Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.
The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
Scott
P H
PH
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Brunn
G J
GJ
Kohn
A D
AD
Roth
R A
RA
Lawrence
J C
JC
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004789:Enzyme Activation; D039642:Eukaryotic Initiation Factors; D007328:Insulin / Q000378:metabolism*; D007329:Insulin Antagonists / Q000494:pharmacology; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D007329:Insulin Antagonists; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011090:Polyenes; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin; C499527:Eif4ebp1 protein, mouse; C546843:mTOR protein, mouse