Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway

Scott, P. H., Brunn, G. J., Kohn, A. D., Roth, R. A. and Lawrence Jr., J. C. (1998) Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proceedings of the National Academy of Sciences of the United States of America, 95(13), pp. 7772-7777. (doi:10.1073/pnas.95.13.7772) (PMID:9636226) (PMCID:PMC22753)

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Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Scott, Dr Pamela
Authors: Scott, P. H., Brunn, G. J., Kohn, A. D., Roth, R. A., and Lawrence Jr., J. C.
College/School:College of Medical Veterinary and Life Sciences > School of Life Sciences
Journal Name:Proceedings of the National Academy of Sciences of the United States of America
Publisher:National Academy of Sciences
ISSN:0027-8424
ISSN (Online):1091-6490
Published Online:23 June 1998

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