In rat hepatocytes glucagon increases mammalian target of rapamycin phosphorylation on serine 2448 but antagonizes the phosphorylation of its downstream targets induced by insulin and amino acids.
The major function of mammalian target of rapamycin (mTOR) is the control of cell growth. Insulin and amino acids regulate the mTOR pathway, and both are needed to promote its maximal activation. To further understand mTOR regulation by insulin and amino acids, we have studied the enzyme in primary cultures of hepatocytes. We show that insulin increases mTOR phosphorylation on Ser2448, a consensus phosphorylation site for protein kinase B (PKB). Ser2448 phosphorylation is also increased by amino acids, although they do not activate PKB. Furthermore, insulin and amino acids have an additive effect, indicating that they act through distinct pathways. We also show that phosphorylation of Ser2448 does not seem to modulate in vitro phosphorylation of eukaryotic initiation factor 4E-binding protein 1 by mTOR. However, stimulation of hepatocytes with insulin and amino acids leads to an increase in mTOR kinase activity. Rapamycin has no effect on insulin-, glucagon-, and 8-(4-chlorophenylthio)adenosine-cAMP-induced amino acid transport. Surprisingly, glucagon and 8-(4-chlorophenylthio)adenosine-cAMP, which do not activate PKB, stimulate the phosphorylation on Ser2448 of mTOR. However, glucagon inhibits amino acid- and insulin-induced activation of ribosomal S6 protein kinase 1 and phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1. Our results demonstrate that glucagon, which is not able to activate but rather inhibits the mTOR pathways, stimulates the phosphorylation of mTOR on Ser2448. This finding suggests that phosphorylation of this site might not be sufficient for mTOR kinase activity but is likely to be involved in other functions.
INSERM, Unité 145, Institut Fédératif de Recherche 50, 06107 Nice, Cédex 02, France. satney@unice.fr
Isabelle
I
Mothe-Satney
Nadine
N
Gautier
Charlotte
C
Hinault
John C
JC
Lawrence
Emmanuel
E
Van Obberghen
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000737:chemistry*; D022781:Hepatocytes / Q000378:metabolism*; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D000596:Amino Acids; D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; D005934:Glucagon; D000242:Cyclic AMP; D011494:Protein Kinases; C546845:mTOR protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus