The PI3K-Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR; also known as the mammalian target of rapamycin) is a highly deregulated pathway in cancers. mTOR exists in two complexes, mTORC1 and mTORC2. Akt phosphorylated at T308 inhibits TSC1/2 complex to activate mTORC1; mTORC2 is recognized as the kinase phosphorylating Akt at S473. Inhibition of autophagy by mTORC1 was shown to rescue disheveled (Dvl) leading to activation of Wnt pathway. Cyclin D1 and the c-Myc are activated by the Wnt signaling. Cyclin D1 is a key player in initiation of cell cycle. c-Myc triggers metabolic reprograming in G1 phase of cell cycle, which also activates the transcription factors like FoxO and p53 that play key roles in promoting the progression of cell cycle. While the role of p53 in cancer cell metabolism in arresting glycolysis and inhibition of pentose phosphate pathway has come to be recognized, there are confusions in the literature on the role of FoxO and that of rictor. FoxO was shown to be the transcription factor of rictor, in addition to the cell cycle inhibitors like p21. Rictor has dual roles; inhibition of c-Myc and constitution of mTORC2, both of which are key factors in the exit of G1-S phase and entry into G2 phase of cell cycle. A model is presented in this article, which suggests that the PI3K-Akt-mTOR and Wnt pathways converge and regulate the progression of cell cycle through G0-G1-S-phases and reprogram the metabolism in cancer cells. This model is different from the conventional method of looking at individual pathways triggering the cell cycle.
mTORC1; autophagy; Wnt; G1-S; cell cycle
Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium-specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours.
clear cell; endometrial carcinoma; mouse model; p53; serous
The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient sensitive protein kinase that is aberrantly activated in many human cancers. However, whether dysregulation of mTORC1 signaling in normal tissues contributes to cancer risk is unknown. Here, we focused on hepatocellular carcinoma because it is a cancer with clear links to environmental factors that affect mTORC1, including dietary influences. Genetic ablation of the mTORC1 inhibitory component Tsc1 results in constitutively elevated mTORC1 signaling, an effect similar to that of obesity on this pathway. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes known to accompany the primary etiologies of this cancer, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling caused unresolved endoplasmic reticulum stress and defects in autophagy, which contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we demonstrate a previously unrecognized role for mTORC1 in carcinogenesis, perhaps representing a key molecular link between cancer risk and environmental factors, such as diet.
The phosphoinositide 3-kinase (PI3K) pathway regulates mammalian cell growth, survival, and motility and plays a major pathogenetic role in human prostate cancer (PCa). However, the oncogenic contributions downstream of the PI3K pathway made by mammalian target of rapamycin complex 1 (mTORC1)–mediated cell growth signal transduction in PCa have yet to be elucidated in detail. Here, we engineered constitutive mTORC1 activation in prostate epithelium by a conditional genetic deletion of tuberous sclerosis complex 1 (Tsc1), a potent negative regulator of mTORC1 signaling. Epithelial inactivation was not immediately tumorigenic, but Tsc1-deficient mice developed prostatic intraepithelial neoplasia (mPIN) in lateral and anterior prostates by 6 months of age, with increasing disease penetrance over time. Lateral prostate lesions in 16- to 22-month-old mutant mice progressed to two types of more advanced lesions, adenomatous gland forming lesion (Type 1) and atypical glands embedded in massively expanded reactive stroma (Type 2). Both Type 1 and Type 2 lesions contained multiple foci of microinvasive carcinoma. Epithelial neoplastic and atypical stromal lesions persisted despite 4 weeks of RAD001 chemotherapy. Rapalogue resistance was not due to AKT or extracellular signal-regulated kinase 1/2 activation. Expression of the homeobox gene Nkx3.1 was lost in Tsc1-deficient mPIN, and it cooperated with TSC1 loss in mPIN initiation in doubly mutant Tsc1:Nkx3.1 prostatic epithelial knockout mice. Thus, TSC1 inactivation distal to PI3K and AKT activation is sufficient to activate a molecular signaling cascade producing prostatic neoplasia and focal carcinogenesis.
Binge drinking often triggers compromised myocardial contractile function while activating AMP-activated protein kinase (AMPK). Given the role of AMPK in the initiation of autophagy through the mammalian target of rapamycin complex 1 (mTORC1) and Unc51-like kinase (ULK1), this study was designed to examine the impact of AMPK deficiency on cardiac function and the mechanism involved with a focus on autophagy following an acute ethanol challenge.
Methods and results
Wild-type (WT) and transgenic mice overexpressing a kinase-dead (KD) α2 isoform (K45R mutation) of AMPK were challenged with ethanol. Glucose tolerance, echocardiography, Langendorff heart and cardiomyocyte contractile function, autophagy, and autophagic signalling including AMPK, acetyl-CoA carboxylase (ACC), mTOR, the mTORC1-associated protein Raptor, and ULK1 were examined. Ethanol exposure triggered glucose intolerance and compromised cardiac contraction accompanied by increased phosphorylation of AMPK and ACC as well as autophagosome accumulation (increased LC3II and p62), the effects of which were attenuated or mitigated by AMPK deficiency or inhibition. Ethanol dampened and stimulated, respectively, the phosphorylation of mTOR and Raptor, the effects of which were abolished by AMPK deficiency. ULK1 phosphorylation at Ser757 and Ser777 was down-regulated and up-regulated, respectively, by ethanol, the effect of which was nullified by AMPK deficiency or inhibition. Moreover, the ethanol challenge enhanced LC3 puncta in H9c2 cells and promoted cardiac contractile dysfunction, and these effects were ablated by the inhibition of autophagy or AMPK. Lysosomal inhibition failed to accentuate ethanol-induced increases in LC3II and p62.
In summary, these data suggest that ethanol exposure may trigger myocardial dysfunction through a mechanism associated with AMPK-mTORC1-ULK1-mediated autophagy.
Ethanol; AMPK deficiency; Autophagy; Cardiac function; ULK1
A major goal of biomedical research has been the identification of molecular mechanisms that can enhance memory. Here we report a novel signaling pathway that regulates the conversion from short- to long-term memory. The mTOR complex 2 (mTORC2), which contains the key regulatory protein Rictor (Rapamycin-Insensitive Companion of mTOR), was discovered only recently, and little is known about its physiological role. We show that conditional deletion of rictor in the postnatal murine forebrain greatly reduces mTORC2 activity and selectively impairs both long-term memory (LTM) and the late (but not the early) phase of hippocampal long-term potentiation (LTP). Actin polymerization is reduced in the hippocampus of mTORC2-deficient mice and its restoration rescues both L-LTP and LTM. More importantly, a compound that selectively promotes mTORC2 activity converts early-LTP into late-LTP and enhances LTM. These findings indicate that mTORC2 could be a novel therapeutic target for the treatment of cognitive dysfunction.
mTOR complexes; actin assembly; spatial memory; fear memory; cognitive enhancer
ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling.1–3 Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.
ULK1; ULK2; Atg5; raptor; mTOR
Signaling downstream of mechanistic target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) controls specific and distinct aspects of insulin action and nutrient homeostasis in an interconnected and as yet unclear way. Mice lacking the mTORC1 substrate S6 kinase 1 (S6K1) maintain proper glycemic control with a high-fat diet. This phenotype is accompanied by insulin hypersensitivity, Akt- and AMP-activated kinase upregulation, and increased lipolysis in adipose tissue and skeletal muscle. Here, we show that, when S6K1 inactivation is combined with the deletion of the mTORC2 substrate Akt2, glucose homeostasis is compromised due to defects in both insulin action and β-cell function. After a high-fat diet, the S6K1−/−
Akt2−/− double-mutant mice do not become obese, though they are severely hyperglycemic. Our data demonstrate that S6K1 is required for pancreatic β-cell growth and function during adaptation to insulin resistance states. Strikingly, the inactivation of two targets of mTOR and phosphatidylinositol 3-kinase signaling is sufficient to reproduce major hallmarks of type 2 diabetes.
