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

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 32 P content of mTOR that was determined after purifying the protein from 32 P-labeled adipocytes with rapamycin⅐FKBP12 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.

DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling

“…It is possible that a component involved in mTOR signalling undergoes irreversible inactivation, perhaps by degradation, in apoptotic cells. A possible candidate here is protein kinase B (PKB) which has been implicated in mTOR signalling (Gingras et al, 1998;Scott et al, 1998;Nave et al, 1999) and has recently been shown to be cleaved in apoptotic cells (Rokudai et al, 2000).…”

Molecular Mechanisms of Insulin Resistance: Serine Phosphorylation of Insulin Receptor Substrate-1 and Increased Expression of p85α

“…In response to insulin and amino acids, mTOR, which is a serine/threonine kinase, phosphorylates and modulates activities of p70 S6 kinase (S6K1 kinase) and an inhibitor of translational initiation, eIF-4E binding protein (46 -48). While insulin activates mTOR and S6K1 kinase via the IRS-1/PI 3-kinase/Akt pathway (49,50), amino acids seem to exert their effect through a direct influence of mTOR (44,51,52). In any event, activation of mTOR and S6K1 kinase causes serine phosphorylation of IRS-1, with a subsequent decline in the IRS-1-associated PI 3-kinase activity (Fig.…”

T Cell Effector Function and Anergy Avoidance Are Quantitatively Linked to Cell Division

“…PKC is normally coupled to the IL-2R through PI 3 K, as PKC is activated by PDK1-dependent phosphorylation (40), and a role for PKC in IL-2-mediated signal transduction has been defined using the IL-2-dependent cell line CTLL (59,60). Interestingly, full activation of the PKC-␦ isoform requires the activity of mTOR/FRAP (61), which is in turn dependent on PKB/Akt (62). These two distinct PI 3 K-dependent pathways leading to PKC activation, which can be bypassed using phorbol esters, represent a set of biochemical events that could potentially be defective in the undivided pool.…”