absence of
10 nM ligands on a rotating disk at 4 °C for 2 h.
Translation reactions were performed in the TNT-coupled rabbit
reticulocyte lysate (Promega) system according to the manufacturer's
instructions. After extensive washes with NENT (20 mM Tris; pH 8.0, 100 mM NaCl, 6 mM
MgCl2, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, 8% glycerol, 1 mM
phenylmethylsulfonyl fluoride) buffer, the bound proteins were
separated on an 8% SDS-polyacrylamide gel and analyzed by Western blot
with anti-AR NH27 antibody.
Immunoprecipitation and Western Blot Analysis--
A standard
protocol was performed for immunoprecipitation. For the
immunoprecipitation of APPL and AR, COS-1 cells (at a cell density of
1 × 106 cells/10-cm dish) were maintained in 10%
CD-FBS medium and transfected with APPL, AR, Akt, or combinations of
these genes. After 16 h, cells were treated with or without DHT
for 24 h. For the immunoprecipitation of APPL and PI3K, COS-1
cells (at a cell density of 1 × 106 cells/10-cm dish)
were maintained in 10% FBS medium and transfected with APPL and
wild-type p85, a regulatory subunit. After 24 h, cells were lysed
by radioimmune precipitation assay buffer containing 1×
phosphate-buffered saline, 1% IGEPAL CA-630 (Sigma), 0.5% sodium deoxycholate, and 0.1% SDS and supplemented with 10 µl/ml protease inhibitor phenylmethylsulfonyl fluoride (10 mg/ml). Lysates were cleared by centrifugation at 12,000 × g for 15 min at
4 °C, and 800 µg supernatants were incubated with 1 µg of
individual antibodies and 30 µl of packed protein A/G PLUS-Sepharose
beads at 4 °C for 1 h. After incubation, the beads were washed
five times with radioimmune precipitation assay buffer. The resulting
immunoprecipitated immunocomplexes were solubilized in 60 µl of
protein sample buffer, resolved by 8% SDS-PAGE, and transferred to a
polyvinylidene difluoride membrane. The protein complex was detected by
Western blot analysis and developed by the AP conjugate substrate kit
(Bio-Rad).
Northern Blot Analysis--
LNCaP cells were cultured following
the methods previously described (28) and treated with or without DHT
for 24 h. Total RNA was isolated from each plate using the TRIZOL
reagent (Invitrogen), and 25 µg of each RNA was loaded onto
denaturing agarose gels. The RNA samples were separated by
electrophoresis and blotted onto a nylon membrane using a vacuum
blotter. PSA cDNA was used as the hybridization probe, and
APPL Expression in Different Cell
Lines--
We first applied RT-PCR
analysis to test the expression of endogenous APPL mRNA in COS-1
and prostate cancer lines, which will be used in the current studies.
As shown in Fig. 1, the amplified APPL transcript of 250 bp was
detected in the androgen-dependent LNCaP cells,
androgen-independent PC-3 cells, and the COS-1 cells.
APPL Suppresses AR Transactivation--
APPL has been
identified as an Akt-interacting protein that contains
phosphotyrosine binding, pleckstrin homology (PH), and leucine
zipper domains (20). Our earlier study showed that Akt could suppress
AR transactivation in prostate cancer cells (21). To investigate the
potential influence of APPL on the AR transactivation, we first
co-transfected APPL with AR in the presence of 10 nM DHT to
see if APPL could influence the activity of the luciferase reporter
linked to three different promoters containing AREs, MMTV 5' promoter,
PSA 5' promoter, and four copies of a synthetic ARE ((ARE)4) in PC-3
cells. As shown in Fig. 2A, 10 nM DHT induces AR transactivation up to 25-fold, and the
addition of APPL can then repress AR transactivation in a
dose-dependent manner in all three different ARE promoters
in PC-3 cells. To reduce the potential artificial effect of exogenous
overexpression of AR, we replaced PC-3 cells with LNCaP cells that
express endogenous, mutated but functional AR. As shown in Fig.
2B, the addition of APPL also represses endogenous
AR-induced (ARE)4-luc activity in a dose-dependent manner.
In contrast, APPL showed only marginal effects on the estrogen receptor
(ER) APPL Enhances Akt Effect on Suppression of AR
Transactivation--
Because we previously showed that Akt could
repress AR transactivation (21), we were interested in seeing if APPL,
an Akt-interacting protein, had any influence on the Akt-repressed AR
transactivation. As shown in Fig.
3A, in PC-3 cells cAkt can
repress AR transactivation in all reporters with three different AREs,
and the addition of APPL can then further suppress AR transactivation.
In contrast, the addition of cAkt enhances the ER Suppression of AR Transactivation by APPL Is Dependent on the
PI3K/Akt Pathway--
An earlier report suggested that APPL
could interact with Akt and PI3K and might function as an adapter
affecting the PI3K/Akt pathway (20). We were interested in seeing if
APPL-mediated suppression of AR transactivation relied on the PI3K/Akt
pathway. Three components, APPL Enhances Akt Phosphorylation Dependent on PI3K
Activity--
APPL was first identified as an Akt interaction protein
(20). However, the potential physiological roles of APPL as well as how
APPL influences Akt function remain largely unknown. As our data show,
because APPL can enhance the Akt-mediated suppression of AR
transactivation and the suppression of AR transactivation by APPL is
dependent on PI3K/Akt pathway, we would like to see if it is possible
that APPL may also be able to influence PI3K/Akt activity. Using a
co-immunoprecipitation assay, we found that APPL could interact with
p85, a subunit of PI3K (Fig.
