Attenuation of mammalian target of rapamycin activity by increased cAMP in 3T3-L1 adipocytes.
Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio)adenosine 3'-5'-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin. These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70(S6K), and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR.
Scott
P H
PH
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Lawrence
J C
JC
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D039642:Eukaryotic Initiation Factors; D007118:Immunoassay; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D013806:Theophylline / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; C499526:Eif4ebp1 protein, rat; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013873:Thionucleotides; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D013806:Theophylline; D000242:Cyclic AMP; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546843:mTOR protein, mouse; C546845:mTOR protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus