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Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
Address all correspondence and requests for reprints to: Shin-Ichiro Takahashi, Ph.D., Laboratory of Cell Regulation, Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: atkshin{at}mail.ecc.u-tokyo.ac.jp
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Abstract |
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Introduction |
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FRTL-5 is a nontransformed line of rat thyroid follicular cells that responds to TSH and IGF-I with cell proliferation and certain differentiated function, including iodide transport and thyroglobulin synthesis (12, 13, 14, 15). TSH stimulates an increase in intracellular cAMP, and all the effects of TSH can be mimicked by agents that increase intracellular cAMP. We and others have shown that TSH and IGF-I stimulate cell growth synergistically and that TSH or cAMP pretreatment is essential for the potentiation of IGF-I-dependent DNA synthesis (7, 8). This interaction between TSH and IGF-I has also been shown to be important in vivo (16, 17). Pretreatment of FRTL-5 cells with TSH, or other agents that increase cAMP concentrations, potentiates IGF-I-dependent tyrosine phosphorylation of multiple substrates (18). We previously reported that TSH or (Bu)2 cAMP produced a time- and concentration-dependent increase in tyrosine phosphorylation of some intracellular proteins such as a 125-kDa protein (18). These increases in tyrosine phosphorylation were well correlated to the amplifying effects of cAMP on IGF-I-dependent DNA synthesis. These results suggested that cAMP- dependent tyrosine phosphorylation may be an essential part of the mechanism by which cAMP amplifies IGF-I-dependent signals for cell growth.
The aim of the present study was, therefore, to examine whether cAMP-dependant tyrosine kinase activity and the increase in tyrosine phosphorylation of specific substrates is essential for the cAMP-dependent amplification of DNA synthesis induced by IGF-I in FRTL-5 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP (
220 Ci/pmol) were purchased
from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Other chemicals were of the reagent grade available commercially.
Cell culture
FRTL-5 cells (ATCC no. CRL8305), a line of rat
thyroid follicular cells, was developed by Ambesi-Impiombato et
al. (12) and kindly provided by Dr. Leonard Kohn (NIDDK) and the
Interthyr Research Foundation (Baltimore, MD). Cells were cultured as
described previously (8). Briefly, cells were routinely cultured in
Coon’s F-12 medium supplemented with 5% NCS and a three-hormone
mixture (3-H) including bovine TSH (1 mU/ml), bovine insulin (10
µg/ml), and human transferrin (5 µg/ml). Cells were cultured in
150-cm2 flasks at 37 C in an atmosphere of 95%
air and 5% CO2 in a humidified incubator. The
medium was replaced to the fresh medium every 3 days, and the cells
were passed every 10 days before reaching confluency.
Tyrosine kinase assay
To measure tyrosine kinase activity, FRTL-5 cells (1 x
106 cells/10 ml) were sparsely seeded in a 100-mm
dish and cultured in Coon’s F-12 supplemented with 5% NCS and 3-H.
Five days later, the cells were washed twice with HBSS, and culture was
continued for 24 h in 10 ml Coon’s F-12 medium including 0.1%
BSA. Twenty-four hours later the cells were quiescent, and the cultures
of the cells were continued for various times in 8 ml of Coon’s F-12
medium with 0.1% BSA without or with TSH (1 nM) or
(Bu)2cAMP (1 mM). The cells were then
harvested in the detergent-free lysis-buffered solution [DF; 50
mM Tris-HCl pH 7.4, 150 mM NaCl, 500
µM Na3VO4,
0.1 mM EDTA, 10 mM NaF, 10 µg/ml leupeptin, 5
µg/ml pepstatin, 20 µg/ml phenylmethylsulfonyl fluoride (PMSF), 100
kallikrein-inactivating units (KIUs)/ml aprotinin, and 10 mg/ml
p-nitrophenyl phosphate (PNPP)] and homogenized by 30 strokes
using a tight pestle of Dounce homogenizer (Wheaton, NJ). The lysates
were centrifuged at 12,000 x g for 10 min at 4 C, and
the supernatant was recovered as a soluble fraction. After being washed
twice with DF buffer, the pellets were dissolved in Tris/Triton
lysis-buffered solution (T/T; DF + 1% Triton X-100) by tumbling for 30
min at 4 C. The supernatant after centrifugation at 12,000 x
g for 10 min at 4 C was recovered as particulate fraction.
