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Endocrinology Vol. 141, No. 7 2429-2438
Copyright © 2000 by The Endocrine Society

ARTICLES

Tyrosine Kinase and Phosphatidylinositol 3-Kinase Activation Are Required for Cyclic Adenosine 3',5'-Monophosphate-Dependent Potentiation of Deoxyribonucleic Acid Synthesis Induced by Insulin-Like Growth Factor-I in FRTL-5 Cells

Taku Nedachi, Masakazu Akahori, Miyako Ariga, Hideki Sakamoto, Naoko Suzuki, Kohta Umesaki, Fumihiko Hakuno and Shin-Ichiro Takahashi

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factors (IGFs) and/or their receptors are essential for the normal mammalian growth and development (1, 2, 3). In many cell types, IGF-I regulates cell proliferation, apoptosis, and a vast variety of differentiated cell functions (4, 5, 6). Despite the diversity of these effects, the biological effects of IGFs in vitro are relatively weak and often not demonstrable except in the presence of other hormones or growth factors (7, 8, 9, 10, 11). Delineation of the mechanisms by which IGFs interact with other hormones and growth factors in model systems is likely to shed further light on the physiological roles of IGFs and their receptors.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Coon’s modified Ham’s F-12 (Coon’s F-12) was purchased from Life Technologies, Inc.(Gaithersburg, MD), and HBSS was obtained from Nissui (Tokyo, Japan). Newborn calf serum (NCS) was obtained from Nichirei Co. (Tokyo, Japan). Transferrin and bovine TSH (bTSH; 1.23 U/mg) for culture were purchased from Sigma (St. Louis, MO). Purified bTSH (30 U/mg) for biological studies was a generous gift from the National Hormone and Pituitary Program (NIDDK). Recombinant human IGF-I (hIGF-I) was kindly donated by Dr. Toshiaki Ohkuma (Fujisawa Pharmaceutical Co., Ltd. Osaka, Japan). Leupeptin and pepstatin were kindly donated by Dr. Takaaki Aoyagi (Institute of Microbial Chemistry, Tokyo, Japan). Antiphosphotyrosine monoclonal antibody was kindly provided by Dr. T. Yamori (Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo). Anti-p85 regulatory subunit of phoshatidylinositol (PI) 3-kinase antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). cDNA of a human p85 regulatory subunit of PI 3-kinase was generously given by Dr. Tomoichiro Asano (Faculty of Medicine, The University of Tokyo, Tokyo). Penicillin was obtained from Ban’yu Pharmaceutical Co. (Tokyo), streptomycin and kanamycin from Meiji Seika Co. (Tokyo), and Amphotericin (fungizone) was from Sankyo Co., Ltd. (Tokyo). All dishes and flasks were from IWAKI (Tokyo). [Methyl-3H]thymidine (6.5 mCi/mmol), and [{gamma}-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 [{gamma}-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 [{gamma}-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 [{gamma}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cAMP on tyrosine kinase or protein-tyrosine phosphatase activities
To determine whether tyrosine kinase activation or PTPase inhibition was observed in response to cAMP treatment, we measured tyrosine kinase or PTPase activities of the cell lysates after (Bu)2cAMP treatment for 24 h. Tyrosine kinase activities increased about 1.3-fold in the particulate fraction, but did not change in the soluble fraction (Fig. 1AGo). Approximately 20–40% of total tyrosine kinase activity was inhibited by genistein, a tyrosine kinase inhibitor (Table 1Go). On the other hand, cAMP pretreatment increased PTPase activities about 2-fold in the soluble fraction but did not affect the particulate fraction (Fig. 1BGo). 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. 2AGo 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. 2Go, A and B. We obtained similar results using TSH instead of (Bu)2cAMP (data not shown).



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  		Figure 1. Effects of (Bu)2cAMP on tyrosine kinase or protein-tyrosine phosphatase activities in FRTL-5 cells. Quiescent FRTL-5 cells were treated with no additives or (Bu)2cAMP (cAMP; 10-3 M) for 24 h. After harvesting, the cell lysates were separated to soluble and particulate fractions and tyrosine kinase activities (A) or protein-tyrosine phosphatase activities (B) were measured in each fraction as described in Materials and Methods. Tyrosine kinase or protein-tyrosine phosphatase activities are expressed as a percentage of the values of cells without (Bu)2cAMP, and the results shown are the mean ± SEM of five independent experiments. **, Significant difference (P < 0.01) between values of the cells treated with no additives and (Bu)2cAMP treatments.

