Disruption of the interface between the pleckstrin homology (PH) and kinase domains of Akt protein is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein

The serine/threonine protein kinase Akt is a critical regulator of cell growth and survival in response to growth factors. A key step in Akt activation is phosphorylation at Ser-473 by the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Although Rictor is required for the stability and activity of mTORC2, little is known about functional regions or post-translational modifications within Rictor that are responsible for regulating mTORC2. Here, we demonstrate that Rictor contains two distinct central regions critical for mTORC2 function. One we refer to as the stability region because it is critical for interaction with Sin1.1 and LST8, and a second adjacent region is required for multisite acetylation. p300-mediated acetylation of Rictor increases mTORC2 activity toward Akt, whereas site-directed mutants within the acetylation region of Rictor exhibit reduced insulin-like growth factor 1 (IGF-1)-stimulated mTORC2 kinase activity. Inhibition of deacetylases, including the NAD+-dependent sirtuins, promotes Rictor acetylation and IGF-1-mediated Akt phosphorylation. These results suggest that multiple-site acetylation of Rictor signals for increased activation of mTORC2, providing a critical link between nutrient-sensitive deacetylases and mTORC2 signaling to Akt.     

Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.     

Autoregulation of the Mechanistic Target of Rapamycin (mTOR) Complex 2 Integrity Is Controlled by an ATP-dependent Mechanism

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mm and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.     

Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site

The function of Akt (protein kinase B) is regulated by phosphorylation on two sites conserved within the AGC kinase family: the activation loop (Thr-308) in the kinase core and a hydrophobic phosphorylation site on the carboxyl terminus (Ser-473). Thr-308 is phosphorylated by the phosphoinositide-dependent kinase-1, (PDK-1), whereas the mechanism of phosphorylation of the hydrophobic site, tentatively referred to as the PDK-2 site, is unknown. Here we report that phosphorylation of the hydrophobic motif requires catalytically competent Akt. First we show that a kinase-inactive construct of Akt fails to incorporate phosphate at Ser-473 following IGF-1 stimulation in vivo but does incorporate phosphate at Thr-308 and a second carboxyl-terminal site, Thr-450; this ligand triggers the phosphorylation of both sites in wild-type enzyme. Neither does a catalytically inactive construct in which phosphorylation at the activation loop is blocked, T308A, become phosphorylated on the hydrophobic site in response to stimulation. Second, we show that Akt autophosphorylates on the hydrophobic site in vitro: phosphorylation of the activation loop by PDK-1 triggers the phosphorylation of the hydrophobic site in kinase-active, but not thermally inactivated, Akt alpha. Thus, Akt is regulated by autophosphorylation at the Ser-473 hydrophobic site.     

mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.     

mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1)

SGK1 (serum- and glucocorticoid-induced protein kinase 1) is a member of the AGC (protein kinase A/protein kinase G/protein kinase C) family of protein kinases and is activated by agonists including growth factors. SGK1 regulates diverse effects of extracellular agonists by phosphorylating regulatory proteins that control cellular processes such as ion transport and growth. Like other AGC family kinases, activation of SGK1 is triggered by phosphorylation of a threonine residue within the T-loop of the kinase domain and a serine residue lying within the C-terminal hydrophobic motif (Ser(422) in SGK1). PDK1 (phosphoinositide-dependent kinase 1) phosphorylates the T-loop of SGK1. The identity of the hydrophobic motif kinase is unclear. Recent work has established that mTORC1 [mTOR (mammalian target of rapamycin) complex 1] phosphorylates the hydrophobic motif of S6K (S6 kinase), whereas mTORC2 (mTOR complex 2) phosphorylates the hydrophobic motif of Akt (also known as protein kinase B). In the present study we demonstrate that SGK1 hydrophobic motif phosphorylation and activity is ablated in knockout fibroblasts possessing mTORC1 activity, but lacking the mTORC2 subunits rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated-protein-kinase-interacting protein 1) or mLST8 (mammalian lethal with SEC13 protein 8). Furthermore, phosphorylation of NDRG1 (N-myc downstream regulated gene 1), a physiological substrate of SGK1, was also abolished in rictor-, Sin1- or mLST8-deficient fibroblasts. mTORC2 immunoprecipitated from wild-type, but not from mLST8- or rictor-knockout cells, phosphorylated SGK1 at Ser(422). Consistent with mTORC1 not regulating SGK1, immunoprecipitated mTORC1 failed to phosphorylate SGK1 at Ser(422), under conditions which it phosphorylated the hydrophobic motif of S6K. Moreover, rapamycin treatment of HEK (human embryonic kidney)-293, MCF-7 or HeLa cells suppressed phosphorylation of S6K, without affecting SGK1 phosphorylation or activation. The findings of the present study indicate that mTORC2, but not mTORC1, plays a vital role in controlling the hydrophobic motif phosphorylation and activity of SGK1. Our findings may explain why in previous studies phosphorylation of substrates, such as FOXO (forkhead box O), that could be regulated by SGK, are reduced in mTORC2-deficient cells. The results of the present study indicate that NDRG1 phosphorylation represents an excellent biomarker for mTORC2 activity.     

Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.     

Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB alpha/Akt-1). Although 3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC(50) of approximately 0.22 microm. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.     

Endoplasmic reticulum is a main localization site of mTORC2

The Akt kinase is a critical effector in growth factor signaling. Activation of Akt driven by the growth factor dependent PI3K (phosphatidylinositol-3-OH kinase) is coupled to the plasma membrane translocation and phosphorylation of Akt on two sites by PDK1 (phosphoinositide-dependent protein kinase-1) on Thr-308 and by mTORC2 (mammalian Target of Rapamycin Complex 2) on Ser-473. In our study we examined the sub-cellular localization of mTORC2 and identified that this kinase complex predominantly resides on endoplasmic reticulum (ER). Our immunostaining analysis did not show a substantial co-localization of the mTORC2 component rictor with Golgi, lysosome, clathrin-coated vesicles, early endosomes, or plasma membrane but indicated a strong co-localization of rictor with ribosomal protein S6 and ER marker. Our biochemical study also identified the mTORC2 components rictor, SIN1, and mTOR as the highly abundant proteins in the ER fraction, whereas only small amount of these proteins are detected in the plasma membrane and cytosolic fractions. We found that growth factor signaling does not alter the ER localization of mTORC2 and also does not induce its translocation to the plasma membrane. Based on our study we suggest that the mTORC2-dependent phosphorylation of Akt on Ser-473 takes place on the surface of ER.     

In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.     

Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.     

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.     

A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.     

Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.     

PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth

Akt/protein kinase B critically regulates the balance between cell survival and apoptosis. Phosphorylation of Akt at two key sites, the activation loop and the hydrophobic motif, activates the kinase and promotes cell survival. The mechanism of dephosphorylation and signal termination is unknown. Here, we identify a protein phosphatase, PH domain leucine-rich repeat protein phosphatase (PHLPP), that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1), triggering apoptosis and suppressing tumor growth. The effects of PHLPP on apoptosis are prevented in cells expressing an S473D construct of Akt, revealing that the hydrophobic motif is the primary cellular target of PHLPP. PHLPP levels are markedly reduced in several colon cancer and glioblastoma cell lines that have elevated Akt phosphorylation. Reintroduction of PHLPP into a glioblastoma cell line causes a dramatic suppression of tumor growth. These data are consistent with PHLPP terminating Akt signaling by directly dephosphorylating and inactivating Akt.     

Identification of Akt-independent Regulation of Hepatic Lipogenesis by Mammalian Target of Rapamycin (mTOR) Complex 2

Mammalian target of rapamycin complex 2 (mTORC2) is a key activator of protein kinases that act downstream of insulin and growth factor signaling. Here we report that mice lacking the essential mTORC2 component rictor in liver (Lrictor(KO)) are unable to respond normally to insulin. In response to insulin, Lrictor(KO) mice failed to inhibit hepatic glucose output. Lrictor(KO) mice also fail to develop hepatic steatosis on a high fat diet and manifest half-normal serum cholesterol levels. This is accompanied by lower levels of expression of SREBP-1c and SREBP-2 and genes of fatty acid and cholesterol biosynthesis. Lrictor(KO) mice had defects in insulin-stimulated Akt Ser-473 and Thr-308 phosphorylation, leading to decreased phosphorylation of Akt substrates FoxO, GSK-3β, PRAS40, AS160, and Tsc2. Lrictor(KO) mice also manifest defects in insulin-activated mTORC1 activity, evidenced by decreased S6 kinase and Lipin1 phosphorylation. Glucose intolerance and insulin resistance of Lrictor(KO) mice could be fully rescued by hepatic expression of activated Akt2 or dominant negative FoxO1. However, in the absence of mTORC2, forced Akt2 activation was unable to drive hepatic lipogenesis. Thus, we have identified an Akt-independent relay from mTORC2 to hepatic lipogenesis that separates the effects of insulin on glucose and lipid metabolism.     

Raptor-rictor axis in TGFbeta-induced protein synthesis

Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.