5A). Because the PI3K/Akt
pathway can down-regulate the gene expression of p27Kip1
(30), we used p27Kip1 as a surrogate gene to monitor the
Akt activity in the presence and absence of APPL. As shown in Fig.
5B, p27Kip1 promoter activity (lane
1) was suppressed by both APPL (lane 2) and cAkt
(lane 3). The simultaneous addition of cAkt and APPL (lane 4) could further suppress p27Kip1 promoter
activity (lane 4). In contrast, the addition of dAkt (lane 5), LY240092 (lane 6), or
To further confirm this notion, we directly assayed the Akt activity by
monitoring the Akt phosphorylation status in the presence and absence
of APPL. As shown in Fig. 5C, IGF-1 can activate the Akt
activity (lane 2), and this effect can be blocked by Inhibition of the PI3K/Akt Pathway Increases the PSA
Expression Level--
Data from Figs. 2-5 suggest that APPL may
suppress AR transactivation via interaction with PI3K to mediate the
growth factor signal for the activation of Akt. To avoid potential
artificial effects from the reporter gene assay on the results in Figs.
2-4, we replaced PC-3 cells with LNCaP cells and tested the influence of Akt and APPL on the mRNA expression of PSA, an androgen-induced target gene that has been used as a marker to monitor the progress of
prostate cancer (31). As shown in Fig.
6A, androgen-induced PSA
mRNA expression (middle panel, lane 2) can be
repressed by the addition of cAkt or APPL (middle panel,
lanes 3 and 5, respectively). As expected, the
simultaneous addition of cAkt and APPL can further suppress the PSA
mRNA expression (middle panel, lane 4).
Quantification of the Northern blot in Fig. 6A is presented
(upper panel). To further prove the PI3K/Akt signaling
repression on AR transactivation, we treated LNCaP cells with LY294002
to determine the effect of the PI3K/Akt pathway on PSA mRNA and
protein expression. As shown in Fig. 6B, 4 h of
treatment of 20 µM LY294002 decreased the PSA mRNA
level (middle panel, lane 4 versus
lane 2) but increased PSA mRNA expression at 24 h
(middle panel, lane 8 versus
lane 6). Quantification of the PSA mRNA expression level
in Fig. 6B is shown in the upper panel. Western
blot results indicated that blockade of PI3K/Akt pathway with LY294002
(24 h) causes a significant increase of protein expression level of PSA
(Fig. 6C, lane 3 versus lane
2), but 4 h of treatment of LY294002 only marginally
influenced PSA protein level (data not shown). We also tested the
effect of androgen ablation on Akt activity by growing LNCaP cells
under serum-starvation conditions for 4 days in the presence and
absence of DHT. Removal of androgens resulted in increased levels of
active phosphorylated Akt (Fig. 6D, lane 2 versus lane 1), but no changes were observed in
Akt protein levels. These data indicate that acute androgen deprivation
of LNCaP cells triggered an increase in PI3K and Akt activity.
APPL C-terminal Domain Suppresses AR Transactivation--
The APPL
C terminus is responsible for binding to Akt (20), suggesting that the
APPL C terminus itself may play a role on the repression of AR
transactivation. Thus, we dissected the full-length APPL into the
N-terminal domain (amino acids 1-428 containing the leucine zipper and
PH domain) and the C-terminal domain (amino acids 443-710 containing
the phosphotyrosine binding domain) (Fig. 7A). As shown in Fig.
7B, the APPL C terminus suppresses AR transactivation similar to the full-length APPL. In contrast, the APPL N terminus shows
little suppression on AR transactivation. The inability of the APPL N
terminus to inhibit AR transactivation was not due to lower levels of
protein expression, since the expression levels of the full-length
APPL, APPL N terminus, and APPL C terminus were comparable (Fig.
7C). Because we demonstrated that APPL repression of AR
transactivation is dependent on the PI3K/Akt pathway by inducing Akt
activity, it may also be possible that the APPL C terminus is
sufficient to enhance Akt activity. Full-length APPL and APPL N and
APPL C termini were transfected into COS-1 cells under serum-starvation
conditions for 24 h, and then cells were treated with IGF-1 for 20 min. As shown in Fig. 7D, full-length APPL and APPL C
terminus both increased the phosphorylated Akt level. The expression of
full-length APPL and APPL N and APPL C termini was confirmed by Western
blot (data not shown). Hence, the results from Fig. 7 demonstrate that
the C-terminal region of APPL may be responsible for APPL suppression
of AR transactivation.
APPL Is Detected in the AR Immunocomplexes--
We used the GST
pull-down assay and Western blot analysis to test whether APPL can
associate with AR. APPL fused with GST was used as bait to incubate
with LNCaP cell lysates, which contain endogenous AR and Akt. Fig.
8A indicates AR could be
detected through GST-APPL but not GST alone. DHT addition did not
significantly enhance the APPL·AR complex formation. We then
used in vitro translated 35S-labeled AR to
replace LNCaP cell extracts to see if AR can interact with APPL
directly. As shown in Fig. 8B, GST-APPL did not pull-down AR
in the presence or absence of DHT. However, GST-ARA70 was able to
interact with AR under the same conditions. These results suggest that
APPL may not directly interact with AR and may require a bridge factor
that exists in the LNCaP cell extract to form an APPL·AR complex.
Because Akt has been proven to be the interacting protein for both AR
(21) and APPL (20), it is likely that Akt may serve as a bridge factor
to bring the APPL and AR together. If Akt is one of the important
bridge factors for the promotion of APPL·AR complex formation,
ectopic expression of Akt should enhance this complex formation. To
test this hypothesis, COS-1 cells co-transfected with AR, FLAG-APPL,
and hemagglutinin (HA)-Akt were immunoprecipitated with anti-FLAG M2
antibody and analyzed by Western blot. The membranes were then probed
individually with the antibodies of anti-AR (G122-77), anti-APPL (FLAG
M2), and anti-Akt (HA). As shown in Fig. 8C, the APPL·AR
complex was not clearly seen (lane 6), and the addition of
Akt markedly enhanced APPL·AR complex formation (lane 7).
These results support the assumption that Akt may be an important
bridge factor for the promotion of APPL·AR complex formation.
APPL contains multiple important regulatory motifs, including a PH
domain, a phosphotyrosine binding domain, and a leucine zippered
coiled-coil domain. The PH domain exists in diverse signaling molecules
and permits anchorage of proteins to the cell membrane via phospholipid
interactions (20). The leucine zippered coiled-coil domain is found in
many transcription factors and has been proposed to play a role in the
dimerization/polymerization of proteins (32). The phosphotyrosine
binding domain was originally identified from mammalian signaling
proteins such as Shc and has been shown to mediate protein-protein as
well as protein-phospholipid interactions (33). Although APPL is
identified as an Akt-associated protein from the yeast two-hybrid
screening using Akt2 as bait (20), the detailed mechanism of how APPL
may affect the Akt-signaling pathway and its biological function
remains largely unknown. In this study, we demonstrate that APPL
suppresses the AR transactivation in a dose-dependent
manner in the androgen-dependent and androgen-independent prostate cancer cells and that this suppressive effect is largely dependent on the PI3K/Akt pathway. We also observed that APPL could
further enhance the Akt-mediated suppression of AR transactivation. Furthermore, we also found that APPL can interact with PI3K and Akt
(Fig. 5A and data not shown). These data suggest that APPL may regulate PI3K/Akt activity by controlling AR transactivation. In
support of this notion, we found that APPL suppresses the reporter gene
expression of p27Kip1, an Akt down-stream target, and that
this effect is blocked by LY294002, Several lines of evidence suggest that APPL regulates the AR
transactivation via the PI3K/Akt-dependent pathway. First,
suppression of AR transactivation by APPL is blocked by LY294002,
AR is a well known critical proliferation factor in prostate cancer.
The study on the role of PI3K/Akt in the progression of prostate cancer
suggests that PI3K/Akt may function as a dominant growth
factor-activated cell survival pathway in LNCaP cells (34). Thus, PI3K/Akt and AR may cooperate with each other to promote the
prostate cancer cell survival and growth. Based on our data and other
reports, PI3K/Akt and AR may also negatively regulate each other to
maintain homeostasis of prostate cancer cells. Supporting this
hypothesis, Lin et al. (34) find that in LNCaP cells
inhibiting the PI3K/Akt pathway will induce apoptosis, but DHT
pretreatment can prevent this apoptosis. Our data further indicate that
inhibition of PI3K/Akt strongly induces AR functioning, suggesting that
LNCaP cells are able to enhance AR activity to rescue
themselves from apoptosis caused by the blockade of PI3K/Akt. Thompson
et al. (35) demonstrate that Akt can suppress AR
transactivation in PC-3 cells, which is also consistent with our
previous report (21), In addition, Murillo et al. (36) show
that androgen ablation can increase PI3K-Akt activity, as our data
suggest (Fig. 6D), implying that enhanced Akt activity may
rescue LNCaP cells from impairment of androgen withdrawal. Taken
together, these findings suggest that the survival factor Akt and the
proliferation factor AR may mutually influence prostate cancer cell
survival and growth in a balanced way.
Interestingly, using reporter gene assays, Thompson et al.
(35) and Lin et al. (21) indicate that Akt can suppress AR transactivation in PC-3 cells, whereas Wen et al. (37) find that Her2/Neu was able to go through Akt to enhance PSA promoter activity in LNCaP cells in the absence of androgen. Manin et
al. (38) show that LY294002 treatment could decrease AR protein and PSA mRNA levels but was unable to influence AR mRNA
expression in LNCaP cells, whereas Sharma et al. (39) find
that LY294002 treatment did not suppress the AR protein level in LNCaP
cells, but 4 h of LY294002 treatment decreased the PSA mRNA
level, which is consistent with our report (Fig. 6B). The
detailed mechanisms for these discrepancies remain unclear. This may be
due to the use of different reagents and different cell lines with
various passage numbers. Furthermore, different experimental
conditions, such as varied concentrations and treatment times of
LY294002, applied by individual investigators, may also influence the
results. We used parental LNCaP cells with passage numbers 23-30 under the ATCC-medium culture condition in our studies. According to our data
and the results from a recent report which show that LNCaP cells with
androgen withdrawal treatment show increased Akt activity
sustained throughout the progression process (36) combined with the
evidence that PI3K/Akt is the dominant factor for the LNCaP cells
survival (34), it is possible that AR activity can be induced by
LY294002 to play a dominant proliferation role to compensate for the
loss of PI3K/Akt signaling. Overall, AR and PI3K/Akt signaling both
appear to be important proliferation and survival factors in prostate
cancer cells and also seem to antagonize each other to maintain cell homeostasis.
