All Roads Lead to mTOR: Integrating Inflammation and Tumor Angiogenesis

Mammalian target of rapamycin (mTOR) is a crucial molecule in the control of cell size and proliferation; dysregulation of the mTOR pathway is commonly found in human cancers. Many cancer-promoting kinases have been identified as regulators of mTOR activity through phosphorylation and inactivation of the TSC1–TSC2 complex. Tumor-associated macrophages (TAMs) are tumor-promoting factors in inflammation-mediated tumor development, and the signaling molecules involved in TAMs-mediated tumor angiogenesis are not well understood. Therefore, it is urgent to elucidate the cross-talk between inflammatory cells and cancers and to explore the precise pathways involved in TAMs-induced tumor angiogenesis. Recently IKKβ was found to activate the mTOR pathway and to promote tumor angiogenesis through inactivation of the TSC1–TSC2 complex by phosphorylating TSC1. This finding provides critical insights into and suggests one mechanism behind inflammation-mediated tumor angiogenesis. In this extra-view, we briefly discuss the possible influence of TAMs-released proangiogenic factors on mTOR activation and propose a model of the cross-talk between tumors and TAMs in tumor angiogenesis.     

Association of focal adhesion kinase with tuberous sclerosis complex 2 in the regulation of s6 kinase activation and cell growth

Tuberous sclerosis complex 1 (TSC1) and TSC2 tumor suppressor proteins have been shown to negatively regulate cell growth through inhibition of the mammalian target of rapamycin (mTOR) pathway. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a critical role in integrin signaling. Here we identify a novel interaction between FAK and TSC2 and show that TSC2 is phosphorylated by FAK. Furthermore, we show that overexpression of FAK kinase dead mutant inhibits the phosphorylation of ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein-1, two key mTOR (mammalian target of rapamycin) downstream targets, and negatively regulates the cell size and that FAK regulation of S6K phosphorylation is through TSC2. Finally, we provide data that FAK plays a positive role in cell adhesion-induced S6K phosphorylation, whereas TSC2 is required for cell suspension-induced S6K inactivation. Together, these results suggest that FAK might regulate S6K activation and cell size through its interaction with and phosphorylation of TSC2 and also provide a previously unappreciated role of TSC2 in the regulation of mTOR signaling by cell adhesion.     

mTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation

The cell-cycle effects of mTORC1 are not fully understood. We provide evidence that mTOR-raptor phosphorylates SGK1 to modulate p27 function. Cellular mTOR activation, by refeeding of amino acid-deprived cells or by TSC2 shRNA, activated SGK1 and p27 phosphorylation at T157, and both were inhibited by short-term rapamycin treatment and by SGK1 shRNA. mTOR overexpression activated both Akt and SGK1, causing TGF-beta resistance through impaired nuclear import and cytoplasmic accumulation of p27. Rapamycin or raptor shRNA impaired mTOR-driven p70 and SGK1 activation, but not that of Akt, and decreased cytoplasmic p27. mTOR/raptor/SGK1 complexes were detected in cells. mTOR phosphorylated SGK1, but not SGK1-S422A, in vitro. SGK1 phosphorylated p27 in vitro. These data implicate SGK1 as an mTORC1 (mTOR-raptor) substrate. mTOR may promote G1 progression in part through SGK1 activation and deregulate the cell cycle in cancers through both Akt- and SGK-mediated p27 T157 phosphorylation and cytoplasmic p27 mislocalization.     

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling,tumor growth,and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.     

IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway

TNFalpha has recently emerged as a regulator linking inflammation to cancer pathogenesis, but the detailed cellular and molecular mechanisms underlying this link remain to be elucidated. The tuberous sclerosis 1 (TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway, and disruption of TSC1/TSC2 complex function may contribute to tumorigenesis. Here we show that IKKbeta, a major downstream kinase in the TNFalpha signaling pathway, physically interacts with and phosphorylates TSC1 at Ser487 and Ser511, resulting in suppression of TSC1. The IKKbeta-mediated TSC1 suppression activates the mTOR pathway, enhances angiogenesis, and results in tumor development. We further find that expression of activated IKKbeta is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. Our findings identify a pathway that is critical for inflammation-mediated tumor angiogenesis and may provide a target for clinical intervention in human cancer.     

Reduced Juvenile Long-Term Depression in Tuberous Sclerosis Complex Is Mitigated in Adults by Compensatory Recruitment of mGluR5 and Erk Signaling

Tuberous sclerosis complex (TSC) is a multisystem genetic disease that manifests with mental retardation, tumor formation, autism, and epilepsy. Heightened signaling through the mammalian target of rapamycin (mTOR) pathway is involved in TSC pathology, however it remains unclear how other signaling pathways are perturbed and contribute to disease symptoms. Reduced long-term depression (LTD) was recently reported in TSC mutant mice. We find that although reduced LTD is a feature of the juvenile mutant hippocampus, heightened expression of metabotropic glutamate receptor 5 and constitutively activated Erk signaling in the adult hippocampus drives wild-type levels of LTD. Increased mGluR5 and Erk results in a novel mTOR-independent LTD in CA1 hippocampus of adult mice, and contributes to the development of epileptiform bursting activity in the TSC2^+/− CA3 region of the hippocampus. Inhibition of mGluR5 or Erk signaling restores appropriate mTOR-dependence to LTD, and significantly reduces epileptiform bursting in TSC2^+/− hippocampal slices. We also report that adult TSC2^+/− mice exhibit a subtle perseverative behavioral phenotype that is eliminated by mGluR5 antagonism. These findings highlight the potential of modulating the mGluR5-Erk pathway in a developmental stage-specific manner to treat TSC.     

Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model

Tuberous sclerosis complex （TSC） is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inacti- vating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamyein （mTOR）, leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase （ERK/MAPK） signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin （sirolimus）, led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients.     

Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity

The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo.     

Pam (Protein associated with Myc) functions as an E3 ubiquitin ligase and regulates TSC/mTOR signaling

The tumor suppressor tuberin, encoded by the Tuberous Sclerosis Complex (TSC) gene TSC2, negatively regulates the mammalian target of rapamycin (mTOR) pathway, which plays a key role in the control of cell growth and proliferation. In addition to naturally occurring mutations, several kinases including Akt, RSK1, and ERK are known to phosphorylate and inactivate tuberin. We demonstrate a novel mechanism of tuberin inactivation through ubiquitination by Pam, a putative RING finger-containing E3 ubiquitin (Ub) ligase in mammalian cells. We show that Pam associates with E2 ubiquitin-conjugating enzymes, and tuberin can be ubiquitinated by Pam through its RING finger domain. Tuberin ubiquitination is independent of its phosphorylation by Akt, RSK1, and ERK kinases. Pam is also self-ubiquitinated through its RING finger domain. Moreover, the TSC1 protein hamartin, which forms a heterodimer with tuberin, protects tuberin from ubiquitination by Pam. However, TSC1 fails to protect a disease-associated missense mutant of TSC2 from ubiquitination by Pam. Furthermore, Pam knockdown by RNA interference (RNAi) in rat primary neurons elevates the level of tuberin, and subsequently inhibits the mTOR pathway. Our results provide novel evidence that Pam can function as an E3 Ub ligase toward tuberin and regulate mTOR signaling, suggesting that Pam can in turn regulate cell growth and proliferation as well as neuronal function through the TSC/mTOR pathway in mammalian cells.     

Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90(RSK1) (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90(RSK1).     

Differential involvement of IkappaB kinases alpha and beta in cytokine- and insulin-induced mammalian target of rapamycin activation determined by Akt

The mammalian target of rapamycin (mTOR) is a mediator of cell growth, survival, and energy metabolism at least partly through its ability to regulate mRNA translation. mTOR is activated downstream of growth factors such as insulin, cytokines such as TNF, and Akt-dependent signaling associated with oncoprotein expression. mTOR is negatively controlled by the tuberous sclerosis complex 1/2 (TSC1/2), and activation of Akt induces phosphorylation of TSC2, which blocks the repressive TSC1/2 activity. Previously, we showed that activation of mTOR in PTEN-deficient cancer cells involves IkappaB kinase (IKK) alpha, a catalytic subunit of the IKK complex that controls NF-kappaB activation. Recently, a distinct IKK subunit, IKKbeta, was shown to phosphorylate TSC1 to promote mTOR activation in an Akt-independent manner in certain cells stimulated with TNF and in some cancer cells. In this study, we have explored the involvement of both IKKalpha and IKKbeta in insulin- and TNF-induced mTOR activation. Insulin activation of mTOR requires Akt in a manner that involves IKKalpha, preferentially to IKKbeta, and TSC2 phosphorylation. TNF, in most cells examined, activates Akt to use IKKalpha to control mTOR activation. In MCF7 cells, TNF does not activate Akt and requires IKKbeta to activate mTOR. The results show that Akt-dependent signaling, induced by cytokines or insulin, alters the IKK subunit-dependent control of mTOR.     

Role of Prolactin Receptors in Lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.     

