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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

2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês

10.1074/jbc.m300805200

1083-351X

Jae Mi Suh, Jung Hun Song, Dong Wook Kim, Ho Kim, Hyo Kyun Chung, Jung Hwan Hwang, Jin‐Man Kim, Eun Suk Hwang, Jongkyeong Chung, Jeung-Hwan Han, Bo Youn Cho, Heung Kyu Ro, Minho Shong,

Protein Kinase Regulation and GTPase Signaling

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85α regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85α regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo. Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85α regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85α regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo. The pituitary glycoprotein hormones ACTH, follicle-stimulating hormone, luteinizing hormone, and TSH 1The abbreviations used are: TSH, thyroid-stimulating hormone; CHO, Chinese hamster ovary; CREB, cAMP-response element-binding protein; 4EBP, eIF-4E-binding protein; MMI, methimazole; mTOR, mammalian target of rapamycin; PDK1, 3′-phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; S6K1, ribosomal S6 kinase 1; TSAb, stimulating type TSH receptor antibody; TSBAb, blocking type TSH receptor antibody; 8-Br-cAMP, 8-bromo-cAMP; TPCK, N-α-tosyl-l-phenylalanyl chloromethyl ketone; MAP, mitogen-activated protein; HA, hemagglutinin; GFP, green fluorescent protein; PtdIns, phosphatidylinositol.1The abbreviations used are: TSH, thyroid-stimulating hormone; CHO, Chinese hamster ovary; CREB, cAMP-response element-binding protein; 4EBP, eIF-4E-binding protein; MMI, methimazole; mTOR, mammalian target of rapamycin; PDK1, 3′-phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; S6K1, ribosomal S6 kinase 1; TSAb, stimulating type TSH receptor antibody; TSBAb, blocking type TSH receptor antibody; 8-Br-cAMP, 8-bromo-cAMP; TPCK, N-α-tosyl-l-phenylalanyl chloromethyl ketone; MAP, mitogen-activated protein; HA, hemagglutinin; GFP, green fluorescent protein; PtdIns, phosphatidylinositol. control the function of specific target cells in the adrenal gland, gonads, and thyroid. All of these hormones are not only important for hormone production but also for maintaining the glandular weight in their target gland. These hormones bind to ligand-specific cell-surface G-protein-coupled receptors and activate adenylyl cyclase to produce cAMP. These glycoprotein hormone receptors also activate the PI3K-dependent signaling pathways (1Richards J.S. Mol. Endocrinol. 2001; 15: 209-218Crossref PubMed Scopus (340) Google Scholar). However, it is still unclear how these glycoprotein hormone receptors are coupled to the PI3K signaling pathways.The TSH receptor (TSHR) has many important functions that regulate growth, proliferation, differentiation, and the survival of thyrocytes and increasing hormone production in the thyroid gland (2Kohn L.D. Shimura H. Shimura Y. Hidaka A. Giuliani C. Napolitano G. Ohmori M. Laglia G. Saji M. Vitam. Horm. 1995; 50: 287-384Crossref PubMed Scopus (136) Google Scholar, 3Nagayama Y. Rapoport B. Mol. Endocrinol. 1992; 6: 145-156Crossref PubMed Scopus (151) Google Scholar). TSHR mediates these activities by activating the diverse signaling pathways including the PI3K pathway. The signaling components downstream of TSHR (Gβγ, cAMP, and PKA) may overlap with the downstream components of the PI3K signaling pathway. The Gβγ subunit of heterotrimeric G-proteins specifically activates PI3Kγ in the myeloid-derived cells (4Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 5Laffargue M. Calvez R. Finan P. Trifilieff A. Barbier M. Altruda F. Hirsch E. Wymann M.P. Immunity. 2002; 16: 441-451Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The role of Gβγ in PI3Kγ-dependent signaling in thyrocytes is not known. cAMP exerts PKA-dependent and -independent effects on PI3K signaling in thyroid cells (6Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar, 7Tsygankova O.M. Saavedra A. Rebhun J.F. Quilliam L.A. Meinkoth J.L. Mol. Cell. Biol. 2001; 21: 1921-1929Crossref PubMed Scopus (109) Google Scholar, 8Ciullo I. Diez-Roux G. Di Domenico M. Migliaccio A. Avvedimento E.V. Oncogene. 