In conclusion, our finding that APPL may suppress AR function via the
PI3K-Akt pathway may represent a unique pathway that further expands
the importance of the PI3K-Akt-AR pathway in prostate cancer. Further
development of blockers to interrupt this pathway may help us to battle
the prostate cancer.
Akt signal pathway. Co-immunoprecipitation
and glutathione S-transferase pull-down assays suggest that
APPL, Akt, and AR may exist in a complex and Akt may serve as an
important bridge factor for the association of APPL with AR. Together,
our data indicate that APPL may suppress AR transactivation via
potentiating Akt activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dihydrotestosterone (DHT), the AR can bind to the
androgen-response-element (ARE) on the 5' promoter of the target gene,
which results in the modulation of cell growth (5, 6). Prostate cancer
is the most common form of cancer in men and the second leading cause
of cancer deaths in men in the United States (7). Androgen ablation is
the mainstream of therapy for progressive prostate cancer; however,
most of prostate cancer patients eventually fail with androgen ablation
therapy and die of recurrent androgen-independent prostate cancer (8). The failure of androgen ablation therapy may be due to an alteration of
the normal androgen axis through mutation of AR, an alteration in the
expression of AR coregulators, or a dysregulation of AR activity
through signal transduction cascades. A substantial body of literature
suggests that the AR can be regulated directly or indirectly by growth
factor signal transduction pathways, which may contribute to the
development and progression of prostate cancer (8).
, the catalytic domain of PI3K, suggests that APPL
may function as an adapter to tether Akt and PI3K (20). The detailed
mechanism of how APPL influences the P13K/Akt signal pathway, however,
remains unclear. Our early reports show that Akt can suppress AR
transactivation (21). In this study, we demonstrate that various AR
functions can be suppressed by a new signal pathway of APPL
Akt
AR, which may influence AR-mediated prostate cancer growth.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85 was kindly
provided by M. Kasuga, Kobe University, Kobe, Japan (23), and p110* was
from L. T. Williams, Chiron Corp., Emeryville, CA (24). The
promoter region of the human p27Kip1 gene (p27PF) was
provided by Dr. Toshiyuki Sakai (25). LY294002 was purchased from
Calbiochem, and DHT was from Sigma. The plasmids pCMV-AR, pSG5-AR,
mouse mammary tumor virus promoter-luciferase (MMTV-luc),
prostate-specific antigen promoter-luciferase (PSA-luc), promoter-luciferase reporters containing four copies of androgen responsible elements ((ARE)4-luc), pRL-SV40, and pRL-TK have been previously described (2, 26). The anti-AR polyclonal antibody, NH27,
was produced as previously described (27). The anti-AR monoclonal
antibody (G122-77) was purchased from Pharmingen. The anti-FLAG M2
monoclonal antibody was from Sigma, and the AP-conjugated secondary
anti-mouse, and anti-rabbit antibodies were from Santa Cruz.
-actin RNA was used as a control for equivalent RNA loading.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
APPL expression in PC-3, LNCaP, and COS-1
cells. Total RNA was extracted from LNCaP cells (lane
2), PC-3 cells (lane 3), and COS-1 cells (lane
4) and analyzed by RT-PCR for APPL mRNA expression using the
primers and RT-PCR conditions as shown under "Experimental
Procedures." The expected fragments of 250 bp were obtained from the
PCR reactions. The PCR products were resolved in 1.0% agarose
gels.
-mediated transactivation, even with high concentrations of
APPL in PC-3 cells (Fig. 2C). These findings suggest that
the APPL effect on AR transactivation is selective and may not be due
to general transcriptional squenching. To further prove the APPL
repression effect on AR transactivation, we tested antisense APPL
effect on AR activity in LNCaP cells with natural promoter
PSA-luc. As shown in Fig. 2D, APPL also suppressed
endogenous AR-induced PSA-luc activity in a dose-dependent manner, and antisense APPL enhanced AR activity and could reverse the
inhibitory effect of transfected APPL. Overall, the results from Fig. 2
suggest that APPL can repress AR transactivation in prostate cancer
cells, such as LNCaP and PC-3 cells.
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Fig. 2.
