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.     

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3beta (GSK-3beta) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.     

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.     

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent,FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.     

Elevated hepatic fatty acid elongase-5 activity affects multiple pathways controlling hepatic lipid and carbohydrate composition

Hepatic fatty acid elongase-5 (Elovl-5) plays an important role in long chain monounsaturated and polyunsaturated fatty acid synthesis. Elovl-5 activity is regulated during development, by diet, hormones, and drugs, and in chronic disease. This report examines the impact of elevated Elovl-5 activity on hepatic function. Adenovirus-mediated induction of Elovl5 activity in livers of C57BL/6 mice increased hepatic and plasma levels of dihomo-gamma-linolenic acid (20:3,n-6) while suppressing hepatic arachidonic acid (20:4,n-6) and docosahexaenoic acid (22:6,n-3) content. The fasting-refeeding response of peroxisome proliferator-activated receptor alpha-regulated genes was attenuated in mice with elevated Elovl5 activity. In contrast, the fasting-refeeding response of hepatic sterol-regulatory element binding protein-1 (SREBP-1)-regulated and carbohydrate-regulatory element binding protein/Max-like factor X-regulated genes, Akt and glycogen synthase kinase (Gsk)-3beta phosphorylation, and the accumulation of hepatic glycogen content and nuclear SREBP-1 were not impaired by elevated Elovl5 activity. Hepatic triglyceride content and the phosphorylation of AMP-activated kinase alpha and Jun kinase 1/2 were reduced by elevated Elovl5 activity. Hepatic phosphoenolpyruvate carboxykinase expression was suppressed, while hepatic glycogen content and phosphorylated Gsk-3beta were significantly increased, in livers of fasted mice with increased Elovl5 activity. As such, hepatic Elovl5 activity may affect hepatic glucose production during fasting. In summary, Elovl5-induced changes in hepatic fatty acid content affect multiple pathways regulating hepatic lipid and carbohydrate composition.     

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.     

Rapamycin regulates the phosphorylation of rictor

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth. mTOR exists in two functional complexes, mTORC1 and mTORC2. mTORC1 is rapamycin-sensitive, and results in phosphorylation of 4E-BP1 and S6K1. mTORC2 is proposed to regulate Akt Ser473 phosphorylation and be rapamycin-insensitive. mTORC2 consists of mTOR, mLST8, sin1, Protor/PRR5, and the rapamycin insensitive companion of mTOR (rictor). Here, we show that rapamycin regulates the phosphorylation of rictor. Rapamycin-mediated rictor dephosphorylation is time and concentration dependent, and occurs at physiologically relevant rapamycin concentrations. siRNA knockdown of mTOR also leads to rictor dephosphorylation, suggesting that rictor phosphorylation is mediated by mTOR or one of its downstream targets. Rictor phosphorylation induced by serum, insulin and insulin-like growth factor is blocked by rapamycin. Rictor dephosphorylation is not associated with dephosphorylation of Akt Ser473. Further work is needed to better characterize the mechanism of rictor regulation and its role in rapamycin-mediated growth inhibition.     

Mammalian target of rapamycin complex 2 (mTORC2) negatively regulates Toll-like receptor 4-mediated inflammatory response via FoxO1

Activation of the PI3K pathway plays a pivotal role in regulating the inflammatory response. The loss of mTORC2 has been shown to abrogate the activation of Akt, a critical downstream component of PI3K signaling. However, the biological importance of mTORC2 in innate immunity is currently unknown. Here we demonstrate that rictor, a key component of mTORC2, plays a critical role in controlling the innate inflammatory response via its ability to regulate FoxO1. Upon LPS stimulation, both rictor-deficient mouse embryonic fibroblasts (MEFs) and rictor knockdown dendritic cells exhibited a hyperinflammatory phenotype. The hyperinflammatory phenotype was due to a defective Akt signaling axis, because both rictor-deficient MEFs and rictor knockdown dendritic cells exhibited attenuated Akt phosphorylation and kinase activity. Analysis of downstream Akt targets revealed that phosphorylation of FoxO1 was impaired in rictor-deficient cells, resulting in elevated nuclear FoxO1 levels and diminished nuclear export of FoxO1 upon LPS stimulation. Knockdown of FoxO1 attenuated the hyperinflammatory phenotype exhibited by rictor-deficient MEFs. Moreover, FoxO1 deletion in dendritic cells attenuated the capacity of LPS to induce inflammatory cytokine expression. These findings identify a novel signaling pathway by which mTORC2 regulates the TLR-mediated inflammatory response through its ability to regulate FoxO1.     

mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. The mTORC2 containing mTOR and rictor is thought to be rapamycin insensitive and it is recently shown that both rictor and mTORC2 are essential for the development of both embryonic and extra embryonic tissues. To explore rictor function in the early development of mouse embryos, we disrupted the expression of rictor, a specific component of mTORC2, in mouse fertilized eggs by using rictor shRNA. Our results showed that one-cell stage eggs that were lack of rictor could not enter into the two-cell stage normally. Recent biochemical studies suggests that TORC2 is the elusive PDK2 (3'-phosphoinositide-dependent kinase 2) for AKT/PKB Ser473 phosphorylation, which is deemed necessary for AKT function, so we microinjected AKT-S473A into mouse fertilized eggs to investigate whether AKT-S473A is downstream effector of mTOR.rictor to regulate the mitotic division. Our findings revealed that the rictor induced phosphorylation of AKT in Ser473 is required for TORC2 function in early development of mouse embryos.     

Ivabradine Prevents Low Shear Stress Induced Endothelial Inflammation and Oxidative Stress via mTOR/eNOS Pathway

Ivabradine not only reduces heart rate but has other cardiac and vascular protective effects including anti-inflammation and anti-oxidation. Since endothelial nitric oxide synthase (eNOS) is a crucial enzyme in maintaining endothelial activity, we aimed to investigate the impact of ivabradine in low shear stress (LSS) induced inflammation and endothelial injury and the role of eNOS played in it. Endothelial cells (ECs) were subjected to LSS at 2dyne/cm², with 1 hour of ivabradine (0.04μM) or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10μM) pre-treatment. The mRNA expression of IL-6, VCAM-1 along with eNOS were measured by QPCR. Reactive oxygen species (ROS) was detected by dihydroethidium (DHE) and DCF, and protein phosphorylation was detected by western blot. It demonstrated that ivabradine decreased LSS induced inflammation and oxidative stress in endothelial cells. Western blot showed reduced rictor and Akt-Ser473 as well as increased eNOS-Thr495 phosphorylation. However, mTORC1 pathway was only increased when LSS applied within 30 minutes. These effects were reversed by ivabradine. It would appear that ivabradine diminish ROS generation by provoking mTORC2/Akt phosphorylation and repressing mTORC1 induced eNOS-Thr495 activation. These results together suggest that LSS induced endothelial inflammation and oxidative stress are suppressed by ivabradine via mTORC2/Akt activation and mTORC1/eNOS reduction.     

Ginsenoside Rb1 inhibits oxidative stress-induced ovarian granulosa cell injury through Akt-FoxO1 interaction

Ginsenoside Rb1 shows a strong antioxidant effect and has potential activation effects on Akt. The aim of the present study was to investigate the protective effect of Rb1 on age-related ovarian granulosa cell injury. Ovarian granulosa cells (GCs) were obtained from 50 young women (≤30 years) and 50 aged women (≥38 years) at an IVF center. Young and aged ICR mice were administered with or without Rb1 (10 mg kg^?1, i.p.) for 2 weeks. The protective effects of Rb1 were investigated and the role of Rb1 on the modulation of Akt-FoxO1 interaction was determined with immunofluorescence, Western blotting, immunoprecipitation, siRNA silencing and pharmacological inhibitor. Rb1 effectively decreased LDH and MDA, and reversed the apoptotic-related protein levels in hGL cells from old patients. Similar results were found in mice. In addition, the mitochondrial membrane potential was restored and the overaccumulation of ROS was reversed by Rb1. Rb1 preserved peroxide-impaired Akt activation, to some extent, by increasing phosphorylation at Ser473. Rb1 also facilitated p-Akt binding to FoxO1 and promoted the phosphorylation of FoxO1. SiRNA silencing of Akt, Akt inhibitor LY294002, and FoxO1 inhibitor AS1842856 attenuated the effects of Rb1. Ginsenoside Rb1 inhibits age-related GCs oxidative damage by activating Akt phosphorylation at Ser473 and by further interaction with FoxO1.

