regulation of the pi3k, akt/pkb, frap/mtor, and s6k1 ... · this pathway may play a role in the...

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Regulation of the PI3K, Akt/PKB, FRAP/mTOR, and S6K1 signaling pathways by thyroid stimulating hormone and stimulating-type TSH receptor antibodies in the thyroid gland 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 Laboratory of Endocrine Cell Biology, Department of Internal Medicine, §Department of Pathology, Chungnam National University, Taejon ,301-721, Korea; ¶ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea; §§Department of Biochemistry, College of Pharmacy, Sungkyunkwan University, Suwon 440-756, Korea *Department of Internal Medicine, Seoul National University, Seoul, 110-744

running title: Regulation of FRAP/mTOR by TSH and TSAb * supported by National Research Laboratory Program (M1-0104-00-0014) and KOSEF Research Grant (2000-2-20500-007-3) Ministry of Science and Technology, Korea + to whom correspondence should be addressed Minho Shong Laboratory of Endocrine Cell Biology Department of Internal Medicine Chungnam National University School of Medicine 640 Daesadong Chungku Taejon 301-040, Korea Tel) 82-42-220-7161 Fax) 82-42-257-5753 email) [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 30, 2003 as Manuscript M300805200 by guest on January 16, 2020

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Abstract

TSH regulates the growth and differentiation of thyrocytes by activating the TSH

receptor. This study investigated the roles of the PI3K, PDK1, FRAP/mTOR and 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-Br-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 PKA-dependent manner. TSH induced the PDK1-dependent

phosphorylation of S6K1, but did not induce Akt/PKB 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-Br-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

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and PI3K, which is stimulated by TSH and cAMP and might involve the downstream

S6K1 but not Akt/PKB. This pathway may play a role in the TSH/TSAb-mediated

thyrocyte proliferation in vitro and in the response to TSH in vivo.


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Introduction

The pituitary glycoprotein hormones ACTH, FSH, LH, and TSH 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 (1). 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 (2, 3). TSHR mediates these activities by activating the

diverse signaling pathways including the PI3K pathway. The signaling components

downstream of TSHR (Gβγ, cAMP, 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 (4, 5). 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 (6-9). The effects

of cAMP on PI3K signaling are cell-type specific (9-11). Both TSH and cAMP induce


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S473 phosphorylation in Akt/PKB, a major phosphorylation site for the regulation of

Akt/PKB by growth factors. TSH and cAMP–induced phosphorylation of S473 is PI3K-

dependent (i.e., wortmannin sensitive) in rat WRT thyroid cells (6). cAMP and TSH also

induce the phosphorylation of ribosomal protein S6 (6) in a PI3K-independent and

PKA-dependent manner in rat WRT thyroid cells (6). 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 (12, 13) 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 (13, 14). S6K1 is a Ser/Thr kinase that is activated at the G0/G1 of the

cell cycle in mammalian cells (15, 16). It phosphorylates five serine residues in the

ribosomal protein S6 in vitro and is the major S6 kinase in vivo in mammalian cells (17,

18). The S6K1 pathway may regulate the translation of some ribosomal proteins and

ribosome biogenesis (19, 20) thereby regulating cell proliferation. PDK1 and mTOR

(also termed FRAP or RAFT) may regulate the phosphorylation/dephosphorylation of

S6K1 (21, 22). PDK1 activates S6K1 by phosphorylating Thr229, an important residue

in the activation loop of S6K1 (23).

The FRAP/mTOR kinase regulates the initiation and elongation of translation,


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ribosome biosynthesis, and amino acid transport (24), which affect the rate of protein

synthesis. FRAP/mTOR participates in mitogen signaling pathways and acts as a

nutrient sensing checkpoint. (25, 26, 27). Both FRAP/mTOR and PI3K signaling are

required to activate several downstream effector proteins (24, 25). FRAP/mTOR may

stimulate the phosphorylation of downstream targets and repress phosphatase activity

(28). Rapamycin-FKBP12 forms a complex that specifically inhibits FRAP/mTOR in

vivo (29).

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.


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Materials and Methods

Materials

The media and cell culture reagents and materials were purchased from Life

Technologies, Inc. (Gaithersburg, MD), Sigma (St. Louis, MO), Fisher Scientific

(Fairlawn, NJ), Corning, Inc (Corning, NY) and Hyclone Laboratories, Inc (Logan, UT).

Wortmannin and H89 were from Calbiochem (La Jolla, CA). LY294002, and PD98059

were obtained from Sigma (St. Louis, MO) and New England Biolabs (Beverly, MA).

Antibodies for cyclin D1 (sc-8396), p85a (sc-423) and p110gamma (H-119) were from

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies for HA (#2363), Akt

(#9272), phospho-Akt-S473 and T308 (#9271 and #9275), phospho-4EBP1-Ser65

(#9451) and 4EBP1 (#9452), Myc (#2276), S6 ribosomal protein (#2212) and phospho-

S6 ribosomal protein-Ser235/236 (#2211), and p44/p42 MAP Kinase (#9102) and

phospho-p44/42 MAP Kinase-Thr202/Tyr204 (#9101) were from Cell Signaling

Technology (Beverly, MA). Mouse anti-human TSHR (MCA1571) was from Serotec

(Kidlington, Oxford) and anti-FLAG antibody (#200472) was from Stratagene (La

Jolla,CA). All other materials, including 8-bromo-cAMP(8-Br-cAMP), N-α-tosyl-L-

phenylalanyl chloromethyl ketone (TPCK) were purchased from Sigma (St. Louis,

MO).


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Preparation of IgG

The control sera were obtained from 5 normal individuals who had no history, 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 RIA, testing positive for TBII (TSH binding inhibitory

immunoglobulins), and a diffuse goiter with increased 99mTcO4- uptake at scintiscan.

IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B

columns; IgG was lyophilized and stored at -20 ℃ 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

mU/mL crude bovine TSH (Sigma, St. Louis, MO) stimulated thymidine incorporation

into DNA by at least 10-fold. The doubling time of the cells with TSH was 36 ± 6 hours;

without TSH, and they did not proliferate. The cells used were diploid and between their


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5th and 20th passage. The cells were grown in 6H medium consisting of Coon's modified

F12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture

of six hormones: bovine TSH (1 mU/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, PDK1 cDNAs were introduced into the FRTL-5 and

CHO cells using the LipofectAMINE Plus reagents according to the manufacturer

instructions (Life Technologies, Inc.).

