FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrest in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (1) strains that lack FKBP proline rotamase, encoded by FPR1, are viable and fully resistant to rapamycin and (2) FK506 antagonizes rapamycin toxicity in vivo. Mutations that confer rapamycin resistance alter conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity have been identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle (Heitman, 1991).
Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. It is proposed that the TORs, two related phosphatidylinositol kinase homologs, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients (Barbet, 1996).
The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon. The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein. Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown. TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2. The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3. Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4. TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2. Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm (Beck, 1999).
Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to human. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. Gene expression in yeast cells exposed to rapamycin has been analyzed using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, these results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability (Cardenas, 1999).
The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. The immediate transcriptional response of yeast grown in rich media and treated with rapamycin was measured to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule (Hardwick, 1999).
The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs). Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubulen assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast. TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. These data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p (Choi, 2000).
Rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restores pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromises pseudohyphal differentiation and renders it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibit a pseudohyphal growth defect and are markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases have no effect on pseudohyphal differentiation. These findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restore filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi is also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients (Cutler, 2001).
The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. gad8, which encodes a Ser/Thr kinase of the AGC family, was isolated as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 causes phenotypes similar to those of tor1 disruption. Gad8p is less phosphorylated and its kinase activity is undetectable in tor1Delta cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, are important for the kinase activity of Gad8p. Tor1p is responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appears to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR-PDK1-S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes (Matsuo, 2003).
The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. Rapamycin and nutrient starvation, conditions inhibiting TOR, are found to lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. Evidence is presented that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, these results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability (Tsang, 2003).
In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. Yeast TOR1 has been shown to have an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. An intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, is toxic in yeast, and overexpression of wild-type TOR1 suppresses this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppresses growth inhibition by the TOR-toxic domains. In conclusion, these findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM (Alarcon, 1999).
The regulation of ribosome biogenesis in response to environmental conditions is a key aspect of cell growth control. Ribosomal protein (RP) genes are regulated by the nutrient-sensitive, conserved target of rapamycin (TOR) signaling pathway. TOR controls the subcellular localization of protein kinase A (PKA) and the PKA-regulated kinase YAK1. However, the target transcription factor(s) of the TOR-PKA pathway are unknown. Regulation of RP gene transcription via TOR and PKA in yeast involves the Forkhead-like transcription factor FHL1 and the two cofactors IFH1 (a coactivator) and CRF1 (a corepressor). TOR, via PKA, negatively regulates YAK1 and maintains CRF1 in the cytoplasm. Upon TOR inactivation, activated YAK1 phosphorylates and activates CRF1. Phosphorylated CRF1 accumulates in the nucleus and competes with IFH1 for binding to FHL1 at RP gene promoters, and thereby inhibits transcription of RP genes. Thus, a signaling mechanism is described linking an environmental sensor to ribosome biogenesis (Martin, 2004).
Calorie restriction increases life span in many organisms, including the budding yeast Saccharomyces cerevisiae. From a large-scale analysis of 564 single-gene-deletion strains of yeast, 10 gene deletions were identified that increase replicative life span. Six of these correspond to genes encoding components of the nutrient-responsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D cells failed to further increase life span and, like calorie restriction, deletion of either SCH9 or TOR1 increased life span independent of the Sir2 histone deacetylase. It is proposed that the TOR and Sch9 kinases define a primary conduit through which excess nutrient intake limits longevity in yeast (Kaeberlein, 2005).
Chronological life span (CLS) in Saccharomyces cerevisiae, defined as the time cells in a stationary phase culture remain viable, has been proposed as a model for the aging of post-mitotic tissues in mammals. A high-throughput assay was developed to determine CLS for approximately 4800 single-gene deletion strains of yeast, and long-lived strains were identified carrying mutations in the conserved TOR pathway. TOR signaling regulates multiple cellular processes in response to nutrients, especially amino acids, raising the possibility that decreased TOR signaling mediates life span extension by calorie restriction. In support of this possibility, removal of either asparagine or glutamate from the media significantly increased stationary phase survival. Pharmacological inhibition of TOR signaling by methionine sulfoximine or rapamycin also increased CLS. Decreased TOR activity also promoted increased accumulation of storage carbohydrates and enhanced stress resistance and nuclear relocalization of the stress-related transcription factor Msn2. It is proposed that up-regulation of a highly conserved response to starvation-induced stress is important for life span extension by decreased TOR signaling in yeast and higher eukaryotes (Powers, 2005).