Both soluble and particulate fractions were stored at -80 C until
tyrosine kinase assay. The protein in each lysate was assayed according
to the method of Bradford (19). Tyrosine kinase activity was measured
as others described previously (20). Briefly, cell-free phosphorylation
was initiated by adding the reaction mixture to give final
concentration of 20 mM HEPES-NaOH pH 7.4, 40
mM MgCl2, 200
µM
Na3VO4, 2% NP-40, 200
µM [-32P]ATP (220
Ci/pmol), and 2 mg/ml poly[Glu:Tyr] (4:1) in the absence or presence
of genistein (30 µg/ml) to the extracts, which contained the same
amount of protein. After 30 min incubation at 30 C, the reactions were
terminated by applying the aliquots to P81 cellulose filter paper
(Advantec, Tokyo). The filter papers were washed at 4 C in 10%
trichloroacetic acid (TCA) and 10 mM sodium
pyrophosphate and then three times in 5% TCA, dried with methanol, and
counted by the liquid scintillation counter. In all experiments, each
experimental point represents the mean of three replicate dishes.
Protein tyrosine phosphatase assay
IGF-I receptor overexpressed NIH 3T3 cells (NIGF-IR cells),
which were a kind gift from Dr. Derek LeRoith (NIDDK), were grown as
described previously (21), and overexpressed IGF-I receptor was
semipurified according to the our previous method (18). Poly[Glu:Tyr]
and semipurified IGF-I receptor were incubated at 30 C overnight in 40
mM imidazole-HCl pH 7.2 containing 50 mM NaCl,
15 mM
Mg(CH3COOH)2, 100
mM MgCl2, 100 µM
Na3VO4, 200
µM EDTA, 0.05% (vol/vol) Triton-X 100, 3% glycerol, and
200 µM [-32P]ATP. Then the
mixture was subjected to gel filtration on a Sephacryl S-100 column (20
ml column volume; Amersham Pharmacia Biotech). Fractions
containing the 32P-labeled poly[Glu:Tyr] (4:1)
were pooled and stored at 4 C before being used for protein-tyrosine
phosphatase (PTPase) assay. The cell extracts were prepared as
described above except that detergent-free and Tris/Triton
lysis-buffered solution did not contain
Na3VO4. The fractionated
cell lysates were incubated with 32P-labeled
poly[Glu:Tyr] (4:1) in 25 mM imidazole, pH 7.4,
containing 1 mg/ml BSA and 0.1%[vol/vol] 2-mercaptoethanol at 30 C
for 15 min. Reaction was stopped by addition of 10% TCA. After
precipitation of proteins in 10% TCA, free
[32P] Pi was extracted by the molybdate
extraction procedure (22) and counted in a liquid scintillation
counter. In all experiments, each experimental point represents the
mean of three replicate dishes.
Analysis of tyrosine phosphorylation of intracellular
proteins
For studies of tyrosine phosphorylation of intracellular
proteins, FRTL-5 cells (5 x 105 cells/2 ml)
were sparsely seeded in a 35-mm dish. Five days later, the cells were
washed twice with HBSS, and culture was continued for 24 h in 1 ml
Coon’s F-12 medium including 0.1% BSA. Twenty-four hours later, the
cultures of the cells were continued for various times in 1 ml of
Coon’s F-12 medium with 0.1% BSA without or with TSH (1
nM) or (Bu)2cAMP (1 mM)
in the absence or presence of genistein or orthovanadate. After this
pretreatment, the cells were washed five times with HBSS and then
treated without or with IGF-I (100 ng/ml) for 1 min. The cells were
then harvested and the lysates were prepared as described previously
(23). The protein assay of the cell lysates was carried out using
protein assay kit (Bio-Rad Laboratories, Inc. Hercules,
CA). Equal amounts of proteins (75 µg protein) of each sample were
subjected to 8% SDS-PAGE, and tyrosine phosphorylated proteins were
detected by immunoblotting using antiphosphotyrosine antibody as
described previously (23). The results were quantified using NIH Image
computer program (Version 1.61).