PI3K Assay

The cell extracts obtained from the FRTL-5 cells were immunoprecipitated with anti-

TSHR monoclonal antibodies (clone 4C1, Serotec, Oxford) and the control IgG

antibodies. The samples were washed twice with 1% Nonidet P-40 and 1 mM sodium


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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 PI/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 thin-layer chromatography (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


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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), 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), and 10 µg each of aprotinin

and leupeptin per milliliter. The total protein lysates were denatured by boiling in a

Laemmli sample buffer, resolved on 7.5% - 15% sodium dodecyl sulfate-

polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The

membranes were blocked in PBS containing 5% (wt/vol) 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, MA)

[3H]-Thymidine Incorporation Assay


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Confluent FRTL-5 thyroid cells in 100 mm dishes were detached by trypsinization,

resuspended in 6H growth medium, seeded at a density of 3 x 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 7days. TSH and/or wortmannin, LY294002,

H89, rapamycin were added to the quiescent cells, which were then incubated for 24 h,

followed by the addition of 2 µCi/mL [3H]thymidine (NEN Life Science Products,

Boston, MA) 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 (4).

Briefly, the cells were washed with 1x phosphate-buffered saline, pH 7.4, and then fixed

with ice-cold 70% ethanol. The samples were washed with 1x phosphate-buffered saline


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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 Becton

Dickinson fluorescence-activated cell analyzer and Cell Quest version 1.2 software

(Becton Dickinson Immunocytometry Systems, Mansfield, MA). 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 (Life Technologies, Inc.,

Gaithersburg, MD). 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-


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agarose (Santa Cruz). 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, 10 mM MnCl2, 1 mM 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; NEN Life Science Products) 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 wks. Rapamycin (Calbiochem, La Jolla, CA) was delivered once daily by an

intraperitoneal injection at a dose of 1.5 mg/kg dissolved in 2% carboxymethylcellulose


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for one 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

(X200) was counted. Further sections were used for immunohistochemistry (IHC). 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, Inc. USA), was diluted (1:200) with a background-reducing diluent (Dako,

Carpinteria, CA) and incubated for 60 minutes. The sections were then incubated with a

rabbit EnVision-HRP detection system reagent (Dako, Carpinteria, CA) for 30 minutes.

The sections were then sequentially incubated with DAB (3,3-diaminobenzidine)

(DAKO) chromogen for 5 minutes, counterstained with Meyer’s hematoxylin, and

mounted. Careful rinses with several changes of TBS-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


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The protein concentration was determined by the Bradford method (Bio-Rad, Hercules,

CA) using recrystallized bovine serum albumin as a standard. The sera were stored -70

oC until immunoglobulin G (IgG) preparation. The IgGs were extracted by affinity

chromatography using protein A-sepharose CL-4B columns (Amersham Pharmacia,

Piscataway, NJ) 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 ± SE of these experiments. Statistical significance was determined by a two-

way analysis of variance.


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Results

Association 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. 1A, lane 1; Fig. 1B, lane 1). Both TSH and 8-Br-

cAMP stimulated the TSHR-associated PI3K activity (Fig. 1B, lane 2, 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, lane 3, 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) (30, 31) and immunoprecipitation

was carried out using anti-TSHR (Fig. 1C, lane 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 vs 3) and was inhibited by 100 nM wortmannin (Fig. 1C, lane 4).


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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).

The following experiments examined the association between TSHR and the

regulatory subunit of PI3K. Human TSHR and HA-tagged p85α were co-expressed in

CHO cells. TSHR was immunoprecipitated and the immune complexes were analyzed

with anti-HA antibodies (Fig. 2B). The TSHR immunoprecipitates from the CHO-

TSHR cells included p85α (Fig. 2B lane 4). TSHR Immunoprecipitates from FRTL-5

cells treated with TSH and p85α (Fig. 2C, lane 2, and 3). The cell extracts were also

immunoprecipitated with control IgG; the results show that the interaction between

TSHR and HA-p85α is specific (Fig. 2C, lanes C vs T).

The immune complexes precipitated with the anti-TSHR antibodies were also


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probed with anti-phosphotyrosine antibodies (4G10). In untreated cells, the TSHR had a

low level of tyrosine phosphorylation. In contrast, the TSHR from the cells treated with

TSH or 8-Br-cAMP had a significantly higher phosphotyrosine content (Fig.2D, lanes 1

to 4). Endogenous p85α was detected in the TSHR immunoprecipitates from the TSH-

or 8-Br-cAMP treated FRTL-5 thyroid cells.

These findings suggest that 1) the TSH receptor is specifically associated with the

p85α regulatory subunit of PI3K in the thyroid cells, and the TSHR-transfected CHO

cells. 2) The association between the endogenous p85α and TSHR depends on the

TSH/8-Br-cAMP treatment and on tyrosine phosphorylation status of TSHR.


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PDK1 subcellular localization and tyrosine phosphorylation - When PI3K is activated

by growth factors, several events follow. These include the increased synthesis of

PtdIns-3,4,5-P3 and PtdIns-3,4-P2, activation of PDK1, and translocation of PDK1 and

Akt/PKB to the plasma membrane (32, 33). Therefore, the localization of PDK1 was

examined in the TSH-treated cells expressing GFP-tagged PDK1. In the cells treated

with TSH or 8-Br-cAMP, PDK1 rapidly redistributes to the plasma membrane and the

perinuclear region. This redistribution was inhibited by wortmannin, LY294002, and

H89 (Fig. 3A). In the control cells, GFP fluorescence was evenly distributed within the

cytoplasm.

Because tyrosine phosphorylation is associated with the activation of PDK1 (14),

the TSH-treated cells were examined for their the phosphorylation state of PDK1 . The

Myc-tagged PDK1 and TSHR were expressed in CHO cells and tyrosine

phosphorylation was analyzed using anti-phosphotyrosine antibodies (4G10). Myc-

PDK1 was constitutively tyrosine phosphorylated (Fig. 3B) in the TSH or 8-Br-cAMP

treated or untreated cells. Therefore, in the CHO cells, the tyrosine phosphorylation of

PDK1 was not correlated with the cellular localization or TSH treatment.

Akt/PKB and S6K1 activities in TSH-treated cells – Akt/PKB and S6K1 are substrates

of PDK1, which are important downstream effectors of PI3K/PDK1 signaling.

Therefore, Akt/PKB and S6K1 phosphorylation were examined in the TSH-treated


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thyroid cells. Previous studies show that Akt/PKB is phosphorylated on T308 and S473

by PDK1 and PDK2, respectively, in insulin-treated cells. Insulin-treatment had similar

effects on the thyroid cells (Fig. 4B and data not shown). However, TSH treatment did

not induce Akt/PKB T308 and S473 phosphorylation in the thyroid cells (Fig. 4A). This

result was confirmed by comparing the phosphorylation of 4EBP-1/PHAS-1, which is

mediated by Akt/PKB, in the insulin- and TSH-treated thyroid cells. Insulin rapidly

stimulated the phosphorylation of S65 of 4EBP-1/PHAS-1 but TSH did not (Fig. 4D

and 4C). Rapamycin, an inhibitor of FRAP/mTOR, inhibited the phosphorylation of

4EBP-1/PHAS-1 in the insulin-treated thyroid cells (Fig 4D).

