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.     

The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene‐induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site‐specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF‐receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus‐selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock‐in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN‐β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1‐mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation.     

mTOR kinase domain phosphorylation promotes mTORC1 signaling, cell growth, and cell cycle progression

The mammalian target of rapamycin complex 1 (mTORC1) functions as an environmental sensor to promote critical cellular processes such as protein synthesis, cell growth, and cell proliferation in response to growth factors and nutrients. While diverse stimuli regulate mTORC1 signaling, the direct molecular mechanisms by which mTORC1 senses and responds to these signals remain poorly defined. Here we investigated the role of mTOR phosphorylation in mTORC1 function. By employing mass spectrometry and phospho-specific antibodies, we demonstrated novel phosphorylation on S2159 and T2164 within the mTOR kinase domain. Mutational analysis of these phosphorylation sites indicates that dual S2159/T2164 phosphorylation cooperatively promotes mTORC1 signaling to S6K1 and 4EBP1. Mechanistically, S2159/T2164 phosphorylation modulates the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1-associated mTOR S2481 autophosphorylation. Moreover, mTOR S2159/T2164 phosphorylation promotes cell growth and cell cycle progression. We propose a model whereby mTOR kinase domain phosphorylation modulates the interaction of mTOR with regulatory partner proteins and augments intrinsic mTORC1 kinase activity to promote biochemical signaling, cell growth, and cell cycle progression.     

mTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Activity Is Associated with Phosphorylation of Raptor by mTOR

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser⁸⁶³, reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser⁸⁶³ mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.Mammalian target of rapamycin (mTOR)² has been shown to function as a critical controller in cellular growth, survival, metabolism, and development (1). mTOR, a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each of which catalyzes the phosphorylation of different substrates (1). The best characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also known as PHAS) and p70 S6 kinase (S6K) (1), whereas mTORC2 phosphorylates the hydrophobic and turn motifs of protein kinase B (Akt/protein kinase B) (2) and protein kinase C (3, 4). mTORC1 constitutively consists of mTOR, raptor, and mLst8/GβL (1), whereas the proline-rich Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that disassociates after growth factor stimulation (5, 6). Raptor is essential for mTORC1 activity by providing a substrate binding function (7) but also plays a regulatory role on mTORC1 with stimuli of growth factors and nutrients (8). In response to insulin, raptor binding to substrates is elevated through the release of the competitive inhibitor PRAS40 from mTORC1 (9, 10) because PRAS40 and the substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus sequence, the TOR signaling (TOS) motif (10–14). In response to amino acid sufficiency, raptor directly interacts with a heterodimer of Rag GTPases and promotes mTORC1 localization to the Rheb-containing vesicular compartment (15).mTORC1 integrates signaling pathways from growth factors, nutrients, energy, and stress, all of which generally converge on the tuberous sclerosis complex (TSC1-TSC2) through the phosphorylation of TSC2 (1). Growth factors inhibit the GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via the PI3K/Akt pathway (16, 17), whereas energy depletion activates TSC2 GTPase-activating protein activity by stimulating AMP-activated protein kinase (AMPK) (18). Rheb binds directly to mTOR, albeit with very low affinity (19), and upon charging with GTP, Rheb functions as an mTORC1 activator (6). mTORC1 complexes isolated from growth factor-stimulated cells show increased kinase activity yet do not contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding to mTOR leads to an increase in mTORC1 activity toward substrates, and what the role of raptor is in this activation is currently unknown. More recently, the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative regulation mechanism independent of TSC2 in response to energy supply (20). RSK-mediated raptor phosphorylation enhances mTORC1 activity and provides a mechanism whereby stress may activate mTORC1 independent of the PI3K/Akt pathway (21). Therefore, the phosphorylation status of raptor can be critical for the regulation of mTORC1 activity.In this study, we investigated phosphorylation sites in raptor catalyzed by mTOR. Using two-dimensional phosphopeptide mapping, we found that Ser⁸⁶³ and Ser⁸⁵⁹ in raptor were phosphorylated by mTOR both in vivo and in vitro. mTORC1 activity in vitro and in vivo is associated with the phosphorylation of Ser⁸⁶³ in raptor.     