The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR, also called DEPDC6, as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1; promotes cell growth and survival; and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of Multiple Myelomas harboring Cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in Multiple Myeloma cells represents a new mechanism for activating PI3K/Akt signaling and promoting cell survival.
The mTORC1 signaling pathway integrates environmental conditions into distinct signals for cell growth by balancing anabolic and catabolic processes. Accordingly, energetic stress inhibits mTORC1 signaling predominantly through AMPK-dependent activation of TSC1/2. Thus, TSC1/2-/- cells are hypersensitive to glucose deprivation and this has been linked to increased p53 translation and activation of apoptosis. Herein, we show that mTORC1 inhibition during glucose deprivation prevented not only the execution of death, but also induction of energetic stress. mTORC1 inhibition during glucose deprivation decreased AMPK activation and allowed ATP to remain high, which was both necessary and sufficient for protection. This effect was not due to increased catabolic activities such as autophagy, but rather exclusively due to decreased anabolic processes, reducing energy consumption. Specifically, TSC1/2-/- cells become highly dependent on glutamate dehydrogenase-dependent glutamine metabolism via the TCA cycle for survival. Therefore, mTORC1 inhibition during energetic stress is primarily to balance metabolic demand with supply.
The cell signaling pathways of the mammalian target of rapamycin (mTOR) are broad in nature, but are tightly integrated through the protein complexes of mTORC1 and mTORC2. Although both complexes share some similar subcomponents, mTORC1 is primarily associated with the regulatory protein Raptor while mTORC2 relies upon Rictor. Pathways of mTOR that partner with Wnt as well as growth factor signaling are vital for endothelial and cardiomyocyte growth. In mature differentiated endothelial cells and cardiac cells, mTOR activation regulates both apoptotic and autophagic pathways during oxidative stress that can be dependent upon the activation of protein kinase B (Akt). These protective pathways of mTOR can promote angiogenesis and limit acute cell death to foster cardiac repair and tissue regeneration. However, under some conditions, blockade of mTOR pathways may be necessary to limit vasculopathy and promote microcirculatory flow. Future work that further elucidates the vital regulatory pathways of mTOR can offer new therapeutic insights for the treatment of cardiovascular diseases.
Akt; cardiac; endothelial; erythropoietin; TORC1; TORC2; Wnt; wingless
Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2.
High levels of the adipocytokine leptin are associated with reduced risk of Alzheimer’s disease (AD). Leptin treatment also reduces β-amyloid (Aβ) levels in in vivo and in vitro models of AD. Aβ and leptin interact with the Akt/mammalian target of rapamycin complex1 (mTORC1) signaling pathway. Akt/mTORC1 activation reduces tau phosphorylation through the inhibition of the downstream enzyme GSK-3β. mTORC1 also regulates translation of many proteins including leptin. While Aβ has been shown to inactivate Akt, inhibit mTORC1, and facilitate the phosphorylation of tau, leptin activates both Akt and mTORC1 and reduces tau phosphorylation. However, the extent to which Aβ may modulate leptin expression and increase tau phosphorylation involving Akt/mTORC1 has not been determined. In this study, we show that incubation of organotypic slices from rabbit hippocampus with Aβ downregulates leptin expression, inhibits Akt, activates GSK-3β, increases tau phosphorylation, and inactivates mTORC1. Leptin treatment reverses Aβ effects by alleviating Akt inhibition, preventing GSK-3β activation, reducing tau phosphorylation, and activating mTORC1. On the other hand, Rapamycin, an allosteric inhibitor of mTORC1, downregulates leptin expression, increases tau phosphorylation, and does not affect Akt and GSK-3β. Our results demonstrate for the first time that Aβ regulates leptin expression and tau phosphorylation through mTORC1.