S6K1 has two protein subunits, p70 and p85, both of which are phosphorylated

in insulin-treated cells. Figure 5 shows that TSH induces the rapid phosphorylation of

p70 and p85, as detected by the slow-migrating hyperphosphorylated protein species on

a Western blot. The appearance of these species correlated with the increased S6 kinase

activity in the immunoprecipites (Figs. 5A and 5B). The phosphorylation of p70 and

p85 increases with increasing TSH (1 mU/ml) or insulin (10 µg/ml) exposure time (Fig.

5A) (Fig. 5C). After 30 min exposure to TSH, the S6K1 activity reached a maximum,

increasing 3.5-fold (Fig. 5B). The insulin-induced phosphorylation of S6K1 (Fig. 5C)

reached a maximum 15 min after treatment (Fig. 5D). These experiments suggest that

TSH activates S6K1 but not Akt/PKB; in contrast, insulin activates Akt/PKB and S6K1.


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Roles of PI3K, PDK1 and PKA in activating S6K1 in TSH-treated cells - The

above results suggest that TSH may stimulate a response in thyroid cells, which

involves TSHR, PI3K, PDK1 and S6K1. The activities upstream of S6K1 were

identified using the inhibitors of PI3K (wortmannin and LY294002) PKA (H89),

FRAP/mTOR (rapamycin) and PDK1 (N-α-tosyl-L-phenylalanyl chloromethyl ketone;

TPCK). LY294002 (500 nM) and wortmannin (300 nM) completely inhibited the TSH-

induced phosphorylation of S6K1 (Figs. 6A and 6B). LY294002 and wortmannin began

to inhibit S6K1 phosphorylation at 500 nM and 100 nM, respectively (Fig. 6A, lane 3;

Fig. 6B, lane 4). Forskolin (10 µM) or 8-Br-cAMP (1 mM) rapidly induced the

phosphorylation of S6K1, and this effect was inhibited by LY294002 (500 nM) and

wortmannin (100 nM) or H89 (Figs. 6D and 6E). The inhibitory effect of H89 was dose-

dependent (Fig. 6F). H89 began to inhibit TSH-induced phosphorylation of S6K1 at 5

µM and completely inhibited S6K1 (and CREB; data not shown) phosphorylation at 50

µM. The PDK-1 inhibitor TPCK also inhibited the TSH-induced phosphorylation of

p70S6K1 (Figs. 7A and 7B) at 20-50 µM and inhibited the phosphorylation of the

ribosomal protein, S6, in a dose-dependent manner (Fig. 7A).

The role of PI3K in TSH-stimulated activation of S6K1 was also examined using

the cells expressing a dominant negative form of HA-tagged-p85α (cell line L5-

∆iSH2p85 expressing pCMV-∆iSH2p85-HA). The cells expressing the wild type p85α


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(cell line L5-WTp85 expressing pCMV-p85-HA) were used as the control (Fig. 6C) (35).

The phosphorylation of S6K1 was measured in the presence and absence of TSH. In the

cells expressing the wild type p85, S6K1 was rapidly phosphorylated in response to

TSH, but S6K1 phosphorylation was strongly reduced in the cells expressing the

dominant negative p85 (Fig. 6C). Similar results were observed in the cells treated

with forskolin or 8-Br-cAMP (data not shown).

Roles of MAPK and FRAP/mTOR in activating S6K1 in TSH-treated cells –

S6K1 is regulated by phosphorylation and has at least eight phosphorylation sites.

PDK1, Akt/PKB, NEK6/7, FRAP/mTOR and MAP kinases act upstream of S6K1. In

vitro, S6K1 is phosphorylated by the MAP kinases (34) and in human thyroid cells,

TSH stimulates the MAP kinase cascade by a cAMP-independent pathway (35). The

thyroid cells were cultured in the presence or absence of TSH and Erk1 and Erk2

phosphorylation were examined. Insulin (10 µg/ml) induced Erk1 and Erk2

phosphorylation at threonine 202 and tyrosine 204 after 15 min (Fig. 8A). In contrast,

TSH did not induce Erk1 and Erk2 phosphorylation until 60 min after treatment (Fig.

8B). In the cells cultured in the absence of TSH, insulin and serum, S6K1 was not

phosphorylated but Erk1/Erk2 were constitutively phosphorylated (Fig. 8). The TSH-

induced S6K1 phosphorylation was not inhibited by a pretreatment with a MEK

inhibitor, PD98059 (Fig. 8C, lanes 2 and 4). These results suggest that the MAP kinase


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pathway does not play a role in the TSH-stimulated phosphorylation of S6K1 in thyroid

cells.

Rapamycin completely inhibited the S6K1 phosphorylation in the cells treated

with TSH (Fig 8C), forskolin or 8-Br-cAMP (Fig. 6D, 6E), suggesting a role for

FRAP/mTOR in the response to TSH in thyroid cells.

Regulation of S6K1 by TSH receptor autoantibodiess – There are two types of

TSH receptor autoantibodies: the stimulating (TSAb) antibodies, which are agonists of

the TSH receptor and associated with Graves’ disease, goiter, and hyperthyroidism; and

the blocking (TSBAb) antibodies, which are antagonists of the TSH receptor and are

associated with primary myxedema, thyroid atrophy, and hypothyroidism. The influence

of the stimulating and blocking TSH receptor autoantibodies on S6K1 phosphorylation

was examined in thyroid cells. Normal pooled IgG (NP) was obtained from normal

adults with no known thyroid disease and were used as the controls. Autoantibodies

from patients with Graves’ disease (GD) significantly enhanced phosphorylation of

S6K1, but the control IgG (NP) did not (Fig. 9A). IgG from 10 patients with Graves’

disease were tested in a radioreceptor assay for TSH-binding inhibitory immunoglobulin

(TBII). Seven patients with TBII-positive Graves’ disease stimulated S6K1

phosphorylation and the ribosomal protein, S6 (Fig. 9A). This activity was completely

inhibited by LY294002 and wortmannin, but not by PD98059 (Fig. 9B). TSBAb IgG


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from patients with primary myxedema (PM) did not induce S6K1 phosphorylation (Fig.

9A, lane PM). TSBAb IgG did not inhibit the insulin-induced phosphorylation of S6K1,

but did inhibit the TSH/TSAb-induced phosphorylation of S6K1 in thyroid cells (Fig.

9C, lane 4, 5, 6).

Effects of S6K1 signaling pathways on thyrocyte proliferation - In thyroid cells,

the cellular proliferation is regulated by TSH, insulin, and other growth factors (36).

The role of signaling kinases in TSH-stimulated proliferation was examined by

measuring the level of DNA synthesis in the presence of wortmannin, LY294002,

rapamycin, or H89. Thyroid cells were cultured in the absence of TSH for 7 days and

then stimulated with TSH in the absence or presence of the kinase inhibitor and 3H-

thymidine. The incorporation of 3H-thymidine increased 17-fold in the cells exposed to

TSH (Fig. 10A). Rapamycin (20 nM), wortmannin (100 nM), LY294002 (500 nM), and

H89 (50 µM) completely inhibited TSH-stimulated DNA synthesis.