2001; 20: 1186-1192Crossref PubMed Scopus (88) Google Scholar, 9Cass L.A. Meinkoth J.L. Endocrinology. 1998; 139: 1991-1998Crossref PubMed Scopus (71) Google Scholar). The effects of cAMP on PI3K signaling are cell type-specific (9Cass L.A. Meinkoth J.L. Endocrinology. 1998; 139: 1991-1998Crossref PubMed Scopus (71) Google Scholar, 10Filippa N. Sable C.L. Filloux C. Hemmings B.A. Obberghen E. Mol. Cell. Biol. 1999; 19: 4989-5000Crossref PubMed Scopus (230) Google Scholar, 11Kim S. Jee K. Kim D. Koh H. Chung J. J. Biol. Chem. 2001; 276: 12864-12870Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Both TSH and cAMP induce Ser-473 phosphorylation in Akt/PKB, a major phosphorylation site for the regulation of Akt/PKB by growth factors. TSH- and cAMP-induced phosphorylation of Ser-473 is PI3K-dependent (i.e. wortmannin-sensitive) in rat WRT thyroid cells (6Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). cAMP and TSH also induce the phosphorylation of ribosomal protein S6 (6Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar) in a PI3K-independent and PKA-dependent manner in rat WRT thyroid cells (6Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). These studies raise the possibility that TSH and cAMP differentially regulate phosphorylation of Akt/PKB and S6 and that this process may be mediated by PI3K signaling.PDK1 is a PI3K-dependent serine/threonine kinase (12Williams M.R. Arthur J.S. Balendran A. van der Kaay J. Poli V. Cohen P. Alessi D.R. Curr. Biol. 2000; 10: 439-448Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 13Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar) whose in vivo substrates include Akt/PKB and S6K1. The mechanisms that regulate PDK1 are poorly characterized but may include cellular localization, substrate conformation, or phosphorylation (13Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 14Park J. Hill M.M. Hess D. Brazil D.P. Hofsteenge J. Hemmings B.A. J. Biol. Chem. 2001; 276: 37459-37471Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). S6K1 is a Ser/Thr kinase that is activated at the G0/G1 of the cell cycle in mammalian cells (15Bell A. Gagnon A. Dods P. Papineau D. Tiberi M. Sorisky A. Am. J. Physiol. 2002; 283: C1056-C1064Crossref PubMed Google Scholar, 16Susa M. Olivier A.R. Fabbro D. Thomas G. Cell. 1989; 57: 817-824Abstract Full Text PDF PubMed Scopus (78) Google Scholar). It phosphorylates five serine residues in the ribosomal protein S6 in vitro and is the major S6 kinase in vivo in mammalian cells (17Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar, 18Blenis J. Chung J. Erikson E. Alcorta D.A. Erikson R.L. Cell Growth Differ. 1991; 2: 279-285PubMed Google Scholar). The S6K1 pathway may regulate the translation of some ribosomal proteins and ribosome biogenesis (19Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (806) Google Scholar, 20Terada N. Takase K. Papst P. Nairn A.C. Gelfand E.W. J. Immunol. 1995; 155: 3418-3426PubMed Google Scholar) thereby regulating cell proliferation. PDK1 and mTOR (also termed FRAP or RAFT) may regulate the phosphorylation/dephosphorylation of S6K1 (21Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar, 22Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). PDK1 activates S6K1 by phosphorylating Thr-229, an important residue in the activation loop of S6K1 (23Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).The FRAP/mTOR kinase regulates the initiation and elongation of translation, ribosome biosynthesis, and amino acid transport (24Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1710) Google Scholar), which affect the rate of protein synthesis. FRAP/mTOR participates in mitogen-signaling pathways and acts as a nutrient-sensing checkpoint (25Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar, 26Gingras A.C. Raught B. Soneberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1167) Google Scholar, 27Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (794) Google Scholar). Both FRAP/mTOR and PI3K signaling are required to activate several downstream effector proteins (24Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1710) Google Scholar, 25Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (598) Google Scholar). FRAP/mTOR may stimulate the phosphorylation of downstream targets and repress phosphatase activity (28Rohde J. Heitman J. Maria E.C. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Rapamycin-FKBP12 forms a complex that specifically inhibits FRAP/mTOR in vivo (29Zhang H. Stallock J.P. Ng J.C. Reinhard C. Neufeld T.P. Genes Dev. 2000; 14: 2712-2724Crossref PubMed Scopus (505) Google Scholar).This study examines the interactions between the TSHR and the regulatory p85α subunit of PI3K in the presence and absence of TSH and cAMP. TSH and 8-Br-cAMP stimulate the interaction between TSHR and PI3K, which leads to a PI3K- and PKA-dependent translocation of PDK1. PDK1 phosphorylation of Akt/PKB and S6K1 appears to be differentially stimulated by TSH and insulin. TSH stimulates S6K1 through the PI3K-, PDK1-, and PKA-dependent but Akt/PKB-independent pathways. The FRAP/mTOR inhibitor rapamycin inhibits the TSH-stimulated phosphorylation of S6K1 and inhibits cell cycle progression in the FRTL-5 thyroid cells. Rapamycin also modulates the thyroid follicle activity, which is induced by elevated endogenous TSH levels in vivo. This study suggests that S6K1 plays an important role in TSHR-activated PI3K signaling, which modulates the thyrocyte proliferation and thyroid follicle activity.EXPERIMENTAL PROCEDURESMaterials—The media and cell culture reagents and materials were purchased from Invitrogen, Sigma, Fisher, Corning Glass, and Hyclone Laboratories, Inc. (Logan, UT). Wortmannin and H89 were from Calbiochem. LY294002 and PD98059 were obtained from Sigma and New England Biolabs (Beverly, MA). Antibodies for cyclin D1 (sc-8396), p85a (sc-423), and p110γ (H-119) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for HA (2363), Akt (9272), phospho-Akt-Ser-473, and Thr-308 (9271 and 9275); phospho-4EBP1-Ser-65 (9451) and 4EBP1 (9452); Myc (2276), S6 ribosomal protein (2212), and phospho-S6 ribosomal protein-Ser-235/236 (2211); and p44/p42 MAP kinase (9102) and phospho-p44/42 MAP kinase-Thr-202/Tyr-204 (9101) were from Cell Signaling Technology (Beverly, MA). Mouse anti-human TSHR (MCA1571) was from Serotec (Kidlington, Oxford, UK), and anti-FLAG antibody (200472) was from Stratagene (La Jolla, CA). All other materials, including 8-bromo-cAMP (8-Br-cAMP and, N-α-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), were purchased from Sigma.Preparation of IgG—The control sera were obtained from 5 normal individuals who had no history or clinical or chemical evidence (abnormal thyroid hormone and TSH levels) of thyroid disease. The diagnosis of the 10 patients with Graves disease was based on conventional clinical and laboratory criteria, including elevated serum thyroid hormone levels, undetectable TSH by a sensitive radioimmunoassay, testing positive for TBII (TSH-binding inhibitory immunoglobulins), and a diffuse goiter with increased 99mTc 99mTcO4−99mTcO4−99mTcO4−99mTcO4− uptake at scintiscan. IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B columns; IgG was lyophilized and stored at–20 °C until assay. The IgGs were extracted by affinity chromatography from normal pooled sera (NP), as well as the sera from the patients with primary myxedema (PM) who tested positive to the blocking TSHR antibody test.Cell Culture and Gene Transfection—A fresh subclone (F1) of FRTL-5 was used in the rat thyroid cells (Interthyr Research Foundation, Baltimore, MD). After 6 days in medium with no TSH, the addition of 1 milliunit/ml crude bovine TSH (Sigma) stimulated thymidine incorporation into DNA by at least 10-fold. The doubling time of the cells with TSH was 36 ± 6 h without TSH, and they did not proliferate. The cells used were diploid and between their 5th and 20th passage. The cells were grown in 6H medium consisting of Coon's modified F-12 supplemented with 5% calf serum, 1 mm nonessential amino acids, and a mixture of six hormones as follows: bovine TSH (1 milliunit/ml), insulin (10 μg/ml), cortisol (0.4 ng/ml), transferrin (5 μg/ml), glycyl-l-histidyl- l-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all cells every 2 or 3 days, and the cells were passaged every 7–10 days. In individual