 

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  		Table 1. Effects of cAMP treatment on genistein-sensitive tyrosine kinase activity

 


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  		Figure 2. Time course of the (Bu)2cAMP stimulation of tyrosine kinase activities and tyrosine phosphorylation in FRTL-5 cells. A, Quiescent FRTL-5 cells were treated with (Bu)2cAMP (cAMP; 10-3 M) for various times. After harvesting, the particulate fraction was prepared and tyrosine kinase activities were measured as described in Materials and Methods. The results shown are the mean ± SEM of five independent experiments. B, Quiescent FRTL-5 cells were treated with (Bu)2cAMP (cAMP; 10-3 M) for the indicated times. After treatment, cells were harvested and immunoblot analysis of p125 was performed with antiphosphotyrosine antibody as described in Materials and Methods. The experiments were performed three times independently and a representative blot is shown. In the lower panel, p125 tyrosine phosphorylation was quantitated by NIH Image program and is expressed as a percentage of the values at 0 time. The results shown are the mean ± SEM of three independent experiments. In panels A and B, * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values at 0 time and indicated times.

 
Effects of genistein or orthovanadate on cAMP-dependent tyrosine phosphorylation
We previously showed that tyrosine phosphorylation of p125 after 24 h TSH or (Bu)2cAMP stimulation was similar in several respects to the cAMP-dependent potentiation of DNA synthesis induced by IGF-I (18). To examine the effects of genistein or orthovanadate on cAMP-induced tyrosine phosphorylation, the cells were treated with TSH or (Bu)2cAMP in the presence or absence of various concentrations of genistein or orthovanadate for 24 h. Tyrosine phosphorylation of p125 in response to (Bu)2cAMP was significantly reduced by genistein in a concentration-dependent manner (Fig. 3Go). 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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  		Figure 3. Effects of genistein during the pretreatment time with (Bu)2cAMP on tyrosine phosphorylation in FRTL-5 cells. Quiescent FRTL-5 cells were treated with no additives or (Bu)2cAMP (cAMP; 10-3 M) in the absence or presence of various concentration of genistein for 24 h. After treatment, cells were harvested and immunoblot analysis was performed with antiphosphotyrosine antibody as described in Materials and Methods. The experiments were performed three times independently and a representative blot is shown. In the lower panel, p125 tyrosine phosphorylation was quantitated by NIH Image program and is expressed as a percentage of the means of cells treated with (Bu)2cAMP in the absence of genistein. The results shown are the mean ± SEM of three independent experiments; * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values of the cells treated with no inhibitor and genistein at indicated concentrations.

 
Effects of genistein on cAMP-dependent potentiation of DNA synthesis induced by IGF-I
We then tested the effects of various concentrations of genistein during the pretreatment time with TSH or (Bu)2cAMP on DNA synthesis induced by IGF-I. As shown in Fig. 4AGo, 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. 4BGo). 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. 3Go and 4AGo). We obtained similar results using TSH instead of (Bu)2cAMP (data not shown).



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  		Figure 4. Effects of genistein during the pretreatment time with (Bu)2cAMP on cAMP-dependent potentiation of DNA synthesis induced by IGF-I in FRTL-5 cells. A, Quiescent FRTL-5 cells were pretreated with no additives or (Bu)2cAMP (cAMP; 10-3 M) in the absence or presence of various concentrations of genistein for 24 h. The cells were washed five times with HBSS and incubated with or without IGF-I (100 ng/ml) for an additional 24 h. [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h as described in Materials and Methods. The results shown are the mean ± SEM of triplicate wells; * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values of the cells treated with no inhibitor and genistein at indicated concentrations in each treatment group. B, Quiescent FRTL-5 cells were pretreated for 24 h with no additives or (Bu)2cAMP (Bt2cAMP; 10-3 M) and at the indicated time genistein (30 µg/ml as final concentrations) was added to the cultures. The cells were washed five times with HBSS and incubated with or without IGF-I (100 ng/ml) for an additional 24 h. [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h as described under in Materials and Methods. The results shown are the mean ± SEM of triplicate wells; * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values at 24 h treatment without genistein and at indicated treatment times.