TSH regulates the expression of cyclins D and E, which are rate limiting for entry

into the S phase. Cyclin D1 is expressed at a low basal level in the absence of TSH. The

expression of cyclin D1 increases approximately 12 h after exposure to TSH and

decreases to the basal level after 24 h post-exposure (Fig. 10B). Rapamycin inhibits

TSH-induced cyclin D1 expression and does not change the basal level of cyclin D1

(Fig. 10C, lanes 1 and 2). This result is consistent with previous observations showing


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that rapamycin inhibits the G1/S transition and prolongs G1 in several types of cells (37,

38).

The cell cycle distribution was also examined in TSH/insulin-treated thyroid cells in the

presence and absence of rapamycin. The cells maintained in 3H medium with 5% calf

serum (3H5%), which lacks insulin, TSH and somatostatin, were quiescent (G1, 82.9±

5%; S, 1.88± 0.7%) (Fig. 10D). The TSH/insulin treatment promoted cell proliferation

(G1, 49.3 ± 5%; S, 19.5 ± 0.9%) (Fig. 10D). Rapamycin partially inhibited this change

in the cell cycle distribution (G1, 72.6 ± 0.3%) (Fig. 10D).

The phosphorylation of S6K1 was also examined in the experiment shown in

Figure 10. TSH stimulated a lower level of S6K1 phosphorylation than the TSH/insulin

(Fig. 10D lane 4), and rapamycin completely inhibited S6K1 phosphorylation in the

presence of TSH or TSH/insulin (lanes 3 or 5).

Regulation of thyroid follicle activity by rapamycin in vivo. The above results

suggest that rapamycin-sensitive S6K1 signaling plays a major role in the TSH-

stimulated thyrocyte proliferation. The following experiments test the in vivo role of

FRAP/mTOR and S6K1 in the TSH-stimulated thyroid follicles. The in vivo

experimental system involved feeding male Sprague-Dawley rats water containing

0.025% methimazole (MMI); this protocol provides a long-term TSH-stimulation of the

thyroid follicles. In the MMI-treated rats, the thyroid gland showed a prominent dark


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red swelling after 2 weeks of treatment. The thyroid follicle histology revealed the

characteristic findings of long-term TSH stimulation. These include the depletion of

colloids, small hyperplastic collapsed follicles, hypertrophic columnar lining cells, and

crowded hyperchromatic nuclei (Fig. 11A 2nd). The histology was similar in rats

receiving intraperitoneal injections of 2% carboxymethylcellulose for 1 week. However,

the histology was altered in the thyroid glands of the MMI-treated animals administered

2% carboxymethylcellulose containing rapamycin (1.5 mg/kg) once daily. In these

animals, colloid-filled follicles were more abundant (Fig. 11A 4th and B).

The S6K1 kinase activity was estimated in these rats by measuring the quantity of

phosphorylated ribosomal protein S6. In the untreated rats, phosphorylated S6 was not

present in the thyroid follicular cells but was abundant in the parafollicular cells (Fig.

11C Lt). In the MMI-treated rats, a significantly higher percentage of thyroid cells

possessed the phosphorylated S6 protein, (Fig. 11C, Middle) and rapamycin completely

inhibited S6 phosphorylation in the thyroid cells (Fig. 11C, Rt). These observations

suggest that rapamycin-sensitive S6K1 signaling plays a role in the TSH-stimulated

follicles in vivo.


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Discussion

The mechanisms of cellular proliferation in the thyroid gland may be relevant to

human thyroid diseases including benign thyroid nodules, differentiated thyroid cancer,

and Graves’ disease (39, 40). Thyrocyte proliferation is regulated by TSH, which is one

of the most important factors regulating the proliferation and function of thyroid cells,

and by other hormones and growth factors that activate the specific signaling pathways

(41). Most studies of thyrocyte proliferation used the rat thyroid cell lines FRTL-5,

WRT, and PC C13 because these cell lines are responsive to TSH (36). TSH activates

TSHR, which activates adenylyl cyclase, elevates cAMP and activates PKA. However,

TSH regulates the thyroid function by activating a complex signaling network.

This study presents evidence showing that PI3K activates S6K1 in response to

TSH in the proliferating thyroid cells (Fig. 12). TSHR interacts with the p85α

regulatory subunit of PI3K and has inducible tyrosine phosphorylation sites (Fig. 2D). It

is possible that a phosphotyrosine residue in the intracellular loop or C-terminal tail of

TSHR interacts with the SH2 domain of p85α. Tyrosine phosphorylation of TSHR is

stimulated by exogenous 8-Br-cAMP and might involve a cAMP-dependent tyrosine

kinase (Fig. 2D). The tyrosine kinase inhibitor genistein inhibits the tyrosine

phosphorylation of TSHR in the cells treated with TSH/8-Br-cAMP and inhibits the

association between PI3K and TSHR (data not shown). These observations suggest that


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TSH/cAMP stimulates the tyrosine phosphorylation of TSHR, which may stimulate the

TSHR-dependent activation of PI3K in thyroid cells. A dominant negative p85α, which

lacks the iSH2 region that binds the p110 catalytic domain of PI3K, strongly inhibits the

TSH-stimulated phosphorylation of S6K1. This suggests that a class I PI3K may be

involved in TSH signaling. To date, there is no evidence showing that the adaptor

molecules Shc, Grb2 and Gab2 facilitate the interaction between TSHR and PI3K,

although these molecules bridge the interactions between other the receptors and PI3K

(42).

This study suggests that PKA may be required for the TSH/cAMP-stimulated

phosphorylation of S6K1 in thyroid cells. LY294002 and wortmannin did not affect the

TSH-stimulated phosphorylation of CREB (data not shown), suggesting that PI3K acts

downstream of PKA. However, H89 inhibits the 8-Br-cAMP/TSH-induced association

between TSHR and PI3K. Therefore, cAMP/PKA may stimulate the formation of an

active TSHR-PI3K complex or modify the subunit composition of PI3K. Recent studies

show that cAMP/PKA phosphorylates p85α and stabilizes the p85α-p110 complex (8,

49). Further investigation is needed to understand the molecular interactions between

TSHR, PI3K and cAMP/PKA in thyroid cells.

cAMP-GEFs play a role in the PKA-independent effects of TSH and FSH in

their target cells (1, 50). The identification of novel cAMP-GEFs (Epacs) raises the


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possibility that cAMP may activate the small GTPases such as Rap1a, Rap2, and

possibly Ras (51, 52). These GTPases activate the downstream signaling kinases

including PI3K (53). TSH activates Rap1 in WRT thyroid cells (7), which induces

Akt/PKB phosphorylation but not S6K1 (7).