DNA synthesis assay
FRTL-5 cells (5 x 104 cells/500
µl/well) were sparsely seeded in a 48-well plate. Five days later,
the cells were washed twice with HBSS and culture was continued for
24 h in 500 µl Coon’s F-12 medium including 0.1% BSA.
Twenty-four hours later, the cultures of the cells were continued for
an additional 24 h in 300 µl of Coon’s F-12 medium with 0.1%
BSA without or with TSH (1 nM) or
(Bu)2cAMP (1 mM) in the absence or
presence of several concentrations of genistein or LY294002. After this
pretreatment, the cells were washed five times with HBSS and then
treated without or with IGF-I (100 ng/ml) for 24 h.
[Methyl-3H]thymidine (0.3 µCi/well; 1
µCi/ml) was added to each well 4 h before the termination of
each experiment.
[Methyl-3H]thymidine
incorporation into DNA was measured as described previously (8). In all
experiments, each experimental point represents the mean of three
replicate wells.
Immunoprecipitation with anti-p85 regulatory subunit of PI 3-kinase
antibody followed by immunoblotting with antiphosphotyrosine
antibody
As described above, the quiescent FRTL-5 cells in a 100-mm dish
were pretreated without or with TSH (1 nM) or
(Bu)2cAMP (1 mM) for 24 h. After
this pretreatment, the cells were washed five times with HBSS and then
treated without or with IGF-I (100 ng/ml) for 1 min. The cells were
then harvested at 0 C in 400 µl of immunoprecipitation buffer
containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl,
1 mM Na3VO4, 10
mM EDTA, 1 mM EGTA, 1.5 mM
MgCl2, 1% Triton X-100, 100 KIU/ml aprotinin, 20
µg/ml PMSF, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 10 mg/ml PNPP.
The lysates were centrifuged at 12,000 x g for 10 min
at 4 C. The supernatant was diluted with immunoprecipitation buffer to
1 mg protein/ml as a final concentration. The protein assay was carried
out as described above. The lysates that contained the same amount of
proteins, were incubated with anti-p85 regulatory subunit of PI
3-kinase antibody (5 µl) for 12 h at 4 C; 40 µl of protein
A-Sepharose [50% (vol/vol); Amersham Pharmacia Biotech]
was then added and incubation was continued for 2 h. Under 4 C,
the immunoprecipitates were collected by centrifugation, washed three
times with immunoprecipitation buffer, and boiled for 5 min in the
mixture of 60 µl of immunoprecipitation buffer and 30 µl of 3x
Laemmli’s sample buffer (9% SDS, 15% glycerol, 30
mM Tris-HCl, pH 7.8, 0.05% bromophenol blue, 6%
2-mercaptoethanol). These samples were then stored at -80 C until
electrophoresis. Each sample was run on 8% SDS-PAGE and immunoblotting
was performed using antiphosphotyrosine antibody as described
above.
Adsorption with the SH2 domain of a p85-regulatory subunit of PI
3-kinase followed by immunoblotting with antiphosphotyrosine
antibody
The amino-terminal SH2 domain of the 85-kDa regulatory subunit
of PI 3-kinase (amino acid residues 333–424) was expressed as
glutathione S-transferase (GST) fusion proteins in
Escherichia coli and purified on glutathione-Sepharose beads
[50% (vol/vol); Amersham Pharmacia Biotech] according
to the manufacturer’s instructions. As described above, the quiescent
FRTL-5 cells in a 100-mm dish were pretreated without or with
(Bu)2cAMP (1 mM) for
24 h. The cells were then harvested at 0 C in immunoprecipitation
buffer, and the lysates were centrifuged at 12,000 x g
for 10 min at 4 C. The protein assay was carried out and the lysates
containing the same amount of proteins (6 mg) were mixed with
immobilized beads that adsorbed the SH2 domain of p85 PI
3-kinase (500 µg of fusion protein) for 18 h and 4 C. The beads
were extensively washed three times with immunoprecipitation buffer.
