cAMP-dependent activation of mammalian target of rapamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4.
How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation.
Blancquaert
Sara
S
Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.
Wang
Lifu
L
Paternot
Sabine
S
Coulonval
Katia
K
Dumont
Jacques E
JE
Harris
Thurl E
TE
Roger
Pierre P
PP
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D056743:Cyclin D3 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism*; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology
D056743:Cyclin D3; D047908:Intracellular Signaling Peptides and Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; C546845:mTOR protein, rat; D017346:Protein Serine-Threonine Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D051358:Cyclin-Dependent Kinase 4; D020123:Sirolimus