This study suggests that PDK1 plays a role in the TSH-stimulated activation of

S6K1 (Fig. 3 and 7). PDK1 is regulated primarily by the substrate conformation,

cellular localization, and tyrosine phosphorylation (13, 14). The PH domain of PDK-1

selectively binds PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (39). PDK1 redistributes from the

cytosol to the plasma membrane and perinuclear region in the TSH-treated cells. This

process is inhibited in the presence of the PI3K inhibitors and might involve the PI3K

lipids since it requires the PH domain of PDK1. PDK1 phosphorylation may also

regulate its activity (14, 43), although TSH did not stimulate the tyrosine

phosphorylation of PDK1 in the thyroid cells overexpressing PDK1 (data not shown) or

in the CHO cells overexpressing TSHR (Fig 3B). S6K1 (p70 and p85) and Akt/PKB are

well known targets of PDK1 in the growth factor signaling pathways (21, 44). It is not

yet understood why TSH does not induce the Thr308 or Ser473 phosphorylated forms of

Akt/PKB (Fig. 4, Fig. 12), but does induce S6K1 phosphorylation. Protein-protein

interactions or structural conformation may influence this specificity. TSH and insulin

also have different effects on these signaling pathways, which may explain why the


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thyroid cells require both TSH and insulin for cellular proliferation.

4E-BP1 (also known as PHAS-1) normally binds eIF4E, initiating cap-

dependent phosphorylation (45). The hyperphosphorylation of 4E-BP1 disrupts this

binding, activating cap-dependent translation. The PI3K/Akt pathway and FRAP/mTOR

kinase regulate 4E-BP1 (46). 4E-BP1 is phosphorylated in vivo on multiple residues.

Phosphorylation by FRAP/mTOR on Thr37 and Thr46 of 4E-BP1 may prime it for the

subsequent phosphorylation sites at Ser65 and Thr70 (47). Insulin induces the

phosphorylation of Ser65 of 4E-BP1 and this phosphorylation is inhibited by rapamycin.

This suggests that insulin induces FRAP/mTOR, which phosphorylates the Ser65 of 4E-

BP1. In contrast, TSH did not induce the phosphorylation of Ser65 of 4E-BP1.

Akt/PKB may also be required for the phosphorylation and activation of 4E-BP1, and

TSH does not activate Akt/PKB, but insulin does. These findings suggest that

FRAP/mTOR is a required downstream effector of Akt/PKB, which phosphorylates 4E-

BP1 (47) .

cAMP exerts different effects on PI3K signaling in the different cell types

(9,38). A recent study on WRT thyroid cells showed that cAMP stimulates S6K1

through a PKA-dependent, PI3K-independent pathway (6). This conclusion was based

on the observation that cAMP-stimulates the phosphorylation of the S6 protein was

repressed by H89 (50 µM) but not by low-dose wortmannin or by a microinjection of


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the N-terminal SH2 domain of the p85 regulatory subunit of PI3K. Cohen et al reported

that H89 (10 µM) is equally effective in inhibiting MSK1, S6K1, ROCK-II and PKA.

Therefore, H89 might inhibit S6K1 directly. However, in this study, H89 inhibited the

association between TSHR and PI3K and the activation of PI3K (Fig. 1), suggesting

that PKA plays a role in the TSHR-stimulated activation of PI3K in thyroid cells.

In contrast to the above results in the WRT thyroid cells, this study shows that

S6K1 activation by cAMP is dependent on PI3K in the FRTL-5 thyroid cells.

Wortmannin and LY294002 are structurally unrelated molecules that, at low

concentrations, are relatively specific, cell-permeable inhibitors of PI3K. Wortmannin

may directly inhibit the FRAP/mTOR autokinase activity at an IC50 approximately 100-

fold higher than its IC50 for inhibiting PI3K (~200 nM in vitro, ~300 nM in vivo).

LY294002 inhibits FRAP/mTOR autokinase activity in vitro with an IC50 of 5 µM (32).

LY294002 and wortmannin inhibit the TSH-stimulated phosphorylation of S6K1 at 500

nM and 100 nM, respectively, which suggests the involvement of PI3K (Fig. 6).

FRAP/mTOR regulates the protein translation by two independent mechanisms

involving the direct or indirect activation of S6K1 and the inactivation of the

translational repressor PHAS-1/4E-BP1. Rapamycin completely inhibits the

phosphorylation of S6K1 induced by TSH, 8-Br-cAMP, forskolin, and insulin in FRTL-

5 thyroid cells (Fig. 6). The mechanisms by which TSH regulates FRAP/mTOR kinase


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is not known. However, FRAP/mTOR senses the cellular ATP level (27) and

mitochondrial dysfunction (54), and 2-deoxyglucose (100mM), which is an inhibitor of

the glycolytic pathway, partially inhibits S6K1 phosphorylation in the TSH-treated cells

(data not shown). Rapamycin may inhibit the S6K1 phosphorylation by activating the

phosphatases in the TSH-treated cells (55). The TSH-induced changes in the cellular

ATP level might affect the TSH -induced phosphorylation of S6K1 in thyroid cells.

These findings suggest that FRAP/mTOR kinase may not act downstream of

PI3K/PDK1 in TSH-treated cells. It is possible that FRAP/mTOR senses the nutrient

signals that change in the TSH-treated cells.

Different thyrocyte cell systems have different requirements for cell

proliferation (i.e., TSH, insulin/IGF-1, serum). TSH and insulin stimulate the

proliferation by mechanisms, which are not completely clear. In dog thyroid cells,

cAMP activates S6K1 but does not promote DNA synthesis (56). TSH also activates

S6K1, and increases the number of cells in the S phase slightly, but does not

significantly increase the number of cells in G2 (Fig. 10). In contrast, TSH/insulin

markedly increases the number of cells in S and G2 and TSH/insulin stimulates the

synergistic prolonged activation of S6K1 (Fig. 10D lane 4). Rapamycin inhibits the

TSH and TSH/insulin-induced cell cycling, suggesting a role for the S6K1 pathways.

However, rapamycin does not inhibit the activation of Akt/PKB in the insulin-treated


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thyroid cells (data not shown), suggesting that the synergistic activation of the S6K1

signaling pathways by TSH/insulin may stimulate the cell cycle progression in FRTL-5

thyroid cells.

Saito et al (57) reported that Akt/PKB was not phosphorylated in the TSH-

treated FRTL-5 thyroid cells. A constitutively active Akt/PKB also promotes the

hormone-independent proliferation in the PC C1 3 thyroid cells (58). These

observations suggest that Akt/PKB might play a role in thyrocyte proliferation.

However, the role of Akt/PKB in TSH and TSH/insulin-treated thyroid cells remains to

be clarified.

This study used Sprague-Dawley rats maintained on MMI-containing drinking

water as an in vivo experimental system to study the long-term stimulation of the

thyroid follicles. The MMI-treated rats had higher TSH levels in response to the

inadequate thyroid hormone production. The thyroid glands from the MMI-treated rats

have a follicular structure that suggests TSH stimulation, and the follicular cells have an

increased phosphorylation of the ribosomal protein S6 (Fig. 11C). These findings

suggest that the endogenous TSH stimulates S6K1 in the thyroid follicles. Follicular

changes induced by sustained TSH stimulation were partially reversed by the short-term

intraperitoneal delivery of rapamycin. This suggests that some of the effects of TSH on

the thyroid follicles are mediated by S6K1.