experiments, the cells were shifted to 3H (which is devoid of insulin, TSH, and somatostatin), 4H (which is devoid of insulin and TSH), or 5H medium (which is devoid of TSH) with or without 5% calf serum before TSH, forskolin (Sigma), or the other agents were added. Chinese hamster ovary (CHO) cells were maintained at 37 °C and 5% CO2 in Ham's F-12 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Expression plasmids carrying hTSHR, p85α, and PDK1 cDNAs were introduced into the FRTL-5 and CHO cells using the LipofectAMINE Plus reagents according to the manufacturer's instructions (Invitrogen).PI3K Assay—The cell extracts obtained from the FRTL-5 cells were immunoprecipitated with anti-TSHR monoclonal antibodies (clone 4C1, Serotec, Oxford, UK) and the control IgG antibodies. The samples were washed twice with 1% Nonidet P-40 and 1 mm sodium orthovanadate in phosphate-buffered saline, twice with washing buffer consisting of 100 mm Tris-HCl (pH 7.5), 500 mm LiCl, and 1 mm sodium orthovanadate, and twice with ST with 150 mm NaCl and 50 mm Tris-HCl (pH 7.2). The samples were resuspended in a PI kinase buffer containing 20 mm Hepes (pH 7.2), 100 mm NaCl, 10 μg/ml leupeptin, and 10 μg/ml pepstatin. A phosphoinositide/EGTA solution consisting of 1 mg/ml phosphoinositide and 2.5 mm EGTA was then added, and the samples were incubated at room temperature for 10 min. A solution containing 20 mm Hepes (pH 7.4), 5 mm MnCl2, 10 μm ATP, and 20 μCi of [γ-32P]ATP was added, and the samples were incubated at 30 °C for 20 min. The reactions were quenched by the addition of 1 m HCl, and the phospholipids were extracted using CHCl3. The dried samples were separated by TLC. The phosphorylated lipids were visualized by autoradiography and quantified using a PhosphorImager (BAS1500, Fuji).Immunoprecipitation and Western Blot Analysis—The following immunoprecipitation procedures were carried out at 4 °C. The cells grown on the 100-mm dishes were washed with phosphate-buffered saline twice prior to lysis. The RIPA buffer containing the protease inhibitors (20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml chymostatin, 2 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) were added for cell lysis. The cell lysate was collected, triturated, and centrifuged at 1000 × g for 10 min. To preclear the cell lysate, the supernatant was mixed with 20 μl of protein A/G beads (Santa Cruz Biotechnology), incubated for 30 min with rocking, and were centrifuged for 15 min at 1000 × g. Precleared samples were incubated with the primary antibodies for 2 h with rocking, and protein A/G beads were then added, incubated overnight, and centrifuged at 1000 × g. The immunoprecipitates were collected and washed three times with RIPA buffer.For Western blot analysis, the cells were lysed at 4 °C in a mixture of 10 mm KPO4, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerophosphate, 2 mm dithiothreitol, 1% Nonidet P-40, 1 mm Pefabloc (Roche Applied Science), and 10 μg each of aprotinin and leupeptin/ml. The total protein lysates were denatured by boiling in a Laemmli sample buffer, resolved on 7.5–15% SDS-PAGE, and transferred to polyvinylidene fluoride membranes. The membranes were blocked in phosphate-buffered saline containing 5% (w/v) milk and 0.1% Tween and then incubated for 2 h with the polyclonal antibodies against p70 S6K (supplied by Dr. J. Blenis, Harvard University, Boston).[3H]Thymidine Incorporation Assay—Confluent FRTL-5 thyroid cells in 100-mm dishes were detached by trypsinization, resuspended in 6H growth medium, seeded at a density of 3 × 104 cells/well in 24-well plates, and incubated for 2–3 days until 80% confluent. The medium was changed to 5H medium and incubated for an additional 7 days. TSH and/or wortmannin, LY294002, H89, and rapamycin were added to the quiescent cells, which were then incubated for 24 h, followed by the addition of 2 μCi/ml [3H]thymidine (PerkinElmer Life Sciences) to pulse the cells for an additional 12 h. The experimental samples were prepared in triplicate. The cells were washed four times with ice-cold phosphate-buffered saline, precipitated twice with ice-cold 10% trichloroacetic acid (30 min each time