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

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.     

mTORC2 is a tyrosine kinase

The mammalian target of rapamycin (mTOR), a protein kinase, is the centre of huge attention due to its importance in intracellular signaling and in health and disease. In their recent study, Yin et al. show that mTOR can regulate signaling through the insulin-like growth factor 1 receptor and that it possesses a new enzymatic activity — the ability to phosphorylate proteins on tyrosine residues.mTOR is a large, multi-domain protein; its catalytic domain resembles that of lipid kinases such as phosphoinositide 3-kinase (PI 3-kinase), but mTOR actually has protein kinase activity, adding phosphate groups to serine or threonine residues in a growing catalog of substrates, many of which are involved in anabolic pathways.mTOR binds to several protein partners in the cell to form two distinct types of complexes, termed mTOR complexes 1 and 2 (mTORC1/2¹). These differ in their protein components, substrate specificity and regulation. For example, mTORC1 is activated by amino acids, and by hormones and growth factors. mTORC1 contains a protein termed Raptor which allows it to phosphorylate substrates such as the ribosomal protein S6 kinases (S6Ks), and this effect is blocked by rapamycin.mTORC2 contains Rictor in place of Raptor and therefore phosphorylates a distinct set of substrates. These include regulatory (so-called ''hydrophobic'') sites in a family of protein kinases which include Akt, also called protein kinase B (PKB). Rapamycin does not directly inhibit mTORC2 function, but can impair it after longer-term treatment². The regulation of mTORC2 activity remains poorly understood.mTOR complexes play multifaceted roles in insulin signaling. For example, Akt plays key roles in insulin signaling, mediating the regulation of various proteins involved in the effects of this hormone on metabolism, e.g., glucose transport. Akt signaling indirectly activates mTORC1. In turn, mTORC1 regulates key anabolic processes including protein, lipid and ribosome synthesis. However, mTORC1 can, via the S6Ks, inhibit insulin signaling. This involves the phosphorylation of insulin receptor substrates 1 or 2 (IRS1/2), a crucial link between insulin (and related) receptors and downstream signalling protein, e.g., Akt.The receptors for insulin (InsR) and insulin-like growth factor I (IGF-IR) are ligand-activated tyrosine kinases, which undergo autophosphorylation allowing them to phosphorylate additional proteins such as IRS1. In turn, phosphorylated IRS1 binds PI 3-kinase; this leads to enhanced production of phosphatidylinositol 3,4,5-trisphosphate, PIP₃, and to activation of Akt.Yin et al.³ found that rapamycin led to increased phosphorylation of InsR and IGF-IR at key autophosphorylation sites, reflecting increased kinase activity of these receptors.Knockdown of mTOR or Rictor, or treatment of cells with an inhibitor of mTOR kinase activity, Torin 2, decreased the rapamycin-induced phosphorylation of InsR or IGF-IR, while Raptor knockdown had the converse effect. This indicates the effect requires mTORC2; indeed, the authors show that mTORC2 binds to these receptors, apparently via IRS1/2. However, mTORC2 does not appear to directly phosphorylate IRS1/2. One possible way in which mTORC2 increases tyrosine phosphorylation of InsR or IGF-IR is by stimulating the kinase activity of the receptors which then catalyse the phosphorylation of the receptors on tyrosine. The authors ruled this out, by using kinase-dead versions of the receptors or mTOR. Therefore, mTORC2 promotes the tyrosine phosphorylation of InsR/IGF-1R, which is required for downstream signaling from these receptors. While these authors clearly show that rapamycin causes increased phosphorylation of the mTORC2 substrate AKT, earlier studies showed that, at similar time points of treatment in the same cell-type, rapamycin inhibited AKT phosphorylation indicating interference with mTORC2 function². It is not clear how rapamycin promotes mTORC2 function under the conditions used in this study. Another study⁴ found that mTORC2 promotes degradation of IRS1, suggesting, in contrast to the conclusions of Yin et al., that mTORC2 can promote insulin resistance. These and other data suggest that the web of interactions between these signaling components is indeed very complex (Figure 1).Open in a separate windowFigure 1Summary of the signalling connections discussed here, including the new link described by Yin et al.³ between mTORC2 and the insulin/IGF-1 receptors. Phosphorylation sites are shown schematically (not all are indicated) as ''P'' in a yellow background; Y, S and T indicate tyrosine, serine and threonine respectively. Green and red arrows show activating and inhibitory phosphorylation events respectively. The gray arrow and ''?'' indicate potential further tyrosine phosphorylation events catalysed by mTORC2. Solid arrows show direct phosphorylation events; dashed lines are indirect signalling links.mTOR has previously only been reported to act on serine or threonine residues; the present report shows that mTOR can efficiently phosphorylate tyrosines in vitro using either recombinant InsR or peptides as substrate. These data reveal that mTORC2 function is a ''dual-specificity'' protein kinase phosphorylating tyrosine as well as serine/threonine sites. Interestingly, mTORC1 was unable to phosphorylate tyrosines.Does the mTORC2-stimulated phosphorylation of the InsR/IGF-1R play a role in the actions of the ligands for these receptors? To test this, the authors examined the Rictor knockdown on HepG2 cell proliferation. While this had no effect in the absence of insulin or IGF-1, depletion of Rictor did inhibit proliferation in IGF-1- or insulin-stimulated conditions. Rictor overexpression increased proliferation, an effect that requires the activity of the InsR/IGF-1R.What are the main implications of these data? First, rapamycin may actually promote signaling from the InsR/IGF-1R through mTORC2 (as well as via Grb10, a target for mTORC1 itself^5,6) both by the mechanism delineated here and by abrogating the feedback loop from mTORC1 via the S6Ks to IRS1. Second, combining Ins/IGF-1R receptor inhibitors with mTOR inhibitors may be a more effective anti-cancer treatment than inhibiting the individual pathways. Third, mTORC2 may phosphorylate additional, so far unidentified proteins on tyrosine, adding to the growing repertoire of mTOR substrates.     

Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity

Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt^T308 and P-Akt^S473, and previous work showed decreased P-Akt^S473 in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt^T308 and P-Akt^S473 in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt^T308. Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt^S473 or P-Akt^T308 upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA–dependent phosphorylation of Akt^S473 and Akt^T308 in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.     

The functions of mTOR in ischemic diseases

Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase and that forms two multiprotein complexes known as the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTOR regulates cell growth, proliferation and survival. mTORC1 is composed of the mTOR catalytic subunit and three associated proteins: raptor, mLST8/GβL and PRAS40. mTORC2 contains mTOR, rictor, mLST8/GβL, mSin1, and protor. Here, we discuss mTOR as a promising anti-ischemic agent. It is believed that mTORC2 lies down-stream of Akt and acts as a direct activator of Akt. The different functions of mTOR can be explained by the existence of two distinct mTOR complexes containing unique interacting proteins. The loss of TSC2, which is upstream of mTOR, activates S6K1, promotes cell growth and survival, activates mTOR kinase activities, inhibits mTORC1 and mTORC2 via mTOR inhibitors, and suppresses S6K1 and Akt. Although mTOR signaling pathways are often activated in human diseases, such as cancer, mTOR signaling pathways are deactivated in ischemic diseases. From Drosophila to humans, mTOR is necessary for Ser473 phosphorylation of Akt, and the regulation of Akt-mTOR signaling pathways may have a potential role in ischemic disease. This review evaluates the potential functions of mTOR in ischemic diseases. A novel mTOR-interacting protein deregulates over-expression in ischemic disease, representing a new mechanism for controlling mTOR signaling pathways and potential therapeutic strategies for ischemic diseases.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

The TSC1-TSC2 complex is required for proper activation of mTOR complex 2

The mammalian target of rapamycin (mTOR) is a protein kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. Both complexes phosphorylate a hydrophobic motif on downstream protein kinases, which contributes to the activation of these kinases. mTOR complex 1 (mTORC1) phosphorylates S6K1, while mTORC2 phosphorylates Akt. The TSC1-TSC2 complex is a critical negative regulator of mTORC1. However, how mTORC2 is regulated and whether the TSC1-TSC2 complex is involved are unknown. We find that mTORC2 isolated from a variety of cells lacking a functional TSC1-TSC2 complex is impaired in its kinase activity toward Akt. Importantly, the defect in mTORC2 activity in these cells can be separated from effects on mTORC1 signaling and known feedback mechanisms affecting insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Our data also suggest that the TSC1-TSC2 complex positively regulates mTORC2 in a manner independent of its GTPase-activating protein activity toward Rheb. Finally, we find that the TSC1-TSC2 complex can physically associate with mTORC2 but not mTORC1. These data demonstrate that the TSC1-TSC2 complex inhibits mTORC1 and activates mTORC2, which through different mechanisms promotes Akt activation.     

Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2

The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.     

PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338-351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor beta (PDGFRbeta). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRbeta expression and signaling.     

ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.     

Role of PRAS40 in Akt and mTOR signaling in health and disease

The proline-rich Akt substrate of 40 kDa (PRAS40) acts at the intersection of the Akt- and mammalian target of rapamycin (mTOR)-mediated signaling pathways. The protein kinase mTOR is the catalytic subunit of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTORC2, that link energy and nutrients to the regulation of cellular growth and energy metabolism. Activation of mTOR in response to nutrients and growth factors results in the phosphorylation of numerous substrates, including the phosphorylations of S6 kinase by mTORC1 and Akt by mTORC2. Alterations in Akt and mTOR activity have been linked to the progression of multiple diseases such as cancer and type 2 diabetes. Although PRAS40 was first reported as substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity. Adding to the complexity is that gene silencing studies indicate that PRAS40 is also necessary for the activity of the mTORC1 complex. This review summarizes the regulation and potential function(s) of PRAS40 in the complex Akt- and mTOR-signaling network in health and disease.     

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.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.