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This study also shows that the TSHR antibodies modulate the S6K1 activity in

thyroid cells. TSAb induces and TSBAb inhibits the phosphorylation of S6K1 and the

S6 protein. The TSAb-stimulated phosphorylation of S6K1 was inhibited by LY294002

and wortmannin but not by PD98059. This suggests that the TSAb-induced activation of

S6K1 requires PI3K. However, it was not possible to determine if the S6K1 and TSAb

activities correlated with each other in this study as a result of insufficient data.

Nevertheless, the TSAb-stimulated activation and TSBAb-stimulated inhibition of

S6K1 might be related to goiter and atrophy in Graves’ disease and primary myxedema,

respectively.


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Acknowledgements

The authors wish to acknowledge Dr. John Blenis for providing the polyclonal

S6K1 antibodies, Dr. Leonard D. Kohn for helpful discussion and critical review of the

manuscript, and Yong Mi Kang for technical assistance.

Footnotes

This work was supported by National Research Laboratory Program (M1-

0104-00-0014) and KOSEF Research Grant (2000-2-20500-007-3) Ministry of Science

and Technology, Korea

Abbreviations

The abbreviations used are: CHO, Chinese hamster ovary; cAMP, cyclic adenosine

monophosphaste; CREB, cAMP respose element binding protein; 4EBP, eIF-4E binding

protein; MAPK, mitogen activated kinase; MMI, methimazole; mTOR, mammalian

target of rapamycin; PDK1, 3’-phosphoinositide dependent kinase; PI3K,

phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; SDS-

PAGE, sodium dodecylsulfate-polyacrylamide gel; S6K1, ribosomal S6 kinase 1;TSAb;

stimulating-type TSH receptor antibody; TSBAb, blocking-type TSH receptor

antibody;TSH, thyroid stimulating hormone;


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Figure Legends

Figure 1. A and B. TSHR-associated PI3K activity in thyroid cells stimulated with

TSH and cAMP. FRTL-5 thyroid cells which were maintained in 4H without serum

condition for 48hr and treated with TSH (1 mU/ml) or 8-Br-cAMP (1mM) for 10 min.

H89 was pretreated for 45min before TSH or 8-Br-cAMP treatment. TSH receptors

were immunoprecipitated with a monoclonal anti-TSH receptor antibody and the

immune complexes were subjected to PI3K lipid kinase assays. Wortmannin and

LY294002 were added in the immune complexes for 10 min. Phosphorylated lipids

were separated by TLC, detected by autoradiography, and quantified with

phosphorimager analyses. Immunoblot analysis showed that similar amounts of human

TSH receptor were used in the assays. W: wortmannin (100 nM), L: LY294002 (500

nM) in vitro, H89 (50 µM) in vivo. C. TSHR associated PI3K activity in CHO and

CHO-TSHR. The activity of TSHR-associated PI3K was measured as above (A, B).

Cell lysates from CHO and CHO-TSHR treated with TSH for 5 min were

immunoprecipitated with anti-TSHR antibody and control IgG for negative control.

Figure 2. A. The expression of p85α regulatory subunit and p110γ catalytic

subunit of class I PI3K isoforms in FRTL5. FRTL-5 cells were grown to 70%


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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 12h with TSH (1 mU/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, C and 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 p85α

(HA- p85α) and pcDNA3-hTSHR and after 24 hr incubation, cells were harvested and

immunoprecipitated with an anti-TSHR antibody (B). FRTL5 cells transfected with

HA-tagged p85α (HA- p85α) and were treated with TSH for 5 min (C) and TSH and 8-

Br-cAMP were treated for 5min in FRTL-5 thyroid cells (D). Cells were

immunoprecipitated with an anti-TSHR antibody. The amount of total protein was

measured as described in ‘Materials and Method’. Immunoprecipitates were separated

with SDS-PAGE on a 10% gel and immunoblotted with anti-HA (B, C), anti-

phosphotyrosine antibodies ( D) and anti-p85α (D).

Figure 3. A Subcellular localization of PDK-1 in TSH-treated thyroid cells. FRTL-5

cells were grown on coverslips in 6-well plates and transfected with pEGFP-PDK-1.

Cells were maintained without TSH, insulin and serum after 50% confluence and in


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48hr after transfection, cells were treated with TSH or 8-Br-cAMP for 5 min or treated

with wortmannin (W), LY294002 (L), and H89 for 45 min and further stimulated with

TSH for 5 min as indicated. Fixed cells were examined under a laser-scanning confocal

microscope. The results shown are representative of three independent experiments. B.

Effects of TSH and 8-Br-cAMP on tyrosine phosphorylation of PDK1. CHO-TSHR

cells were transfected with pCDNA3-PDK-1-Myc with lipofection and the cells treated

with TSH and 8-Br-cAMP for 15 min after 24h of transfection. PDK1-Myc was

immunoprecipitated with anti-Myc antibodies and its tyrosine phosphorylation was

analyzed with anti-phosphotyrosine antibody (4G10).

Figure 4. Effects of TSH and insulin on phosphorylation of Akt/PKB and 4E-

BP1/PHAS1 in FRTL-5 thyroid cells. 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 with TSH (1 mU/ml) (A, C), insulin (10 µg/ml) (B, D) or insulin (10

µg/ml) and rapamycin (20 nM) and analyzed by Western blot using anti-Akt antibodies

(total and phosphospecific) and anti-4E-BP1 antibodies (total and phosphospecific).

Figure 5. Effects of TSH and insulin on the phosphorylation and activation of


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S6K1 in FRTL-5 thyroid cells. 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 with TSH (1 mU/ml) (A) or insulin (10 µg/ml) (C) and analyzed by

Western blot using a polyclonal anti-S6K1 antibody and an anti-phospho-S6 ribosomal

protein (Ser235/236) antibody. To measure S6K1 activity, cells were transfected with

HA-tagged p70 S6K1 and immunoprecipitated using an anti-HA monoclonal antibody.

Phosphotransferase activity of S6K1 was assayed using recombinant S6 peptide as a

substrate (B, D). The values represent the mean ± standard errors of assays carried out

with three independent cell preparations.

Figure 6. A and B. Effects of PI3K inhibitors on TSH-induced phosphorylation of

S6K1 in FRTL-5 thyroid cells. FRTL-5 cells were prepared as described in the legend

to Figure 3. Total cell lysates were prepared from cells treated with TSH (45 min) and/or

LY294002 and wortmannin and analyzed by Western blot using a polyclonal S6K1

antibody. LY294002 and wortmannin were pretreated for 45 min before TSH addition.