     

Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice

The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.     

Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.     

TBK1 has a new Akt

TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley et al. demonstrated that TBK1-dependent activation of downstream kinase Akt is mediated via mechanistic target of rapamycin complex 2. This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.

TANK-binding kinase 1 (TBK1), a serine/threonine kinase that belongs to the noncanonical IκB kinase family, plays an essential role in the innate immune response to viral and bacterial pathogens by regulating the type I interferon–mediated T cell response (1). Although TBK1 has been most widely studied in this context, more recent investigations using tissue-specific KO mice and drugs that inhibit kinase activity have revealed novel roles for this kinase in nonimmune cells, particularly at the intersection of immunity and metabolism. For example, TBK1 expression and activity are induced in adipose tissue in obesity by elevated expression of proinflammatory cytokines such as tumor necrosis factor α (2). TBK1 contributes to obesity by repressing energy expenditure and increasing anabolic functions as determined from analysis of mice with conditional adipose cell KO of TBK1 (3). TBK1 has also been reported to promote activation of Akt, a central kinase involved in metabolic regulation (4). However, the mechanism by which TBK1 regulates Akt has remained unclear.Akt is an essential regulator of glucose metabolism and plays an important role in controlling cellular glucose uptake and utilization through both positive and negative regulatory actions (4). Phosphorylation of Akt on T308 in its activation loop stimulates kinase activity, and phosphorylation on S473 further enhances activity and determines substrate specificity (4). Although it had been previously reported that TBK1 can directly phosphorylate Akt at S473 and T308 in in vitro kinase assays, the ability of TBK1 to mediate these phosphorylation events under physiological conditions was not known (5). In a recent study, Tooley et al. (6) contributed to the mechanistic understanding of TBK1 function in metabolic regulation by demonstrating a role for TBK1 in mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) activation and subsequent phosphorylation of Akt.To investigate how TBK1 regulates Akt activation, mouse embryonic fibroblasts (MEFs) were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and Akt-T308 phosphorylation (6). The intensity and duration of Akt phosphorylation at both sites was diminished significantly, both in the absence of TBK1 and in the presence of the TBK1 inhibitor amlexanox. Restoration of endogenous levels of TBK1, but not kinase-dead TBK1, rescued EGF-stimulated Akt-S473 phosphorylation. The stimulation of Akt-S473 phosphorylation by EGF, as well as by other growth factors and the hormone insulin, was found to be dependent upon mTOR activity. Together, these results validate the ability of TBK1 to regulate Akt-S473 phosphorylation and show that in response to normal growth regulatory signaling, this regulation is mediated through mTOR kinase.The kinase mTOR is the core catalytic kinase of two multisubunit complexes, mTOR complex 1 (mTORC1) and mTORC2, which are distinguished by the scaffolding proteins Raptor and Rictor, respectively (7). mTORC1 is regulated by the combination of growth factor/hormone signaling and nutrient availability to drive anabolic metabolism. mTORC2, on the other hand, is regulated by growth factor/hormone signaling to activate Akt. Together, mTORC1 and mTORC2 are key signaling nodes in the regulation of cell growth and proliferation, and dysregulation of these signaling pathways contributes to metabolic disease and cancer. In previous investigations, the authors had demonstrated that phosphorylation of mTOR on S2159 by TBK1 enhanced mTORC1 activation and downstream signaling to promote cell growth and proliferation (8). To investigate if TBK1 acts upstream of mTORC2 to regulate Akt-S473 phosphorylation through a similar mechanism, MEFs derived from mice with an alanine knock-in at S2159 (Mtor^A/A) were stimulated with EGF. A marked reduction of Akt-S473 phosphorylation was observed in Mtor^A/A MEFs compared with WT MEFs (Mtor^+/+). Using immunoprecipitation of Rictor to isolate the mTORC2 complex, TBK1 was observed to interact with mTORC2 and directly phosphorylate mTOR-S2159 to activate mTORC2 intrinsic kinase activity toward Akt-S473. TBK1 activity is increased by phosphorylation of S172 in its activation loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al. (6) found that EGF stimulation did not enhance S172 phosphorylation, supporting that it is the basal activity of TBK1 that is important for mTORC2 signaling downstream of growth factors. However, when RAW264.7 macrophages and primary bone marrow–derived macrophages were stimulated with the dsRNA mimetic poly(I:C), which induces TBK1-S172 phosphorylation, TBK1 and mTOR-S2159 were also found to be required for mTORC2-dependent phosphorylation of Akt-S473. Finally, the physiological regulation of mTORC2 activity by TBK1 was assessed by injection of Mtor^A/A and Mtor^+/+ mice with poly(I:C). Spleen tissue isolated from Mtor^A/A mice showed diminished Akt-S473 phosphorylation. Therefore, the authors conclude that under both basal and activated states, the activation of Akt by TBK1 is mediated through mTORC2 (Fig. 1) (6).Open in a separate windowFigure 1TBK1 promotes AKT activation through mTORC2. TBK1 interacts with and phosphorylates mTORC2 on S2159 of mTOR in response to either growth factor stimulation or innate immune agonists to promote AKT activation. Created using BioRender.com. mTORC2, mTOR complex 2; SGK, serum/glucocorticoid-regulated kinase; TBK1, TANK-binding kinase 1.TBK1 regulation of mTORC2-dependent phosphorylation of Akt shown in this study adds to the growing role of TBK1 as a signaling node in the regulation of cellular metabolism and growth by diverse environmental inputs. In response to foreign pathogens or inflammatory cytokines that stimulate TBK1 activation, or growth factor/hormone signaling that requires basal TBK1 activity, mTORC2 is activated to promote Akt-S473 phosphorylation and its downstream functions. Given that TBK1 expression and activity are enhanced in metabolic diseases and cancer, and the important role that Akt plays in these pathological conditions, identifying TBK1 as an upstream regulator of Akt reveals a potential novel approach to disrupt this signaling axis for therapeutic benefit (4, 9). In this regard, drugs such as amlexanox and other compounds are under investigation for their potential clinical use (10). Of note, the study by Tooley et al. (6) only examined the TBK1-dependent phosphorylation of Akt-S473 by mTORC2; mTORC2 also has additional substrates, including serum/glucocorticoid-regulated kinase and members of the PKC family (Fig. 1) (4). These kinases regulate unique cellular functions, such as regulation of the actin cytoskeleton. It will be important to determine if TBK1 regulates the activation of these kinases through mTORC2 as well, to understand the full impact of inhibiting TBK1 function therapeutically.The mechanism by which TBK1 regulates mTORC2 function has not been established. Although the kinase activity of TBK1 is required for Akt-S473 phosphorylation, neither phosphorylation of S172 in the activation loop of TBK1 nor phosphorylation of mTOR-S2159 was increased by growth factor stimulation in this study. Phosphorylation of S172 stabilizes the active confirmation of TBK1 and it is possible that additional uncharacterized phosphorylation sites could serve a similar function. Alternatively, the interaction of TBK1 with mTORC2 could impact TBK1 conformation, or multimerization, to enhance activity. Intracellular localization of mTORC2 could also be determined by TBK1 interaction, which could affect substrate availability. As little is known about the upstream regulation of mTORC2, the next acts should be elucidating further the mechanism of its activation by TBK1 to reveal novel approaches for targeting the mTORC2-Akt signaling pathway.     

RAD 001 (everolimus) prevents mTOR and Akt late re‐activation in response to imatinib in chronic myeloid leukemia

The mammalian target of rapamycin (mTOR) is one target of BCR‐ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug‐resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR‐phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re‐activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de‐phosphorylation of Akt at Ser⁴⁷³ in the hydrophobic motif of C‐terminal tail required for Akt full activation and precludes Akt re‐phosphorylation in response to IM. Moreover, RAD 001‐induced inhibition of Akt causes the de‐phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14‐3‐3 binding sites, TSC2 release from 14‐3‐3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin‐dependent kinase [CDK] inhibitor p27^Kip1, the stabilization of p27^Kip1 ligand with CDK2, and the G₀/G₁ arrest). RAD 001 cytotoxicity on cells not expressing the BCR‐ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect. J. Cell. Biochem. 109: 320–328, 2010. © 2009 Wiley‐Liss, Inc.