 
Association of a phosphotyrosyl 125-kDa protein with a p85-regulatory subunit of PI 3-kinase
As it has been shown that many phosphotyrosyl proteins directly interact with signaling molecules containing the SH2 domain, we tested whether the phosphotyrosyl p125 induced by cAMP binds to the p85-regulatory subunit of PI 3-kinase (Fig. 5BGo). 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. 5Go, 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. 5CGo). In addition, an increase in tyrosine phosphorylation of p125 associated with p85 PI 3-kinase was dependent on cAMP treatment time (Fig. 6AGo). 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. 6BGo). 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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  		Figure 5. Interaction between phosphotyrosyl p125 and a p85-regulatory subunit of PI 3-kinase in FRTL-5 cells. A and B, Quiescent FRTL-5 cells were pretreated with no additives or (Bu)2cAMP (10-3 M) for 24 h. The cells were washed three times with HBSS and incubated with or without IGF-I (100 ng/ml) for an additional 2 min. After treatment, cells were harvested and immunoblotting was performed using antiphosphotyrosine antibody (A). In the case of panel B, cells were harvested after treatment and proteins were immunoprecipitated with anti-p85 regulatory subunit of PI 3-kinase antibody followed by immunoblotting with antiphosphotyrosine antibody as described in Materials and Methods. C, Quiescent FRTL-5 cells were pretreated with no additives or (Bu)2cAMP (10-3 M) for 24 h. After treatment, cells were harvested and cell lysates were applied to glutathione-Sepharose beads that adsorbed GST alone (GST) or GST-amino-terminal SH2 domain of p85 PI-3 kinase fusion proteins (GST-SH2). Proteins associated with immobilized beads were analyzed by immunoblotting with antiphosphotyrosine antibody as described in Materials and Methods.

 


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  		Figure 6. Time course of the (Bu)2cAMP stimulation of interaction between p125 and a p85-regulatory subunit of PI 3-kinase in FRTL-5 cells. A and B, Quiescent FRTL-5 cells were treated with (Bu)2cAMP (cAMP; 10-3 M) for the indicated times. After treatment, cells were harvested and proteins were immunoprecipitated with anti-p85 regulatory subunit of PI 3-kinase antibody followed by immunoblotting with antiphosphotyrosine antibody or anti-p85 PI-3 kinase antibody (A) or by blotting with GST-amino-terminal SH2 domain of p85 PI 3-kinase (B), as described in Materials and Methods. The experiments were performed three times independently, and a representative blot is shown in the upper panel. In the lower panels, tyrosine phosphorylation of p125 (A) or the amount of the SH2 domain of p85 PI 3-kinase associated with p125 (B) was quantitated by the NIH Image program and is expressed as a percentage of the values at 24 h. The results shown are the mean ± SEM of three independent experiments; * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values at 0 time and indicated times.

 
Effects of cAMP on PI 3-kinase activity
To determine whether cAMP treatment affects PI 3-kinase activity, we measured PI 3-kinase activity bound to p85-regulatory subunit of PI 3-kinase. Treatment with (Bu)2 cAMP for 12 h or 24 h caused a significant increase in PI 3-kinase activity (Fig. 7Go).



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  		Figure 7. Effects of (Bu)2cAMP on PI 3-kinase activity bound to p85 PI 3-kinase in FRTL-5 cells. Quiescent FRTL-5 cells were treated with (Bu)2cAMP (cAMP; 10-3 M) for 0 h, 12 h, or 24 h. After treatment, cells were harvested and proteins were immunoprecipitated with anti-p85 regulatory subunit of PI 3-kinase antibody followed by measurement of PI 3-kinase activity in the immunoprecipitates, as described in Materials and Methods. PI 3-kinase activities are expressed as a percentage of the values at 0 time and the results shown are the mean ± SEM of three independent experiments; * indicates a significant difference (P < 0.05) between values at 0 time and the indicated times.