The bound proteins were eluted with 100 µl Laemmli’s sample buffer
by boiling for 5 min and subjected to immunoblotting using
antiphosphotyrosine antibody as described above.
Immunoprecipitation with anti-p85-regulatory subunit of PI 3-kinase
antibody followed by far-Western blotting with the SH2 domain of a
p85-regulatory subunit of PI 3-kinase
Quiescent FRTL-5 cells in a 100-mm dish were treated with
(Bu)2cAMP (1 mM) for various times.
The immunoprecipitates with anti-p85 PI 3-kinase antibody were
prepared, run on 8% SDS-PAGE, and transferred to a nitrocellulose
membrane as described above. Far-Western blots were performed with
GST-amino- terminal SH2 domain of p85 PI 3-kinase fusion proteins (1
mg/ml), which purified on glutathione-Sepharose according to the
manufacturer’s directions, followed by immunoblotting using anti-GST
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
according to methods previously reported (24). In addition, the
immunoblotting of the same membranes was also performed with
antiphosphotyrosine antibody or anti-p85 PI 3-kinase antibody as
described previously (25).
PI 3-kinase activity assay
As described above, quiescent FRTL-5 cells in a 100-mm dish were
pretreated without or with (Bu)2cAMP (1
mM) for 24 h. Cells were lysed at 4 C in 300 µl of
NP-40 buffer [10 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 0.5 mM
Na3VO4, 1 mM
EDTA, 1% NP-40, 100 KIU/ml aprotinin, 20 µg/ml PMSF, 10 µg/ml
leupeptin, 5 µg/ml pepstatin, 10 mg/ml PNPP]. The lysates were
centrifuged at 12,000 x g for 10 min at 4 C. The
supernatant that contained the same amount of proteins was incubated
with anti-p85 regulatory subunit of PI 3-kinase antibody (1 µl) for
12 h at 4 C; 10 µl of protein A-Sepharose [50% (vol/vol)] was
then added and incubation was continued for 2 h. Under 4 C, the
immunoprecipitates were collected by centrifugation, washed once with
NP-40 buffer, LiCl buffer [100 mM Tris-HCl pH
7.5, 500 mM LiCl], distilled water, washed with
TNE buffer [10 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1 mM EDTA], and
finally resuspended in 45 µl of reaction buffer [20
mM Tris-HCl, pH 7.5, 100 mM
NaCl, 0.5 mM EGTA]. PI 3-kinase assay was
carried out according to the method of Whitman et al. (26)
with slight modifications. Briefly, PI 3-kinase assay was initiated by
incubation of immunocomplex in 45 µl reaction buffer with 5 µl of
the mixture to give a final concentration of 20
µM [-32P]ATP (4
µCi/mmol), 20 mM MgCl2
and 20 µg phosphatidylinositol (Avanti, Amersham Pharmacia Biotech, Uppsala, Sweden) at 25 C for 20 min. After
incubation, 100 µl of chloroform-methanol-HCl (10:20:1) were added to
the reaction mixture to stop a reaction. A lipid product was extracted,
spotted onto a silica gel plate, and developed with a solvent
containing chloroform-methanol-ammonia water-water (43:38:6:6).
32P radioactivity incorporated into
phosphatidylinositol was measured by autoradiography as PI 3-kinase
activity. Each experimental point represents the mean of three
replicate dishes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Approximately 20–40% of total
tyrosine kinase activity was inhibited by genistein, a tyrosine kinase
inhibitor (Table 1). On the other hand,
cAMP pretreatment increased PTPase activities about 2-fold in the
soluble fraction but did not affect the particulate fraction (Fig. 1B).
Orthovanadate (500 µM), a PTPase inhibitor, completely
inhibited PTPase activities (data not shown). The kinase activity in
response to (Bu)2cAMP increased in both a time
and concentration-dependent manner (Fig. 2A and data not shown). In addition, this
increase in tyrosine kinase activity was well correlated with the
chronic increase in tyrosine phosphorylation of a 125-kDa protein
(p125) as shown in Fig. 2, A and B. We obtained similar results using
TSH instead of (Bu)2cAMP (data not shown).