The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor (regulatory associated protein of mTOR: potential Drosophila homolog CG4320) to relay nutrient signals to downstream translation machinery. Raptor associates in a near stoichiometric ratio with mTOR to form a complex that functions as the nutrient sensor. It was proposed that raptor acts as a scaffold to bridge TOR with its putative phosphorylation targets. In C. elegans, mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, DAF-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span (Jia, 2004).
FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. The AAA+ ATPase homolog ruvb-1 has been identified as a potent suppressor of pha-4 mutations. This study shows that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. This study found that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. In conclusion this data suggests that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. A model is suggested in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease (Sheaffer, 2008).
Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. Mutations have been identified in the C. elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span (Soukas, 2009).
The structurally related natural products rapamycin and FK506 bind to the same intracellular receptor, FKBP12, yet the resulting complexes interfere with distinct signalling pathways. FKBP12-rapamycin inhibits progression through the G1 phase of the cell cycle in osteosarcoma, liver and T cells as well as in yeast, and interferes with mitogenic signalling pathways that are involved in G1 progression, namely with activation of the protein p70S6k and cyclin-dependent kinases. A mammalian FKBP-rapamycin-associated protein (FRAP) has been isolated whose binding to structural variants of rapamycin complexed to FKBP12 correlates with the ability of these ligands to inhibit cell-cycle progression. Peptide sequences from purified bovine FRAP were used to isolate a human cDNA clone that is highly related to the DRR1/TOR1 and DRR2/TOR2 gene products from Saccharomyces cerevisiae. Although it has not been previously demonstrated that either of the DRR/TOR gene products can bind the FKBP-rapamycin complex directly, these yeast genes have been genetically linked to a rapamycin-sensitive pathway and are thought to encode lipid kinases (Brown, 1994).
The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. A protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. It is proposed that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins (Sabatini, 1994).
The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity is clearly observed when immunoprecipitations are conducted with the mTOR antibody, mTAb2. Insulin also increases by severalfold the 32P content of mTOR, determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affects neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreases the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity are abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimics insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. These findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB (Scott, 1999).
Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1; see Drosophila Thor). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Protein kinase Cdelta (PKCdelta) associates with RAFT1 and PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation (Kumar, 2000a).
The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase [p70(S6k)] and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation (Kumar, 2000b).
Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Tuberous sclerosis 1 (Tsc1) and Tsc2, tumor suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. It is proposed that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. These studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumor suppressors (Gao, 2002).
TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. These data indicate a molecular mechanism for TSC2 in insulin signalling, tumor suppressor functions and in the inhibition of cell growth (Inoki, 2002).
The small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, it is shown that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. It is concluded that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells (Tee, 2003).
To examine whether Rheb overexpression could modulate S6K1 activity, S6K1 was coexpressed with Rheb at two different expression levels in HEK293E cells and kinase activity was assayed by using GST-S6 as a substrate. Coexpression of Rheb significantly increased the basal and insulin-stimulated activity of S6K1. Higher levels of Rheb expression enhanced the basal and insulin-stimulated activity of S6K1 by 5.6- and 1.7-fold, respectively, and this activity level is more potent than the S6K1 activity observed in the presence of lower levels of Rheb protein. These results indicate that Rheb activates signaling cascades that result in S6K1 activation (Tee, 2003).
Given that the activity of S6K1 is enhanced upon cell signaling through mTOR, PI3K, and mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)-mediated pathways, it was determined which signaling pathway was activated upon Rheb overexpression. To investigate PI3K-mediated signaling, Rheb, along with Akt, a downstream target of PI3K, was expressed within HEK293E cells and kinase activity was assayed. Whereas EGF stimulation led to a 4-fold increase in Akt activity, Rheb overexpression did not enhance basal or EGF-stimulated Akt activity. In contrast, Rheb potently activated S6K1 by 11-fold when assayed in parallel. Wortmannin was used as a control to show that PI3K-mediated activation of Akt upon stimulation with EGF was specifically measured. To examine whether Rheb enhanced MAPK-mediated signaling, Rheb was coexpressed with RSK1, a known downstream signaling component of MAPK, within HEK293E cells. Although EGF stimulation led to a 12-fold increase in RSK1 activity, Rheb did not augment the basal or EGF-induced activation of RSK1 assayed with GST-S6 as a substrate. In contrast, Rheb overexpression drastically increased S6K1 activity when assayed in parallel. To inhibit activation of ERK (extracellular signal-regulated kinase) through MEK (MAPK/ERK-kinase)-mediated signaling, cells were treated with the U0126 compound to specifically inhibit MEK. These findings suggest that Rheb does not function upstream of either PI3K/Akt or ERK/RSK1 signaling pathways (Tee, 2003).