APPL suppressed AR transactivation in a
dose-dependent manner. A,
dose-dependent inhibition of AR activity by APPL in PC-3
cells. Cells were transfected with 50 ng of pCMV-AR, 150 ng of
MMTV-luc, PSA-luc, or (ARE)4-luc, 2.5 ng of pRL-SV40, and 50-300 ng of
APPL (+, 50 ng; ++, 150 ng; +++, 300 ng). The parent vector pCDNA-3
was used to balance equal amounts of plasmid transfection. Transfected
cells were treated for 24 h with 10 8 nM
DHT or ethanol as vehicle control. Duplicate samples were analyzed for
each data point. B, APPL repression of endogenous AR
activity on (ARE)4 promoter in LNCaP cells. Cells were transfected with
250 ng of (ARE)4-luc, 5 ng pRL-TK, and 50-300 ng of APPL (+, 50 ng;
++, 150 ng; +++, 300 ng). Empty vector was used to balance the total
DNA amount. C, APPL did not repress ER activity on ERE-luc
promoter in PC-3 cells. Cells were transfected with 50 ng of
pSG5-ER
, 250 ng of (ERE)4-luc, 5 ng of pRL-TK, and 150-300 ng of
APPL (++, 150 ng; +++, 300 ng). Transfected cells were treated for
24 h with 10
8 nM estradiol
(E2) or ethanol as vehicle control. Empty vector was used to
balance the total DNA amount. D, antisense-APPL
(APPL-as) enhanced AR transactivation. LNCaP cells were
transfected with 250 ng of PSA-luc, 5 ng of pRL-TK, 300 ng of APPL-as,
300-600 ng of APPL (+, 300 ng; ++, 600 ng), and 300 ng of APPL-as plus
300 ng of APPL. Empty vector was used to balance the total DNA amount.
ERE, estrogen response element.
-mediated
transactivation (Fig. 3B), which is consistent with a recent
publication (29). Together, results from Fig. 3 clearly indicate that
both APPL and Akt can repress AR transactivation and that APPL can
further enhance the Akt effect on AR transactivation.
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Fig. 3.
APPL cooperated with Akt to further suppress
AR transactivation. A, APPL cooperated with Akt to
further suppress AR activity in PC-3 cells. Cells were transfected with
50 ng of pCMV-AR, 150 ng of MMTV-luc, PSA-luc, or (ARE)4-luc, 2.5 ng of
pRL-SV40, 50-150 ng of APPL (+, 50 ng; ++, 150 ng), and 50 ng of cAkt.
pCDNA3 vector was added to balance the total DNA amount.
Transfected cells were treated for 24 h with 10 8
nM DHT or ethanol as the vehicle control. Duplicate samples
were analyzed for each data point. B, cAkt, but not APPL,
enhances the ER
-mediated transactivation in PC-3 cells. Cells were
transfected with 50 ng of pCMV-ER
, 150 ng of ERE-luc, 2.5 ng of
pRL-SV40, 150 ng of APPL, and 150 ng of cAkt. Transfected cells were
treated for 24 h with 10
8 nM E2 or
ethanol as vehicle control. Duplicate samples were analyzed for each
data point.
p85 (a dominant negative form of PI3K),
LY294002 (a selective PI3K inhibitor), and dAkt (a dominant negative
form of Akt), were applied to examine potential effects on
APPL-mediated AR transactivation. As shown in Fig.
4, in PC-3 cells the addition of APPL
(lane 3) or cAkt (lane 4) alone suppresses AR
transactivation (lane 2). Simultaneous addition of APPL and
cAkt further suppresses AR transactivation (lane 5). As
expected, the addition of dAkt (lane 6), LY294002
(lane 8), and
p85 (lane 10) alone enhance AR
transactivation, which is consistent with a previous report (21). Furthermore, the suppression effect of APPL on AR transactivation can be reversed after adding either dAkt (lanes 3 versus 7), LY294002 (lanes 3 versus 9), or
p85 (lanes 3 versus 11). These results suggest that the
PI3K/Akt pathway plays a major role in the APPL-mediated inhibition of
AR transactivation. Taken together, the results from Fig. 4 clearly
demonstrate that the suppression of AR transactivation by APPL is
dependent on the PI3K/Akt pathway.
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Fig. 4.
Suppression of AR transactivation by APPL is
via PI3K/Akt pathway. Dominant-negative Akt (dAkt) and
LY294002 blocked the effect of APPL on AR activity in PC-3 cells. Cells
were transfected with 50 ng of pSG5-AR, 150 ng of MMTV-luc or
(ARE)4-luc, 5 ng of pRL-TK, 150 ng of APPL, 150 ng cAkt, 150 ng of
dominant-negative Akt, and 20 µM LY294002. Transfected
cells were treated for 24 h with 10 8 nM
DHT or ethanol as vehicle control. Duplicate samples were analyzed for
each data point.
p85
(lane 7) could reverse the APPL-repressed
p27Kip1 promoter activity, implying that APPL may influence
p27Kip1 promoter activity via activating the PI3K/Akt
pathway.
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Fig. 5.
APPL enhances Akt activity.