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). In contrast, orthovanadate did not
cause tyrosine phosphorylation of p125 in the absence of
(Bu)2cAMP and had little effects on a
cAMP-dependent increase in p125 tyrosine phosphorylation (data not
shown). We obtained similar results using TSH instead of
(Bu)2cAMP (data not shown). These results
suggested that tyrosine kinases play more important roles for a
cAMP-dependent increase in tyrosine phosphorylation of p125.
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, genistein significantly
abolished the cAMP-priming effects in a concentration-dependent manner.
On the other hand, significant effects of genistein were not observed
on DNA synthesis induced by IGF-I without
(Bu)2cAMP pretreatment at higher concentrations
of genistein. These results suggested that tyrosine kinase activity was
essential for cAMP-dependent enhancement in DNA synthesis induced by
IGF-I. In addition, chronic genistein treatment during
(Bu)2cAMP pretreatment was necessary to suppress
a cAMP-dependent enhancement of DNA synthesis induced by IGF-I (Fig. 4B). Based on densitometric analyses of immunoblotting using
antiphosphotyrosine antibody, the inhibitory effects of genistein on
cAMP-dependent tyrosine phosphorylation of p125 correlate well with
cAMP-dependent potentiation of DNA synthesis induced by IGF-I (Figs. 3
and 4A). We obtained similar results using TSH instead of
(Bu)2cAMP (data not shown).
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). We treated FRTL-5 cells with
(Bu)2cAMP for 24 h, and the proteins were
coimmunoprecipitated with an antibody against p85 PI 3-kinase, followed
by immunoblotting with an antiphosphotyrosine antibody. Of all of the
phosphotyrosyl proteins resulting from cAMP pretreatment, only
phosphotyrosyl p125 was clearly bound to p85 PI 3-kinase
(Fig. 5, A and B). As a control, we showed that IGF-I treatment caused
association of the tyrosine-phosphorylated 175–185 kDa proteins with
p85 PI 3-kinase. To assess whether the association of p125 to p85 PI
3-kinase could be mimicked by the SH2 domain of p85 PI 3-kinase alone,
cell lysates prepared after (Bu)2cAMP treatments
were adsorbed with GST fusion proteins containing amino-terminal SH2
domain of p85 PI 3-kinase bound to glutathione-Sepharose. Adsorbed
proteins were subsequently immunoblotted with antiphosphotyrosine
antibody. Phosphotyrosyl p125 specifically associated with the
amino-terminal SH2 domain of p85 PI 3-kinase in response to
(Bu)2cAMP treatments (Fig. 5C). In addition, an
increase in tyrosine phosphorylation of p125 associated with p85 PI
3-kinase was dependent on cAMP treatment time (Fig. 6A). Immunoprecipitates from
cAMP-stimulated cell lysates by anti-p85 PI 3-kinase antibody were
subjected to far-Western blotting analysis using GST-SH2 domain of p85
PI 3-kinase. Association of the amounts of the SH2 domain of p85 PI
3-kinase with p125 increased in a cAMP treatment time-dependent manner
(Fig. 6B). All of these results suggested that cAMP stimulation
increases association of phosphotyrosyl p125 and p85 PI 3-kinase
through its SH2 domain.
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).
