To elucidate the functions of the serine/threonine kinase Akt/PKB in vivo, we generated mice lacking both akt1 and akt2 genes. Akt1/Akt2 double-knockout (DKO) mice exhibit severe growth deficiency and die shortly after birth. These mice display impaired skin development because of a proliferation defect, severe skeletal muscle atrophy because of a marked decrease in individual muscle cell size, and impaired bone development. These defects are strikingly similar to the phenotypes of IGF-1 receptor-deficient mice and suggest that Akt may serve as the most critical downstream effector of the IGF-1 receptor during development. In addition, Akt1/Akt2 DKO mice display impeded adipogenesis. Specifically, Akt1 and Akt2 are required for the induced expression of PPAR␥, the master regulator of adipogenesis, establishing a new essential role for Akt in adipocyte differentiation. Overall, the combined deletion of Akt1 and Akt2 establishes in vivo roles for Akt in cell proliferation, growth, and differentiation. These functions of Akt were uncovered despite the observed lower level of Akt activity mediated by Akt3 in Akt1/Akt2 DKO cells, suggesting that a critical threshold level of Akt activity is required to maintain normal cell proliferation, growth, and differentiation.

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.

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...

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G 2 /M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G 2 /M to the G 1 phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G 2 arrest that is alleviated by activated Akt. Furthermore, the transition from the G 2 /M to the G 1 phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G 2 /M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G 2 /M cell cycle checkpoint and apoptosis in MMRdeficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.Akt, or protein kinase B (PKB), is a serine/threonine kinase that has been implicated in the control of major cellular functions such as transcription, protein synthesis, and carbohydrate and lipid metabolism, and it is a downstream effector of growth factor-mediated cell survival. Normally, Akt is activated by growth factors that activate phosphoinositol-3-kinase (PI3K). Upon activation, PI3K phosphorylates the inositol ring at the D3 position, which in turn serves to anchor Akt to the plasma membrane, where it is phosphorylated and fully activated by the 3-phosphoinositide-dependent kinases PDK1 and PDK2. Phospholipid phosphatases such as PTEN and SHIP decrease the pool of available phospholipids and therefore are negative regulators of Akt. Activated Ras, at least in certain circumstances, can up-regulate PI3K and therefore is a potential activator of Akt as well (13,22). Overall, positive regulators of Akt are commonly up-regulated in human cancers, while PTEN is frequently lost or inactivated by mutations (7, 36). Furthermore, heterozygous deletion of PTEN in mice elicits a wide range of spontaneous tumors; this has been attributed mainly to activation of Akt (12,34,38). Finally, activated forms of Akt induce cellular transformation (4). ...

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.