 
Effects of LY294002 on cAMP-dependent potentiation of DNA synthesis induced by IGF-I
Lastly, we studied the effects of LY294002, a PI 3-kinase inhibitor, during cAMP pretreatment on DNA synthesis in the cells pretreated with TSH or (Bu)2 cAMP for 24 h followed by retreatment with IGF-I for additional 24 h (Fig. 8AGo). 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. 8BGo). 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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  		Figure 8. Effects of LY294002 during the pretreatment time with (Bu)2cAMP on cAMP-dependent potentiation of DNA synthesis induced by IGF-I in FRTL-5 cells. A, Quiescent FRTL-5 cells were pretreated with no additives or (Bu)2cAMP (cAMP; 10-3 M) in the absence or presence of various concentrations of LY294002 for 24 h. The cells were washed five times with HBSS and incubated with or without IGF-I (100 ng/ml) for an additional 24 h. [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h as described in Materials and Methods. The results shown are the mean ± SEM of triplicate wells; * and ** indicate a significant difference (P < 0.05 and P < 0.01, respectively) between values of the cells treated with no inhibitor and LY294002 at indicated concentration in each treatment group. B, Quiescent FRTL-5 cells were pretreated for 24 h with (Bu)2cAMP (cAMP; 10-3 M) and at the indicated time LY294002 (50 µM as final concentration) was added to the cultures. The cells were washed five times with HBSS and incubated with or without IGF-I (100 ng/ml) for an additional 24 h. [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h as described in Materials and Methods. The results shown are the mean ± SEM of triplicate wells; * and ** indicate a significant difference (P < 0.05 or P < 0.01, respectively) between values at 24 h treatment without LY294002 and indicated treatment times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously showed that cAMP pretreatment results in potentiation of DNA synthesis induced by IGF-I in FRTL-5 cells (8). We further found that cAMP pretreatment causes the increase in tyrosine phosphorylation of a 125-kDa protein (p125), which was well correlated with an enhancement in IGF-I-dependent DNA synthesis in response to cAMP (18). Accordingly, in the present study we investigated whether and how cAMP-dependent tyrosine phosphorylation is required for cAMP-dependent amplification of DNA synthesis induced by IGF-I. Our new observations have demonstrated that 1) chronic cAMP stimulus increases tyrosine kinase activity followed by enhancement in tyrosine phosphorylation of p125, which associates with a 85-kDa regulatory subunit (p85) of PI 3-kinase through its SH2 domain, and 2) tyrosine kinase and PI 3-kinase activities during cAMP pretreatment are required for potentiation of IGF-I-dependent DNA synthesis in response to cAMP in FRTL-5 cells. The present findings provide an explanation for physiological processes of interaction between tropic hormones and IGFs by highlighting a point of convergence of cAMP-dependent events on IGF-I-dependent signals.

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. 1AGo). 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. 2Go) and genistein inhibited cAMP-dependent enhancement of p125 tyrosine phosphorylation (Fig. 3Go). In contrast, we demonstrated that long-term cAMP treatment increased PTPase activities in the soluble fraction, rather than decreased (Fig. 1BGo), 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. 2Go) 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. 3Go and 4AGo). 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. 4AGo). In addition, genistein inhibits only 20–40% of total tyrosine kinase activity (Table 1Go) 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. 5Go, 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. 6Go). As shown in Fig. 7Go, (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. 8AGo). 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. 4BGo and 8BGo). 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.


   Acknowledgments
 
We wish to thank Dr. Leonard Kohn (NIDDK, Bethesda, MD) and Interthyr Corporation (Baltimore, MD) for the kind gift of FRTL-5 cells and National Hormone and Pituitary Program (NIDDK) for providing bovine TSH (Lot AFP-3950B). We acknowledge Dr. Derek LeRoith (NIDDK) for donating NIGF-IR cells. Recombinant human IGF-I was kindly donated by Dr. Toshiaki Ohkuma, Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). We also express our appreciation to Dr. Takaaki Aoyagi (Institute of Microbial Chemistry, Tokyo, Japan) for leupeptin and pepstatin. In addition, we acknowledge Dr. Takao Yamori (Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan) for donating antiphosphotyrosine monoclonal antibody. We thank Dr. Tomoichiro Asano for giving us cDNA of a human p85-regulatory subunit of PI 3-kinase. Finally, we appreciate a helpful discussion during writing this paper with Dr. Judson J. Van Wyk (University of North Carolina, Chapel Hill, NC), Dr. Steven C. Boyages (Westmead Hospital, University of Sydney, New South Wales, Australia), and Dr. Marco Conti (Stanford University, Stanford, CA).

Received November 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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