Alzheimer’s disease; β-amyloid; Leptin; mTOR; Tau; Organotypic slices
mTORC1 inhibitors, including rapamycin and its analogs, have been actively studied both pre-clinically and clinically. However, the single treatment of mTORC1 inhibitors has been modest in most cancer types. We have previously demonstrated that the activation of PI3K/Akt and MEK/ERK signaling pathways attenuates the anticancer efficacy of mTORC1 inhibitors. In this study, we report that mTORC1 inhibition also phosphorylates and inactivates GSK3β, which is a tumor suppressor in lung cancer. Moreover, we show that perifosine, as an Akt inhibitor, decreases rapamycin-induced phosphorylation of GSK3β and elevated p-GSK3β levels in rapamycin-resistant cell lines. Combination of perifosine with mTORC1 inhibitors showed enhanced anticancer efficacy both in cell cultures and in a xenograft mouse model. In addition, perifosine inhibits the growth of both rapamycin sensitive and resistant A549 cells. However, inhibition of GSK3β by a selective inhibitor- LiCl, or downregulation of GSK3β expression by siRNA, reverses the growth inhibitory effects of perifosine on rapamycin resistant cells, suggesting the important role of GSK3β activation in enhancing mTORC1 inhibitors efficacy by perifosine. Thus, our results provide a potential therapeutic strategy to enhance mTORC1-targeted cancer therapy by using perifosine or targeting GSK3β.
GSK3β; lung cancer; mTOR; perifosine; rapamycin
The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin’s ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.
Integrin signaling promotes, through p21-activated kinase, phosphorylation and inactivation of the tumor suppressor merlin, thus removing a block to mitogenesis in normal cells. However, the biochemical function of merlin and the effector pathways critical for the pathogenesis of malignant mesothelioma and other NF2-related malignancies are not known. We report that integrin-specific signaling promotes activation of mTORC1 and cap-dependent mRNA translation. Depletion of merlin rescues mTORC1 signaling in cells deprived of anchorage to a permissive extracellular matrix, suggesting that integrin signaling controls mTORC1 through inactivation of merlin. This signaling pathway controls translation of the cyclin D1 mRNA and, thereby, cell cycle progression. In addition, it promotes cell survival. Analysis of a panel of malignant mesothelioma cell lines reveals a strong correlation between loss of merlin and activation of mTORC1. Merlin-negative lines are sensitive to the growth-inhibitory effect of rapamycin, and the expression of recombinant merlin renders them partially resistant to rapamycin. Conversely, depletion of merlin restores rapamycin sensitivity in merlin-positive lines. These results indicate that integrin-mediated adhesion promotes mTORC1 signaling through the inactivation of merlin. Furthermore, they reveal that merlin-negative mesotheliomas display unregulated mTORC1 signaling and are sensitive to rapamycin, thus providing a preclinical rationale for prospective, biomarker-driven clinical studies of mTORC1 inhibitors in these tumors.
mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling.
Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1β, IFN-γ and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways.
We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation and mortality in response to endotoxin, and may occur via multiple pathways.
Prostate cancer (PCa) is dependent on androgen receptor signaling and aberrations of the PI3K-Akt-mTORC1 pathway mediating excessive and sustained growth signaling. The nutrient-sensitive kinase mTORC1 is upregulated in nearly 100% of advanced human PCas. Oncogenic mTORC1 signaling activates key subsets of mRNAs that cooperate in distinct steps of PCa initiation and progression. Epidemiological evidence points to increased dairy protein consumption as a major dietary risk factor for the development of PCa. mTORC1 is a master regulator of protein synthesis, lipid synthesis and autophagy pathways that couple nutrient sensing to cell growth and cancer. This review provides evidence that PCa initiation and progression are promoted by cow´s milk, but not human milk, stimulation of mTORC1 signaling. Mammalian milk is presented as an endocrine signaling system, which activates mTORC1, promotes cell growth and proliferation and suppresses autophagy. Naturally, milk-mediated mTORC1 signaling is restricted only to the postnatal growth phase of mammals. However, persistent consumption of cow´s milk proteins in humans provide highly insulinotropic branched-chain amino acids (BCAAs) provided by milk´s fast hydrolysable whey proteins, which elevate postprandial plasma insulin levels, and increase hepatic IGF-1 plasma concentrations by casein-derived amino acids. BCAAs, insulin and IGF-1 are pivotal activating signals of mTORC1. Increased cow´s milk protein-mediated mTORC1 signaling along with constant exposure to commercial cow´s milk estrogens derived from pregnant cows may explain the observed association between high dairy consumption and increased risk of PCa in Westernized societies. As well-balanced mTORC1-signaling plays an important role in appropriate prostate morphogenesis and differentiation, exaggerated mTORC1-signaling by high cow´s milk consumption predominantly during critical growth phases of prostate development and differentiation may exert long-term adverse effects on prostate health. Attenuation of mTORC1 signaling by contemporary Paleolithic diets and restriction of dairy protein intake, especially during mTORC1-dependent phases of prostate development and differentiation, may offer protection from the most common dairy-promoted cancer in men of Western societies.