C. Effects of p85α lacking a binding site (iSH2) for the p110 catalytic subunit of

PI3K on the phosphorylation of S6K1 in TSH-treated thyroid cells. FRTL-5 thyroid

cell lines were transfected with constructs that express mutant HA tagged-p85α


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deficient in binding of p110 catalytic subunit of PI3K (L5-∆iSH2p85) or wild-type

p85α (L5-Wp85). Total cell lysates were prepared from cells treated with TSH and

analyzed by Western blot using a polyclonal S6K1 antibody. D and E. Effects of

forskolin and 8-Br-cAMP on the phosphorylation of S6K1 in FRTL-5 thyroid cells.

Total cell lysates were prepared from cells pretreated with rapamycin, wortmannin,

LY294002 or H89 for 45 min and then treated with forskolin (10 µM) or 8-Br-cAMP (1

mM) and analyzed by Western blot using a polyclonal S6K1 antibody. F. Dose-

dependent effects of H89 on TSH-induced phosphorylation of S6K1 in FRTL-5

thyroid cells. Total cell lysates were prepared from cells treated with TSH and/or H89

and analyzed by Western blot using a polyclonal S6K1 antibody. R: rapamycin (20 nM),

W: wortmannin (100 nM), L: LY294002 (500 nM), H89 (50 µM)

Fig. 7. A and B. Effects of N-α-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

on TSH-induced phosphorylation and activation of S6K1. FRTL-5 cells were

pretreated with TPCK for 45min, and treated with TSH for 30 min before total cell

lysates were prepared and analyzed by Western blot using a polyclonal S6K1 antibody

(A). S6K1 activity was measured as described in the legend to Figure 5 (B).

Figure 8. A and B. Effects of TSH on phosphorylation of Erk1/Erk2 in FRTL-5


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thyroid cells. 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 with

TSH (1 mU/ml) or insulin (10 µg/ml) and analyzed by Western blot using

phosphospecific and total anti-Erk1/Erk2 antibodies. C. Effects of rapamycin and

PD98059 on TSH-induced phosphorylation of S6K1 in FRTL-5 thyroid cells.

FRTL-5 thyroid cells were cultured as described above. Total cell lysates were prepared

from cells treated with TSH (1 mU/ml) for 45 min and pretreated with rapamycin (20

nM) or PD98059 for 45 min before TSH treatment and analyzed by Western blot using a

polyclonal S6K1 antibody.

Fig. 9. Effects of stimulating (TSAb) and blocking (TSBAb) TSH receptor

antibodies on phosphorylation of S6K1 in thyroid cells. (A) 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. The IgGs

were extracted by affinity chromatography from normal pooled sera (NP), as well as the

sera from the patients with primary myxedema (PM) and Graves’ disease (GD) who

tested positive to the TSH binding inhibitory immunoglobulins (TBII) test. The total

cell lysates were prepared from the cells treated with the IgGs (10 mg/ml) and analyzed


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by Western blot using polyclonal anti-S6K1 antibodies and an anti-phospho-S6

ribosomal protein (Ser235/236) antibodies. CON; treated with the medium. (B) The

total cell lysates were prepared from the cells treated with TSH (1 mU/ml) or the IgGs

from the patients with Graves’ disease with or without the inhibitors as indicated. The

PI3K inhibitors, wortmannin (W) and LY294002 (L) in addition to the MEK inhibitor,

PD98059 (PD) were preincubated for 45 min and then either TSH or IgG. Which

stimulate the TSH receptor antibody, TSAb, obtained from Graves’ disease patients

were added to the medium for 45 min. (C) The IgG (blocking type TSH receptor

antibody, TSBAb) obtained from the primary myxedema were preincubated for 1h,

which was followed by the addition of insulin (10 µg/ml), TSAb (10 mg/ml) and TSH

(1 mU/ml) for 45 min. The total cell lysates were prepared and analyzed by Western blot

using a polyclonal anti-S6K1 antibodies.

Fig. 10. A. Effects of wortmannin, LY294002, rapamycin, and H89 on TSH-induced

[3H]-thymidine incorporation. FRTL-5 thyroid cells were cultured in medium lacking

TSH for 7days. 80% confluent cells grown in 96-well plates were treated with TSH

and/or wortmannin (0.1 µM), LY294002 (0.5 µM), rapamycin (20 nM), and H89 (50

µM) for 24 h, followed by the addition of 2 µCi/mL [3H]thymidine for an additional 2 h.

Cells were washed and precipitated with ice-cold 10% trichloroacetic acid.


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Radioactivity was determined by liquid scintillation spectrometry. 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. B and C. Effects of

TSH and rapamycin on the level of cyclin D1. 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. The medium was

replaced with fresh medium including TSH (1 mU/mL). Total cell lysates were prepared

and analyzed by Western blot using an anti-cyclin D1 antibody (B). Total cell lysates

were prepared from cells treated with TSH and/or 20 nM rapamycin for 12 h, and

analyzed by Western blot using anti-cyclin D1 and anti-actin antibodies (C). D. Effects

of rapamycin on TSH/insulin-induced cell-cycle progression and cellular

proliferation. Confluent FRTL-5 thyroid cells in 100 mm dishes were detached by

trypsinization, resuspended in 6H growth medium, seeded at a density of 3 x 104

cells/well in 6-well plates, and incubated for 2-3 days until 80% confluent. The medium

was changed to 3H medium with 5% calf serum and the cells were incubated for an

additional 7 days. TSH and/or insulin plus rapamycin were added to the quiescent cells

and incubated for 48h. Cell cycle analysis was performed using a Becton Dickinson

fluorescence-activated cell analyzer and Cell Quest version 1.2 software. At least 10,000

cells were analyzed per sample, and cell cycle distribution was quantified using the


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ModFit LT version 1.01 software. Representative patterns of cell cycle analysis are

presented. The values represent the mean ± SE of cell counts assays carried out with

three independent cell preparations.

Fig. 11. A and B. Effects of rapamycin on follicle activity in MMI-treated Sprague-

Dawley rats. Male Sprague-Dawley rats (120-130 g) were fed with water containing

0.025% methimazole for 2 wks. Rapamycin dissolved in 2% carboxymethylcellulose

was delivered once daily by intraperitoneal injection at a dose of 1.5 mg/kg for a week

before histologic examination. Cross-sections of the rat thyroid stained with H-E. The

number of colloid-filled follicles were counted in 2 fields at a magnification of x200. C.

Effects of rapamycin on thyroid S6 phosphorylation in MMI treated Sprague-

Dawley rats. Further sections were used for immunohistochemistry (IHC). The primary

antibody, rabbit anti-phospho-S6 ribosomal protein, was diluted (1:200) with a

background-reducing diluent (Dako, Carpinteria, CA) and incubated for 60 minutes,

followed by incubation in the EnVision-HRP reagent. The sections were then

sequentially incubated with DAB (3,3-diaminobenzidine) chromogen for 5 minutes,

counterstained with Meyer’s hematoxylin, and mounted. Careful rinses with several

changes of TBS-0.3% Tween buffer were performed between each step. A negative

control excluded the primary antibody. Cells with cytoplasmic granular staining were


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considered positive.