MAPK and mTOR pathways are involved in cadmium-induced neuronal apoptosis

Cadmium (Cd) may be accumulated in human body through long-term exposure to Cd-polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd-induced neurodegeneration, PC12 and SH-SY5Y cells were exposed to Cd. We observed that Cd-induced apoptosis in the cells in a time- and concentration-dependent manner. Cd rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c -Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd-induced apoptosis. Consistently, over-expression of dominant negative c- Jun or down-regulation of Erk1/2, but not p38 MAPK, partially prevented Cd-induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)-mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd-induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd-induced apoptosis. Down-regulation of mTOR by RNA interference also in part, rescued cells from Cd-induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd-induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd-induced neurodegeneration.     

AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2alpha

Prostaglandin F2alpha (PGF2alpha) is an important mediator of corpus luteum (CL) regression, although the cellular signaling events that mediate this process have not been clearly identified. It is established that PGF2alpha binds to a G-proteincoupled receptor (GPCR) to stimulate protein kinase C (PKC) and Raf-MEK-Erk signaling in luteal cells. The present experiments were performed to determine whether PGF2alpha stimulates the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway in steroidogenic luteal cells. We demonstrate that PGF2alpha treatment results in a timeand concentration-dependent stimulation of the phosphorylation and activation of S6K1. The stimulation of S6K1 in response to PGF2alpha treatment was abolished by the mTOR inhibitor rapamycin. Treatment with PGF2alpha did not increase AKT phosphorylation but increased the phosphorylation of Erk and the tumor suppressor protein tuberous sclerosis complex 2 (TSC2), an upstream regulator of mTOR. The effects of PGF2alpha were mimicked by the PKC activator PMA and inhibited by U0126, a MEK1 inhibitor. The activation of mTOR/S6K1 and putative down stream processes involving the translational apparatus (i.e. 4EBP1 phosphorylation, release of 4EBP1 binding in m(7)G cap binding assays, and the phosphorylation and synthesis of S6) were completely sensitive to treatment with rapamycin, implicating mTOR in the actions of PGF2alpha. Taken together, our data suggest that GPCR activation in response to PGF2alpha stimulates the mTOR pathway which increases the translational machinery in luteal cells. The translation of proteins under the control of mTOR may have implications for luteal development and regression and offer new strategies for therapeutic intervention in PGF2alpha-target tissues.     

AMPK phosphorylation of raptor mediates a metabolic checkpoint

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.     

Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.     

Testosterone-induced hypertrophy of L6 myoblasts is dependent upon Erk and mTOR

Testosterone increases the size and strength of skeletal muscle. This study further characterized the molecular mechanisms of the anabolic actions of testosterone on a rat myoblast cell line (L6 cells). Testosterone did not induce hypertrophy in L6 cells lacking the androgen receptor (AR). Hypertrophy was prevented by the AR antagonist bicalutamide and the mTOR inhibitor rapamycin. Testosterone induced Erk phosphorylation by 2 h, and mTOR autophosphorylation was elevated within 20 min; phosphorylation of p70S6 kinase was increased by 2 h. Inhibitors of Erk or PI3K blocked tesotosterone-induced hypertrophy. Erk phosphorylation returned to baseline when media containing testosterone was replaced at 16 h with fresh media lacking testosterone; when bicalutamide was added to testosterone-enriched media at 16 h, Erk phosphorylation remained elevated. Autophosphorylation of the IGF-1 receptor was minimally altered by testosterone at 20 min and unaffected at later time points; PI3K/PDK1-dependent phosphorylation of Akt was not altered by testosterone. These findings indicate that testosterone stimulates hypertrophy of L6 myoblasts through a mechanism that requires its binding to the AR and involves a signaling cascade dependent upon Erk and mTOR which is likely activated by substances released into the extracellular space which are not IGF-1 or other ligands for receptor tyrosine kinases.     

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.     

p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling

The tumor suppressor p53 is activated upon genotoxic and oxidative stress and in turn inhibits cell proliferation and growth through induction of specific target genes. Cell growth is positively regulated by mTOR, whose activity is inhibited by the TSC1:TSC2 complex. Although genotoxic stress has been suggested to inhibit mTOR via p53-mediated activation of mTOR inhibitors, the precise mechanism of this link was unknown. We now demonstrate that the products of two p53 target genes, Sestrin1 and Sestrin2, activate the AMP-responsive protein kinase (AMPK) and target it to phosphorylate TSC2 and stimulate its GAP activity, thereby inhibiting mTOR. Correspondingly, Sestrin2-deficient mice fail to inhibit mTOR signaling upon genotoxic challenge. Sestrin1 and Sestrin2 therefore provide an important link between genotoxic stress, p53 and the mTOR signaling pathway.