Cancer prevention; Dairy; Estrogens; IGF-1; Insulin; Leucine; Metformin; Milk signaling; Morphogenesis; mTORC1; Prostate cancer
Recent work suggests a link between endocytic trafficking and mTORC1 signaling. This paper demonstrates a specific requirement for the integrity of the late endosomal compartment for amino acid and insulin-stimulated mTORC1 signaling to downstream effectors.
The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid–stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2−/− MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid– and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.
The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the “In Cell Western” (ICW) technique using the Aerius infrared imaging system (LI-COR® Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius® scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling.
The mechanistic target of rapamycin (mTOR) plays a central role in cellular growth and metabolism. mTOR forms two distinct protein complexes, mTORC1 and mTORC2. Much is known about the regulation and functions of mTORC1 due to availability of a natural compound, rapamycin, that inhibits this complex. Studies that define mTORC2 cellular functions and signaling have lagged behind. The development of pharmacological inhibitors that block mTOR kinase activity, and thereby inhibit both mTOR complexes, along with availability of mice with genetic knockouts in mTOR complex components have now provided new insights on mTORC2 function and regulation. Since prolonged effects of rapamycin can also disrupt mTORC2, it is worth re-evaluating the contribution of this less-studied mTOR complex in cancer, metabolic disorders and aging. In this review, we focus on recent developments on mammalian mTORC2 signaling mechanisms and its cellular and tissue-specific functions.
mTOR; mTORC2; rictor; cancer; metabolism; ribosomes; protein synthesis; protein maturation; AGC kinases; growth factor signaling
Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S473), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase and the founding member of a signaling pathway that regulates many fundamental features of cell growth and division. In cells, mTOR acts as the catalytic subunit of two functionally distinct complexes, called mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Together, these complexes coordinate a variety of processes that include protein translation, autophagy, proliferation, survival and metabolism in response to nutrient, energy and growth factor signals. Consistent with its role as a growth-promoting pathway, numerous studies have found that Mtor signaling is hyper-activated in a broad spectrum of human cancers. In particular, mTORC2 is considered a primary effector of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, which is mutated in a majority of human cancers, in part through its ability to phosphorylate and regulate the proto-oncogene Akt/PKB. Many biological functions of mTOR have been pharmacologically explored using the natural product rapamycin, an allosteric inhibitor that has been reviewed extensively elsewhere. This review will focus specifically on the development of small molecule ATP-competitive inhibitors of mTOR and their prospects as a targeted therapy.
mTOR; PI3K; Cancer
Mammalian target of rapamycin (mTOR) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles 1, 2 intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates 3-8. Here we report that lysosomal positioning coordinates anabolic and catabolic responses to changes in nutrient availability by orchestrating early plasma membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, while starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH (pHi). Lysosomal positioning regulates mTORC1 signalling, which, in turn, influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting both at the initiation and termination stages of the process. Our findings provide a fundamental physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1–JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1–JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE–JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1–JNK pathway.
Akt; endoplasmic reticulum stress; IRE1; mTOR; rapamycin