A, APPL associates with PI3K in vivo. The COS-1
cell were transfected with FLAG-APPL and wild-type p85, a regulatory
subunit of PI3K for 24 h, followed by harvesting for
co-immunoprecipitation (IP) as described under
"Experimental Procedures." B, suppression effect of APPL
via Akt on the human p27Kip1 promoter activity in PC-3
cells. Cells were transfected with 300 ng of APPL in the presence or
absence of 300 ng of cAkt, 300 ng of dominant-negative Akt
(dAkt), or 300 ng of p85 in combination with 50 ng of
pRL-TK and 150 ng of promoter p27PF-luc. Luciferase activity was
analyzed after 36 h. C, the COS-1 cells were seeded in
100-mm dishes and transfected with 10 µg of empty vector, 5 µg of
APPL, 5 µg
p85, or combinations as indicated. Empty vector was
used to balance the DNA amount to 10 µg. After 24 h, the cells
were serum-starved for another 24 h then treated with 20 µM LY294002 10 min before treatment with vehicle or 50 ng/ml IGF-1 for 20 min, and the cells were harvested by radioimmune
precipitation assay buffer. The Akt activity was determined by the
Western blot using the phospho-(Ser-473) Akt (pAkt)
antibody. D, the COS-1 cells were seeded in 100-mm dishes
and transfected with 10 µg of empty vector, 5 µg of
pCDNA3-FLAG-APPL, 5 µg of antisense APPL or co-transfected with 5 µg of pCDNA3-FLAG-APPL and 5 µg of antisense APPL followed by
serum starvation for another 24 h. Empty vector was used to
balance the DNA amount to 10 µg. The cells were treated with or
without 50 ng/ml IGF-1 for 20 min before harvesting cells. Cell lysates
were immunoblotted with anti-phosphorylation Akt antibody (Ser-473),
anti-FLAG M2 antibody, or anti-
-actin antibody.
p85 (lane 8) or LY294002 (lane 12), suggesting that
PI3K serves as an upstream activator for IGF-1 mediated Akt activation.
As expected, APPL significantly enhanced the IGF-1 mediated Akt
activation (lane 4). The enhancement effect of APPL on Akt
activation was abrogated by
p85 (lane 6) or LY294002
(lane 10), indicating that APPL may affect Akt activation by
modulation of PI3K activity or PI3K upstream activators. Furthermore,
we used the antisense APPL construct to block endogenous APPL
expression to see if APPL was required for the mitogenic signal
activating Akt. As shown in Fig. 5D, overexpression of APPL
significantly enhanced Akt activity (lane 3), but the
phosphorylated Akt became undetectable after co-transfection with
antisense APPL, even under the treatment of IGF-1 (lane 6 versus lane 2). These findings suggest that APPL is an important adapter protein that may be necessary for Akt activation upon mitogenic stimulation.
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Fig. 6.
APPL and Akt suppressed endogenous PSA
mRNA expression and inhibition of PI3K/Akt signaling enhanced PSA
protein expression level. A, APPL cooperated with Akt
to further reduce the PSA mRNA expression level in LNCaP cells. The
LNCaP cells were transfected with 10 µg of empty vector (lane
1), 5 µg of APPL plus 5 µg of empty vector (lanes 2 and 3), 5 µg of APPL plus 5 µg of cAkt (lane
4), and 5 µg of cAkt plus 5 µg of empty vector (lane
5) for 24 h followed by treatment with ethanol or 10 nM DHT for another 24 h. The cells were harvested and
lysed by TRIZOL. 20 ng of total RNA was loaded for the Northern blot
analysis. Quantification of the relative PSA mRNA level was done by
ImageQuant software (upper panel). B,
LY294002 influenced PSA mRNA expression. The LNCaP cells were
cultured in 10% (CD-FBS)-containing medium for 24 h and then
treated with vehicle, DHT, 20 µM LY294002, or
combinations. After 4 h (lanes 1-4) and 24 h
(lanes 5-8), cells were harvested for Northern blot
analysis. Quantification of the relative PSA mRNA level was done by
ImageQuant software (upper panel). C, LY294002
influenced PSA protein expression level. The LNCaP cells were cultured
in 10% (CD-FBS)-containing medium for 24 h and then treated with
vehicle, DHT, or DHT plus 20 µM LY294002. After 4 h
and 24 h, cells were harvested for Western blot analysis with the
indicated antibodies, and the relative fold change in PSA signal was
analyzed by QuantityOne software (Bio-Rad) (upper panel).
D, androgen ablation of LNCaP cells increases Akt
activation. LNCaP cells were cultured under serum-starvation conditions
with or without 1 nM DHT for 4 days. Cell lysates were
analyzed by immunoblotting with anti-phospho-Ser-473 Akt
(pAkt) and whole Akt protein antibodies, and the relative
fold change in phospho-Ser-473-Akt signal was analyzed by QuantityOne
software (Bio-Rad) (upper panel).
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Fig. 7.
The APPL C terminus is responsible for the
suppression of AR transactivation. A, full-length APPL
and truncated mutants of APPL encompassing various functional domains
were cloned into pCDNA3 vector with a FLAG tag. B, APPL
C terminus suppressed AR activity in PC-3 cells. Cells were transfected
with 50 ng of pCMV-AR, 150 ng of (ARE)4-luc, 2.5 ng of pRL-SV40, and
300 ng of APPL or APPL N or APPL C termini. Transfected cells were
treated for 24 h with 10 8 nM DHT or
ethanol as vehicle control. Duplicate samples were analyzed for each
data point. C, the PC-3 cells were transfected with 10 µg
of pCDNA3-FLAG, pCDNA3-FLAG-APPL, or pCDNA3-FLAG-APPL N or
C termini in 100-mm dishes, treated with DHT or vehicle for 24 h,
and harvested for Western blot assay by using anti-FLAG antibody.