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). LY294002 inhibited cAMP-dependent
potentiation of DNA synthesis induced by IGF-I in a
concentration-dependent manner; however, the presence of LY294002
during pretreatment time did not affect IGF-I-induced DNA synthesis. In
addition, chronic treatment with LY294002 completely abolished
cAMP-priming effects on DNA synthesis induced by IGF-I (Fig. 8B). Under
these conditions, we verified that LY294002 did not affect
cAMP-dependent tyrosine phosphorylation (data not shown). These results
correspond well to the results using genistein. Again, we obtained the
same results using TSH instead of (Bu)2cAMP (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First of all, we found that chronic cAMP stimulation increases tyrosine
kinase activity in the particulate fraction of FRTL-5 cells and that
genistein partially inhibited this activity (Fig. 1A). cAMP-dependent
tyrosine kinase activity as well as cAMP-dependent tyrosine
phosphorylation of p125 increased in proportion to the duration of
exposure to TSH or (Bu)2cAMP (Fig. 2) and
genistein inhibited cAMP-dependent enhancement of p125 tyrosine
phosphorylation (Fig. 3). In contrast, we demonstrated that long-term
cAMP treatment increased PTPase activities in the soluble fraction,
rather than decreased (Fig. 1B), and orthovanadate did not affect
tyrosine phosphorylation of p125 (data not shown). From these results,
we hypothesize that cAMP signaling induces specific tyrosine kinases
and/or their substrates through new protein synthesis to increase
tyrosine phosphorylation of p125. Determination of specific tyrosine
kinases is in progress in our laboratory.
We demonstrate that cAMP-dependent increases in tyrosine kinase
activities were well correlated with increases in tyrosine
phosphorylation of p125 (Fig. 2) and with the potentiation by cAMP of
DNA synthesis induced by IGF-I (18). Further, genistein at high
concentration was able to completely abolish cAMP-dependent enhancement
of tyrosine phosphorylation of p125 as well as DNA synthesis induced by
IGF-I (Figs. 3 and 4A). These results suggested that cAMP-dependent
tyrosine kinase activation followed by phosphorylation of p125 is
important for cAMP-dependent potentiation of DNA synthesis induced by
IGF-I.
The inhibitory effects of genistein on tyrosine phosphorylation were
not due to cell toxicity, since this inhibitor does not change
viability of the cells and does not affect general protein
synthesis at the concentrations that we used. Presence of genistein
during pretreatment with no additives did not affect IGF-I-induced
tyrosine phosphorylation of p180 (data not shown) and DNA synthesis
(Fig. 4A). In addition, genistein inhibits only 20–40% of total
tyrosine kinase activity (Table 1) and does not affect tyrosine
phosphorylation of some other proteins (data not shown), suggesting
that genistein inhibits specific tyrosine kinases. The degree of
inhibition of tyrosine kinase activity, which in part is related to the
concentration of genistein used, may also be another important
determinant. Takano et al. (27) showed no effect of
genistein at concentrations of 1 µg/ml on IGF-I-induced DNA synthesis
in contrast to the present studies in which 10–30 µg/ml were
used.
Identification of the p125 substrate that is phosphorylated in response
to cAMP may provide some further clues as to the nature of the
convergence signal between the cAMP-dependent and IGF-I-dependent
signaling pathways. It has been shown that many phosphotyrosyl proteins
directly interact with the signaling molecules containing SH2 or PTB
domains (28, 29, 30). Interestingly, phosphotyrosyl p125 was the only major
protein specifically bound to p85 PI 3-kinase through its SH2 domain in
response to chronic cAMP treatment (Fig. 5, B and C). We have used
various antibodies against tyrosine-phosphorylated proteins, including
GAP, Gab-1, FAK, and Jak2, with a molecular mass around 120–130 kDa.
To date, any of these antibodies cannot recognize phosphotyrosyl p125.
Purification of a novel phosphotyrosyl p125 is being performed in our
laboratory.
Because phosphotyrosyl p125 cannot be selectively be immunoprecipitated
with any antibodies at present, we are not able to measure PI 3-kinase
activity associated with phosphotyrosyl p125 directly. However, reports
have accumulated recently showing that binding of p85 PI 3-kinase to
tyrosine-phosphorylated proteins caused stimulation of p110 catalytic
subunit of PI 3-kinase (31, 32, 33). We showed that the amounts of p85 PI
3-kinase bound to phosphotyrosyl p125 increased in a cAMP treatment
time-dependent manner (Fig. 6). As shown in Fig. 7, (Bu)2cAMP treatment for 12 h or 24 h
caused an increase in PI 3-kinase activity bound to p85 PI 3-kinase. In
our preliminary experiments, we also found that long-term cAMP
treatment caused activation of Akt/PKB, which is reported to reflect PI
3-kinase activity (34, 35, 36, 37), implying that cAMP stimulation
activates PI 3-kinase. Taken together, these results suggest that
chronic cAMP stimulation causes PI 3-kinase activation by a
novel mechanism.
