EVIDENCE OF INSULIN-STIMULATED PHOSPHORYLATION AND ACTIVATION OF THE MAMMALIAN TARGET OF RAPAMYCIN MEDIATED BY A PROTEIN-KINASE-B SIGNALINGPATHWAY

The effects of insulin on the mammalian target of rapamycin, mTOR, wer e 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 immunop recipitations were conducted with the mTOR antibody, mTAb2. Insulin al so increased by severalfold the P-32 content of mTOR that was determin ed after purifying the protein from P-32-labeled adipocytes with rapam ycin.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, T he effects of insulin on increasing mTOR protein kinase activity and o n decreasing mTAb1 reactivity were abolished by incubating mTOR with p rotein phosphatase I. 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), Experi ments were performed in MER-Akt cells to investigate the role of PKB i n controlling mTOR These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydro xytamoxifen. Activating Pk;B 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 insul in activates mTOR by promoting phosphorylation of the protein via a si gnaling pathway that contains PKB.