on ice), briefly washed once with ice-cold ethanol, lysed with 0.2 n NaOH in 0.5% SDS, and incubated at 37 °C for at least 30 min. The level of radioactivity was determined by liquid scintillation spectrometry (Beckman Instruments). The results were measured as the number of counts/min in each well. Each experimental data point represents triplicate wells from at least four different experiments.Flow Cytometry—The samples were prepared for flow cytometry essentially as described previously (4Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar). Briefly, the cells were washed with 1× phosphate-buffered saline, pH 7.4, and then fixed with ice-cold 70% ethanol. The samples were washed with 1× phosphate-buffered saline and stained with propidium iodide 60 μg/ml (Sigma) containing 100 μg/ml RNase (Sigma) for 30 min at 37 °C. Cell cycle analysis was performed using a BD Biosciences fluorescence-activated cell analyzer and Cell Quest version 1.2 software (BD Biosciences). At least 10,000 cells were analyzed per sample. The cell cycle distribution was quantified using the ModFit LT version 1.01 software (Verity Software House Inc., Topsham, ME).Confocal Microscopy—The FRTL-5 thyroid cells were grown on coverslips and transfected with pEGFP-PDK-1 and pCDNA3-PDK-1-myc using the LipofectAMINE method (Invitrogen). Quiescent cells stimulated with TSH were washed three times with cold phosphate-buffered saline and fixed in 3.7% formaldehyde for 40 min. Fixed cells were mounted on glass slides with phosphate-buffered saline and observed using laser-scanning confocal microscopy (Leica TCS SP2).Protein Kinase Assays—The FRTL-5 thyroid cells were transiently transfected with HA-tagged p70 S6K1 and immunoprecipitated with an anti-HA monoclonal antibody coupled to protein A/G-agarose (Santa Cruz Biotechnology). The samples were washed twice with Buffer A (containing 20 mm Tris-HCl (pH 7.5), 0.1% Nonidet P-40, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerophosphate, 1 mm sodium orthovanadate, 2 mm dithiothreitol, 40 μg/ml phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin) and then twice with Buffer B containing 500 mm NaCl. Finally the immunocomplexes were washed, in succession, with Buffer C (containing 20 mm Hepes (pH 7.2), 10 mm MgCl2, 0.1 mg/ml bovine serum albumin, and 3 mm β-mercaptoethanol), Buffer D (containing 20 mm Hepes (pH 7.2), 10 mm MgCl2,10mm MnCl2,1mm dithiothreitol, and 0.2 mm EGTA), and Buffer E (containing 50 mm Tris (pH 7.5), 10 mm NaCl, 1 mm dithiothreitol, and 10% glycerol). The S6 phosphotransferase activities were assayed in a reaction mixture consisting of 1× Buffer C, 1 μg of S6 peptide, 20 μm ATP, and 5 μCi of [γ-32P]ATP (specific activity: 3000 Ci/mmol; PerkinElmer Life Sciences) at 30 °C for 20 min. The samples were subjected to liquid scintillation counting (Hewlett-Packard).Preparation of Thyroid Gland and Immunohistochemistry—Male Sprague-Dawley rats (120–130 g) were fed with water containing 0.025% MMI for 2 weeks. Rapamycin (Calbiochem) was delivered once daily by an intraperitoneal injection at a dose of 1.5 mg/kg dissolved in 2% carboxymethylcellulose for 1 week before histologic examination. Tissue samples of the rat thyroid gland were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Three-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E). The number of follicles in 2 medium power fields (×200) was counted. Further sections were used for immunohistochemistry. All immunostaining steps were carried out at room temperature. After deparaffinization and antigen retrieval by autoclaving in 10 mm sodium citrate buffer (pH 6.0) at full power for 10 min, the tissue sections were treated with blocking rabbit serum for 15 min. The primary antibody, polyclonal rabbit anti-phospho-S6 ribosomal protein (Cell Signaling Technology), was diluted (1:200) with a background-reducing diluent (Dako, Carpinteria, CA) and incubated for 60 min. The sections were then incubated with a rabbit EnVision-HRP detection system reagent (Dako, Carpinteria, CA) for 30 min. The sections were then sequentially incubated with 3,3-diaminobenzidine (Dako) chromogen for 5 min, counterstained with Meyer's hematoxylin, and