Akt Activates the Mammalian Target of Rapamycin by Regulating Cellular ATP Level and AMPK Activity

Abstract

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...

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis

“…In agreement, overexpression of activated Akt in Tsc2-null cells inhibits AMPK activity (Hahn-Windgassen et al, 2005). These studies suggest that the low levels of active Akt found in Tsc2-null cells are responsible for the elevated activity of AMPK.…”

“…Previous studies have shown that Akt inhibits AMPK, likely through a decrease of the AMP to ATP ratio through activation of glycolysis and oxidative phosphorylation (Gottlob et al, 2001;Kovacic et al, 2003;Hahn-Windgassen et al, 2005). In agreement, overexpression of activated Akt in Tsc2-null cells inhibits AMPK activity (Hahn-Windgassen et al, 2005).…”

“…We found that depletion of Rheb under serum-free conditions inhibited AMPK activity, as indicated by a reduction of the levels of both phospho-AMPK (p-T172), and phospho-acetyl-CoA carboxylase (p-ACC), an AMPK downstream target (Figure 1b). As inhibition of mTORC1 by depletion of Rheb may eliminate negative feedback signaling to Akt and because it was shown that Akt can inhibit AMPK (Hahn-Windgassen et al, 2005;Short et al, 2008), we analyzed whether Rheb depletion can affect Akt activity, measured by the phosphorylation levels at both S473 and T308. Phosphorylation of Akt was barely detected, and Rheb depletion only modestly affected Akt phosphorylation at T308.…”

“…AMPK phosphorylates TSC2 and the mTOR partner Raptor to inhibit mTORC1 signaling (Inoki et al, 2003b;Gwinn et al, 2008). However, AMPK and mTORC1 were both activated in Tsc2-null cells (Hahn-Windgassen et al, 2005), indicating that AMPK phosphorylation of Raptor is not sufficient to inhibit mTORC1 signaling and cell growth when Tsc2 is absent.…”

Stress and mTORture signaling

“…Cells with elevated Akt/PKB activity showed lowered AMPactivated protein kinase (AMPK) activity, blunting its stimulating effect on TSC. Conversely, in the PKB1/ PKB2 DKO cells, AMPK activity was significantly elevated, contributing to enhanced TSC2 function (Hahn-Windgassen et al, 2005). Furthermore, it is possible that the regulation of the Akt/PKB phosphorylation sites in TSC2 -although dispensable for normal development -becomes important in a context of aberrantly elevated insulin pathway activity as in PTEN mutant situations.…”