We, therefore, analyzed the roles of PI 3-kinase in cAMP-dependent
priming to IGF-I. LY294002, a specific PI 3-kinase inhibitor, abolished
cAMP-dependent potentiation of DNA synthesis induced by IGF-I. However,
the presence of this inhibitor during pretreatment time with no
additives did not affect IGF-I-induced DNA synthesis (Fig. 8A). From
these results, we demonstrated that PI 3-kinase also had important
roles in the cAMP-dependent priming effect on potentiation of DNA
synthesis induced by IGF-I. Since PI 3-kinase is shown to play
important roles in various actions of IGF-I (38, 39, 40), we can speculate
that cAMP-dependent and IGF-I-dependent signals converge into a common
signaling pathway.
In the present study, we have shown that chronic treatment with
genistein and LY294002 is necessary to abolish cAMP-dependent
potentiation of DNA synthesis induced by IGF-I (Figs. 4B and 8B). These
results suggest that accumulation of the effects of cAMP-dependent
activation of tyrosine kinases as well as PI 3-kinase is necessary to
prime the cells to respond to IGF-I. Recently, we found that genistein
as well as LY294002 inhibits cAMP-dependent increases in
G1 cyclins such as cyclin D and E in FRTL-5 cells
(41). We suspect that activation of kinases sensitive to these
inhibitors also contributes to regulation of cyclin/cyclin-dependent
kinase/cyclin-dependent kinase inhibitor system.
In conclusion, we have shown that cAMP stimulation activates a set of genistein-sensitive tyrosine kinases and causes tyrosine phosphorylation of p125 followed by association of phosphotyrosyl p125 with the p85-regulatory subunit of PI 3-kinase. These changes appear to play an important role in the potentiation of DNA synthesis induced by IGF-I in the presence of TSH. The present study suggests that tyrosine phosphorylation of p125 may be an important point of convergence for multiple signaling pathways to act on or influence the IGF-I signal response.
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Acknowledgments |
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Received November 5, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. M. Suh, J. H. Song, D. W. Kim, H. Kim, H. K. Chung, J. H. Hwang, J. M. Kim, E. S. Hwang, J. Chung, J.-H. Han, et al. Regulation of the Phosphatidylinositol 3-Kinase, Akt/Protein Kinase B, FRAP/Mammalian Target of Rapamycin, and Ribosomal S6 Kinase 1 Signaling Pathways by Thyroid-stimulating Hormone (TSH) and Stimulating type TSH Receptor Antibodies in the Thyroid Gland J. Biol. Chem., June 6, 2003; 278(24): 21960 - 21971. [Abstract] [Full Text] [PDF]
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 T. Kagawa, L. Varticovski, Y. Sai, and I. M. Arias Mechanism by which cAMP activates PI3-kinase and increases bile acid secretion in WIF-B9 cells Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1655 - C1666. [Abstract] [Full Text] [PDF]
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A. Bell, A. Gagnon, P. Dods, D. Papineau, M. Tiberi, and A. Sorisky TSH signaling and cell survival in 3T3-L1 preadipocytes Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1056 - C1064. [Abstract] [Full Text] [PDF]
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 T. Kimura, A. Van Keymeulen, J. Golstein, A. Fusco, J. E. Dumont, and P. P. Roger Regulation of Thyroid Cell Proliferation by TSH and Other Factors: A Critical Evaluation of in Vitro Models Endocr. Rev., October 1, 2001; 22(5): 631 - 656. [Abstract] [Full Text] [PDF]
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M. C. Martin, I. Dransfield, C. Haslett, and A. G. Rossi Cyclic AMP Regulation of Neutrophil Apoptosis Occurs via a Novel Protein Kinase A-independent Signaling Pathway J. Biol. Chem., November 21, 2001; 276(48): 45041 - 45050. [Abstract] [Full Text] [PDF]
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