Summary Akt-deficiency causes resistance to replicative senescence, oxidative stress- or oncogenic Ras-induced premature senescence, and to reactive oxygen species (ROS)-mediated apoptosis. Akt activation induces premature senescence and sensitizes cells to ROS-mediated apoptosis by increasing intracellular ROS through increased oxygen consumption and by inhibiting the expression of ROS-scavengers downstream of FoxO, particularly sestrin3 expression. This uncovers an Achilles’ heel of Akt, since in contrast to its ability to inhibit apoptosis induced by multiple apoptotic stimuli; Akt could not inhibit ROS-mediated apoptosis. Furthermore, treatment with rapamycin that led to further Akt activation and resistance to etoposide, hypersensitized cancer cells to ROS-mediated apoptosis. Given that rapamycin alone is mainly cytostatic, this constitutes a strategy for cancer therapy that selectively eradicates cancer cells via Akt activation.

The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo.The serine/threonine protein kinase Akt, also known as protein kinase B, a downstream effector of phosphoinositide-3-OH kinase, has emerged as a critical mediator of the mammalian target of rapamycin (mTOR) 2 activity. Mammalian cells express three separate Akt proteins (Akt1-3), which share Ͼ80% amino acid sequence identity and are encoded by different genes. The rate-limiting step in Akt activation is the binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of Akt and the subsequent translocation of Akt to the plasma membrane. Akt is then phosphorylated by 3-phosphoinositide-dependent kinase-1 and by another as yet unknown phosphoinositide-3-OH kinase-dependent kinase. Both phosphorylation events are required for full activation of Akt (for reviews see Refs. 1-3). Biochemical and genetic data show that Akt is a positive regulator of mTOR that mediates the activation of mTOR by growth factors (reviewed in Ref. 4). mTOR controls mRNA translation by phosphorylating and activating S6 kinase 1 (S6K1) and by phosphorylating and inactivating the eukaryotic initiation factor 4E-binding proteins (4E-BPs), which repress mRNA translation. Thus, the phosphorylation status of S6K1 and one of the 4E-BPs members, 4E-BP1, is often used as readout for mTOR activity in vivo. mTOR is activated by the small GTPase Rheb, which is inhibited by its GAP protein TSC2 that heterodimerizes with tuberous sclerosis complex 1 (TSC1) (5-7). Genetic studies and biochemical analyses in mammalian cells (8 -12) and Drosophila (11, 13), show that TSC2 is an upstream negative regulator of mTOR. Akt...

Summary FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here we unraveled regulatory circuits underlying interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 binds the promoter region of Sestrin3 (Sesn3) gene and directly elevates Sesn3 expression, which in turn inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt, while inhibiting mTORC1. FoxO may act as rheostat that maintains homeostatic balance between Akt and mTOR complexes activities. Indeed, in response to physiological stresses, FoxO is required to maintain high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1-a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.

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