mounted. Careful rinses with several changes of Tris-buffered saline, 0.3% Tween buffer were performed between each step. A negative control that excluded the primary antibody was used. Cells with cytoplasmic granular staining were considered positive.Other Assays—The protein concentration was determined by the Bradford method (Bio-Rad) using recrystallized bovine serum albumin as a standard. The sera were stored–70 °C until IgG preparation. The IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B columns (Amersham Biosciences) followed by dialysis. The purity of the IgG preparation was confirmed by the documentation of undetectable TSH levels with immunoradiometric assay.Statistical Analysis—All experiments were repeated at least three times with different cells. The values are the mean ± S.E. of these experiments. Statistical significance was determined by a two-way analysis of variance.RESULTSAssociation of PI3K Activities with TSHR—TSHR was immunoprecipitated from extracts from FRTL-5 thyroid cells exposed to TSH or 8-Br-cAMP using monoclonal anti-TSHR antibodies, and the immunoprecipitate was tested for PI3K activity. The phosphatidylinositol phosphotransferase activity was detected in the immunoprecipitate, which was inhibited by the PI3K inhibitors wortmannin (100 nm) or LY294002 (0.5 μm) (Fig. 1A). The TSHR-associated PI3K activity was barely detectable in the untreated FRTL-5 thyroid cells (Fig. 1, A, lane 1, and B, lane 1). Both TSH and 8-Br-cAMP stimulated the TSHR-associated PI3K activity (Fig. 1B, lanes 2 and 4, respectively). H89 did not inhibit the PI3K kinase activity in vitro (data not shown), but the H89 treated cells had a lower level of TSHR-associated PI3K activity in the TSH- or 8-Br-cAMP-treated cells (Fig. 1B, lanes 3 and 5).The specificity of the interaction between TSHR and PI3K was determined in the following experiment. The extracts were prepared from the wild-type CHO cells or the CHO cells expressing human TSHR (CHO-TSHR) (30Kim W.B. Chung H.K. Lee H.K. Kohn L.D. Tahara K. Cho B.Y. J. Clin. Endocrinol. Metab. 1997; 82: 1953-1959PubMed Google Scholar, 31Park E.S. Kim H. Suh J.M. Park J. You S.H. Chung H.K. Lee K.W. Kwon O.-Y. Cho B.Y. Kim Y.K. Ro H.K. Chung J. Shong M. Mol. Endocrinol. 2000; 14: 662-670Crossref PubMed Scopus (46) Google Scholar), and immunoprecipitation was carried out using anti-TSHR (Fig. 1C, lanes 1–4) or the control IgG (Fig. 1C, lane 5). A low level of PI3K activity was detected in the anti-TSHR immunoprecipitates from the CHO-TSHR cells (Fig. 1C, lane 2). This activity increased in the cells treated with TSH (Fig. 1C, lanes 2 versus 3) and was inhibited by 100 nm wortmannin (Fig. 1C, lane 4). No PI3K activity was detected in the experiments using the wild-type CHO cells (Fig. 1C, lane 1) or the control IgG (Fig. 1C, lane 5). These findings suggest that TSHR interacts specifically with PI3K and that TSH and cAMP stimulate this interaction.TSHR Interacts with the p85α Regulatory Subunit of PI3K— Western blot analysis was carried out to identify the specific regulatory and catalytic subunits of class I PI3K in FRTL-5 thyroid cells. The p85α regulatory subunit of class I PI3K (p85α) was expressed strongly in the cells treated with or without TSH, 8-Br-cAMP, and insulin (Fig. 2A). In contrast, the p110γ catalytic subunit of class I PI3K, which is activated by the Gβγ subunits of the heterodimeric G-proteins, was not expressed at a detectable level in these cells (Fig. 2A).Fig. 2A, the expression of p85α regulatory subunit and p110γ catalytic subunit of class I PI3K isoforms in FRTL5. FRTL-5 cells were grown to 70% confluence in complete 6H medium with 5% serum and then maintained for 6 days in 4H0% medium, which does not contain TSH, insulin, and serum. Total cell lysates were prepared from cells treated for 12 h with TSH (1 milliunit/ml), insulin (10 μg/ml), or 8-Br-cAMP (1 mm) and analyzed by Western blot using an anti-p85α regulatory subunit of PI3K antibody and an anti-p110γ catalytic subunit of PI3K antibody. B–D, interactions between the p85 regulatory subunit of PI3K and TSHR in FRTL5 and CHO cells. CHO cells were transfected with constructs that express HA-tagged p8