D, APPL C terminus increased phosphorylated Akt protein
level. The COS-1 cells were serum-starved for 24 h and then
transfected with 10 µg of pCDNA3-FLAG, pCDNA3-FLAG-APPL, or
pCDNA3-FLAG-APPL N or C termini in 100-mm dishes. After 24 h,
cells were treated with 50 ng/ml IGF-1 for 20 min and harvested for
Western blot assay by using phospho-(Ser-473)-Akt (pAkt) and
Akt antibodies.
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Fig. 8.
APPL was detected in the AR immunocomplexes
but did not directly interact with AR. A, GST-APPL can
pull down endogenous AR in LNCaP cells. GST-APPL was used as bait to
incubate with LNCaP cell lysate with or without DHT treatment. The
incubation and washing were performed as described under
"Experimental Procedures," and then the lysates were analyzed with
anti-AR (NH27) antibody. B, APPL does not interact with AR
directly. GST-APPL or GST-ARA70 was incubated with
S35-labeled AR for 2 h in the presence or absence of
10 nM DHT. C, Akt markedly enhances the
APPL·AR complex formation. COS-1 cells were co-transfected with
FLAG-APPL, AR, and HA-Akt. The cell lysates were immunoprecipitated
(IP) with anti-FLAG M2. The immunoprecipitated complexes
were immunoblotted (WB) with anti-AR antibody (G122-77),
anti-FLAG M2 antibody, or anti-HA antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85, or dAkt. Thus, APPL may
affect the activity of PI3K or the PI3K upstream activator to control
p27Kip1 expression. In addition, APPL markedly enhances the
IGF-1-mediated Akt activation. This effect is abrogated by LY294002,
p85, and antisense APPL. These data indicate that APPL modulates the
Akt activity via a PI3K-dependent manner.
p85, or dAkt. Second, APPL down-regulates gene expression of
p27Kip1, which is blocked by LY294002,
p85, or dAkt.
Finally, APPL markedly enhances the IGF-1-mediated Akt activation and
the destruction of endogenous APPL by antisense APPL, leading to the
blockade of Akt activation upon mitogenic stimulation. How APPL affects the Akt phosphorylation is currently unclear. The fact that the enhancement of Akt activation by APPL is blocked by LY294002,
p85,
or antisense APPL suggests that APPL may affect PI3K. Because APPL
contains the PH domain and interacts with PI3K (Fig. 5A), it
is likely that APPL serves as an adapter protein that recruits the PI3K
to the cell membrane, where it can be activated by growth factor
receptors like the IGF-1 receptor, followed by subsequent Akt activation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. R. Freeman and T. Sakai for reagents. We thank Dr. Mei Li for technique assistance and Karen L. Wolf and Debby Chuang for helpful reading of the manuscript. We also thank the members of Dr. Chang's lab for technical support and insightful discussion.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK60948 and DK60905.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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 585-275-9994; Fax: 585-756-4133; E-mail: chang@urmc.rochester.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M213163200
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ABBREVIATIONS |
---|
The abbreviations used are:
AR, androgen
receptor;
PI3K, phosphatidylinositol 3(OH)-kinase;
IGF-1, insulin-like
growth factor;
DHT, 5-dihydrotestosterone;
PH, pleckstrin homology;
GST, glutathione S-transferase;
HA, hemagglutinin antigen;
MMTV, mouse mammary tumor virus;
PSA, prostate-specific antigen;
luc, luciferase;
ARE, androgen-responsive element;
ER, estrogen
receptor;
AP, adapter protein;
RT, reverse transcription;
FBS, fetal bovine serum;
CD, charcoal-dextran;
PDK, 3-phosphoinositide-dependent kinase;
PTB, phosphotyrosine binding
domain.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Chang, C. S., Kokontis, J., and Liao, S. T. (1988) Science 240, 324-326[Medline] [Order article via Infotrieve] |
2. | Yeh, S., Chang, H. C., Miyamoto, H., Takatera, H., Rahman, M., Kang, H. Y., Thin, T. H., Lin, H. K., and Chang, C. (1999) Keio J. Med. 48, 87-92[Medline] [Order article via Infotrieve] |
3. |
Kang, H. Y.,
Yeh, S.,
Fujimoto, N.,
and Chang, C.
(1999)
J. Biol. Chem.
274,
8570-8576 |
4. |
Fujimoto, N.,
Yeh, S.,
Kang, H. Y.,
Inui, S.,
Chang, H. C.,
Mizokami, A.,
and Chang, C.
(1999)
J. Biol. Chem.
274,
8316-8321 |
5. | Chang, C., Saltzman, A., Yeh, S., Young, W., Keller, E., Lee, H. J., Wang, C., and Mizokami, A. (1995) Crit. Rev. Eukaryotic Gene Expression 5, 97-125[Medline] [Order article via Infotrieve] |
6. | Hsiao, P. W., Thin, T. H., Lin, D. L., and Chang, C. (2000) Mol. Cell. Biochem. 206, 169-175[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Greenlee, R. T.,
Murray, T.,
Bolden, S.,
and Wingo, P. A.
(2000)
CA-Cancer J. Clin.