Fig. 12. Schematic of TSHR-mediated PI3K, PDK-1, and S6K1 activation

pathways. The TSHR-associated PI3K activity is increased by TSH and 8-Br-cAMP.

TSH is able to translocate PDK1 into the plasma membrane via PI3K and PKA

dependent manner and PDK-1 preferentially phosphorylates S6K1, not Akt/PKB.

However, insulin phosphorylates Akt/PKB and 4E-BP1/PHAS1 in a PI3K- and

FRAP/mTOR-dependent manner. Rapamycin inhibits the cooperative actions of 4E-

BP1/PHAS1 and S6K1 and results in the inhibition of TSH-mediated follicle

proliferation and activity. The solid lines represent the pathways preferentially activated

by TSH and the dashed lines represent pathways preferentially activated by insulin in

thyroid cells.


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origin

PIP2

1 2 3 4

TSH - + - -

IP: anti-TSHR

A

IB: anti-TSHR

W L

Figure 1

1 2 3 4 5IP: anti-TSHR

- - + - + H89 - + + - -- - - + +

TSH cAMPB

origin

PIP2

C CHO CHO-TSHR0 0 5 5 (W) 5 (min)TSH

PIP2

IP: anti-TSHR IgG IgG1 2 3 4 5

origin

by guest on January 16, 2020 http://www.jbc.org/ Downloaded from

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Figure 2

p85αp110γ

165105

7655

1 2 3 4 5 6

TSH - + - + - +

cAMP Insulin

A

M(kDa)

IgG

HA-p85α

TSHRHA-p85

- + - + - - + +

FRTL-5

CHO-TSHR1 2 3 4

B

0 5 15 5 5 min

IgG

FRTL-51 2 3 C T

CTSH

p85α

TSHR

TSH cAMP- + - +

FRTL-5

D

HA-p85α

TSHR

pY TSHR

1 2 3 4


http://www.jbc.org/

A

Buffer TSH

Myc-PDK1

IB:anti-pY

IB:anti-MycIP:anti-myc

- - + + +

- + - + -TSH- - - - + cAMP

B

TSH +Wortmannin

TSH +LY294002

TSH+H89

Figure 3

cAMP


http://www.jbc.org/

TSH 0 5 15 30 60 minpT308

pS473

Total

A

B

Figure 4

Insulin 0 15 30 60 120 minpS473

Total

1 2 3 4 5

1 2 3 4 5

Total

pS65

Insulin 0 5 15 15 (min)Rapamycin

1 2 3 4

D

Akt/PKB

Akt/PKB4E-BP1/PHAS1

- - - +

Total

pS65TSH 0 5 15 30 (min)

1 2 3 4

C

4E-BP1/PHAS1


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TSH 0 5 15 30 60 (min)

1

2

3

4

5

0

2

4

6

10

12

0

TSH (1 mU/ml) 0 5 15 30 60 (min) 0 5 15 30 60 (min)

Insulin (10 µg/ml)

S6K

1 A

ctiv

ity (f

old)

A C

S6K

1 A

ctiv

ity (f

old)medium

TSHmedium Insulin

Figure 5

pS6 pS6

B D

Insulin 0 5 15 30 60 (min)

1 2 3 4 5 1 2 3 4 5

pp70 S6K1

pp85 S6K1

pp70 S6K1

pp85 S6K1


http://www.jbc.org/

Forskolin - + + + + +

R W L H89

R W L H89

cAMP - + + + + +

TSH - + + + + +

H89 (µM)5 10 30 50

D

E

F

pp70 S6K1

pp85 S6K1

pp70 S6K1

pp85 S6K1

pp70 S6K1

pp85 S6K1

pp70 S6K1

pp85 S6K1L5-WTp85 L5-∆iSH2 p85

0 5 15 30 60 0 5 15 30 60 min

TSH - + + + + + 0.5 1 2 5LY294002 (µM)

TSH - + + + + + 0.01 0.1 0.3 0.5Wortmannin (µM)

pp70 S6K1

pp85 S6K1

A

B

1 2 3 4 5 6

1 2 3 4 5 6

pp70 S6K1

pp85 S6K1

C

Figure 6TSH


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S6K

1 A

ctiv

ity (f

old)

1

2

TSH - + + + +

TPCK (µM)10 20 50

TSH - + - + - - + + TPCK(20 µM)

B

Figure 7

pS6

pp70 S6K1

pp85 S6K1

A


http://www.jbc.org/

- + - + - + Rapamycin

PD98059

1 2 3 4 5 6

0 5 15 30 60 (min)

Insulin

Total

pT202/Y204

pT202/Y204

Total

TSH

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A

B

C

Figure 8

pp70 S6K1

pp85 S6K1

ERK1/ERK2

ERK1/ERK2


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1 2 3 4 5 61 : control 2 : TSH3 : TSBAb4 : Insulin + TSBAb (10 mg/ml)5 : TSAb + TSBAb (10 mg/ml)6 : TSH + TSBAb (10 mg/ml)

TSAb(10 mg/ml)

TSH

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Figure 9

CON NP PM 1 2 3 4 5 6 7 8 9 10

GD

A

pS6

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pp70 S6K1

pp85 S6K1 pp70 S6K1


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TSH - - + +- + - +rapamycin

cyclin D1β-actin

TSH 0 12 24 48 (h)

cyclin D1

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S: 1.88 %G2: 11.8 %

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G2: 9.80 %

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G2: 11.8 %

TSH + Rapa (4)

TSH/Insulin (3)

TSH/Insulin + Rapa (5)

D

G1: 49.3 %S: 19.5 %

G2: 19.1 %

G1: 72.6 %S: 13.1 %

G2: 10.5 %

Relative Fluorescence

Cel

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1 2 3 4 5

S6K1


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ControlMMI

(2 wks)MMI (2 wks)

Vehicle(1 wk)MMI (2 wks)

rapamycin (1wk)

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Figure 11

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MMI (2 wk) rapamycin (3Days)

MMI (2 wk)vehicle (3Days)Control

C


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cAMP/PKA

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Figure 12

S6

FRAP/mTOR

INSULIN

ATP


http://www.jbc.org/

Cho, Heung Kyu Ro and Minho ShongYounHwang, Jin Man Kim, Eun Suk Hwang, Jongkyeong Chung, Jeung-Hwan Han, Bo

Jae Mi Suh, Jung Hun Song, Dong Wook Kim, Ho Kim, Hyo Kyun Chung, Jung Hwanthyroid gland

thyroid stimulating hormone and stimulating-type TSH receptor antibodies in the Regulation of the PI3K, Akt/PKB, FRAP/mTOR, and S6K1 signaling pathways by

published online March 30, 2003J. Biol. Chem.

10.1074/jbc.M300805200Access the most updated version of this article at doi:

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M300805200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/03/30/jbc.M300805200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/03/30/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/03/30/jbc.M300805200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

regulation of the pi3k, akt/pkb, frap/mtor, and s6k1 ... · this pathway may play a role in the...

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