50,
7-33 |
8. | Feldman, B. J., and Feldman, D. (2001) Nat. Rev. Cancer 1, 34-45[CrossRef][Medline] [Order article via Infotrieve] |
9. | Carpenter, C. L., and Cantley, L. C. (1996) Curr. Opin. Cell Biol. 8, 153-158[CrossRef][Medline] [Order article via Infotrieve] |
10. | 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] |
11. |
Cardone, M. H.,
Roy, N.,
Stennicke, H. R.,
Salvesen, G. S.,
Franke, T. F.,
Stanbridge, E.,
Frisch, S.,
and Reed, J. C.
(1998)
Science
282,
1318-1321 |
12. | Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve] |
13. | Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P., Downward, J., and Evan, G. (1997) Nature 385, 544-548[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Andjelkovic, M.,
Jakubowicz, T.,
Cron, P.,
Ming, X. F.,
Han, J. W.,
and Hemmings, B. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5699-5704 |
15. | Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435-437[Medline] [Order article via Infotrieve] |
16. | Nakatani, K., Sakaue, H., Thompson, D. A., Weigel, R. J., and Roth, R. A. (1999) Biochem. Biophys. Res. Commun. 257, 906-910[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Masure, S.,
Haefner, B.,
Wesselink, J. J.,
Hoefnagel, E.,
Mortier, E.,
Verhasselt, P.,
Tuytelaars, A.,
Gordon, R.,
and Richardson, A.
(1999)
Eur. J. Biochem.
265,
353-360 |
18. | Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem. J. 335, 1-13[Medline] [Order article via Infotrieve] |
19. | Bellacosa, A., Franke, T. F., Gonzalez-Portal, M. E., Datta, K., Taguchi, T., Gardner, J., Cheng, J. Q., Testa, J. R., and Tsichlis, P. N. (1993) Oncogene 8, 745-754[Medline] [Order article via Infotrieve] |
20. | Mitsuuchi, Y., Johnson, S. W., Sonoda, G., Tanno, S., Golemis, E. A., and Testa, J. R. (1999) Oncogene 18, 4891-4898[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Lin, H. K.,
Yeh, S.,
Kang, H. Y.,
and Chang, C.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
7200-7205 |
22. |
Crowder, R. J.,
and Freeman, R. S.
(1998)
J. Neurosci.
18,
2933-2943 |
23. |
Sakaue, H.,
Hara, K.,
Noguchi, T.,
Matozaki, T.,
Kotani, K.,
Ogawa, W.,
Yonezawa, K.,
Waterfield, M. D.,
and Kasuga, M.
(1995)
J. Biol. Chem.
270,
11304-11309 |
24. | Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995) Science 268, 100-102[Medline] [Order article via Infotrieve] |
25. |
Inoue, T.,
Kamiyama, J.,
and Sakai, T.
(1999)
J. Biol. Chem.
274,
32309-32317 |
26. |
Yeh, S.,
and Chang, C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5517-5521 |
27. | Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang, C. (1998) Biochem. Biophys. Res. Commun. 248, 361-367[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Kang, H. Y.,
Lin, H. K.,
Hu, Y. C.,
Yeh, S.,
Huang, K. E.,
and Chang, C.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3018-3023 |
29. |
Campbell, R. A.,
Bhat-Nakshatri, P.,
Patel, N. M.,
Constantinidou, D.,
Ali, S.,
and Nakshatri, H.
(2001)
J. Biol. Chem.
276,
9817-9824 |
30. | Medema, R. H., Kops, G. J., Bos, J. L., and Burgering, B. M. (2000) Nature 404, 782-787[CrossRef][Medline] [Order article via Infotrieve] |
31. | Mulders, T. M., Bruning, P. F., and Bonfrer, J. M. (1990) Eur. J. Surg. Oncol. 16, 37-41[Medline] [Order article via Infotrieve] |
32. |
Bresnick, E. H.,
and Felsenfeld, G.
(1994)
J. Biol. Chem.
269,
21110-21116 |
33. | Kavanaugh, W. M., and Williams, L. T. (1994) Science 266, 1862-1865[Medline] [Order article via Infotrieve] |
34. |
Lin, J.,
Adam, R. M.,
Santiestevan, E.,
and Freeman, M. R.
(1999)
Cancer Res.
59,
2891-2897 |
35. | Thompson, J., Koskinen, P. J., Janne, O. A., and Palvimo, J. J. (2002) The Endocrine Society 84th Annual Meeting, San Francisco, CA, June 19-22, 2002 , The Endocrine Society |
36. |
Murillo, H.,
Huang, H.,
Schmidt, L. J.,
Smith, D. I.,
and Tindall, D. J.
(2001)
Endocrinology
142,
4795-4805 |
37. |
Wen, Y.,
Hu, M. C.,
Makino, K.,
Spohn, B.,
Bartholomeusz, G.,
Yan, D. H.,
and Hung, M. C.
(2000)
Cancer Res.
60,
6841-6845 |
38. | Manin, M., Baron, S., Goossens, K., Beaudoin, C., Jean, C., Veyssiere, G., Verhoeven, G., and Morel, L. (2002) Biochem. J. 366, 729-736[Medline] [Order article via Infotrieve] |
39. |
Sharma, M.,
Chuang, W. W.,
and Sun, Z.
(2002)
J. Biol. Chem.
277,
30935-30941 |