Because Rheb enhances the activity of S6K1, a downstream component of mTOR, the effects were investigated of Rheb-mediated signaling on 4E-BP1, another downstream component of mTOR. Dephosphorylated species of 4E-BP1 bind to and inhibit eIF4E-driven cap-dependent translation. Phosphorylation of 4E-BP1 at multiple Ser/Thr-Pro residues upon mitogenic stimulation led to the release of 4E-BP1 from eIF4E, which is blocked by both rapamycin and nutrient starvation. Three different phosphorylated species of 4E-BP1 resolve on SDS-PAGE, with gamma- and alpha-isoforms being the most and least phosphorylated species, respectively. To determine whether Rheb activates mTOR- or PI3K-mediated signaling, Rheb was coexpressed with hemagglutinin (HA)-tagged 4E-BP1 in the presence of either rapamycin or wortmannin to inhibit mTOR or PI3K, respectively. Insulin-induced Akt phosphorylation on Ser473 was blocked by wortmannin, revealing that the concentration of wortmannin used in this study efficiently inhibits PI3K-mediated signaling. Insulin-induced phosphorylation of 4E-BP1 was also blocked by wortmannin, as observed by the reduced mobility shift of 4E-BP1 to the less-phosphorylated isoforms and by decreased Ser65 phosphorylation. Rheb overexpression within serum-starved cells potently enhances 4E-BP1 phosphorylation, which was still sensitive to rapamycin. In contrast, treatment of cells with wortmannin was modestly effective at reducing 4E-BP1 phosphorylation upon Rheb overexpression, indicating that PI3K signaling is not essential for Rheb-induced 4E-BP1 phosphorylation. These data suggest that Rheb signals by using an mTOR-dependent rather than a PI3K-dependent mechanism. Rheb-induced 4E-BP1 phosphorylation should promote the release of 4E-BP1 from eIF4E. To confirm this, endogenous eIF4E was purified on m7GTP-Sepharose, which mimics the cap-structure found at the extreme 5' terminus of most cytoplasmic mRNAs, and how much HA-tagged 4E-BP1 was bound to eIF4E was examined. As expected, 4E-BP1 is released from eIF4E upon Rheb overexpression, implying that Rheb activates cap-dependent translation (Tee, 2003).
The above data imply that Rheb may modulate mTOR signaling. Given that the loss of Rheb in yeast mimics nutrient starvation, Rheb overexpression may promote mTOR signaling through a nutrient-regulated signaling pathway. To address this possibility, whether Rheb promotes S6K1 activation was investigated in the absence of amino acids. During conditions of amino acid withdrawal, Rheb overexpression potently activated S6K1, which was completely blocked by rapamycin but only partially inhibited by wortmannin. Importantly, insulin stimulation of these amino acid-deprived cells potently activated Akt (as observed by Akt phosphorylation on Ser473) but only weakly activated S6K1. Therefore, unlike Rheb-mediated signaling, acute stimulation of PI3K and Akt is not sufficient to fully activate S6K1 during nutrient insufficiency. The modest insulin-induced activation of S6K1 during amino acid insufficiency was blocked by wortmannin, revealing that this activation is completely dependent on PI3K. Activation of S6K1 upon readdition of amino acids was enhanced when Rheb was overexpressed or when cells were stimulated with insulin). Interestingly, Rheb-induced S6K1 activation upon readdition of amino acids was completely inhibited by rapamycin but only partially inhibited by wortmannin. In contrast, insulin-induced S6K1 activity was markedly impaired by both rapamycin and wortmannin. These data convincingly reveal that Rheb potently activates S6K in the absence of nutrients through mTOR rather than PI3K-mediated signaling. Therefore, it is likely that Rheb enhances nutrient-mediated signaling through mTOR (Tee, 2003).
To decisively determine whether Rheb positively activates mTOR signaling, use was made of a rapamycin-resistant mutant of S6K1 (S6K1-F5A-DeltaCT). Unlike treatment with wortmannin, rapamycin treatment was unable to prevent insulin-induced activation of S6K1-F5A-DeltaCT, demonstrating that this mutant is responsive to PI3K signaling but not mTOR signaling. As a positive control, PDK1 and PKCζ, which are known to activate S6K1 through a PI3K-dependent input, were coexpressed. Overexpression of Rheb potently activates wild-type S6K1 basally (by 11-fold) and during insulin stimulation (by 17-fold) but does not enhance the activity of the S6K1-F5A-DeltaCT mutant. In contrast, increased PI3K-mediated signaling toward S6K1 by coexpression of PDK1 and PKCζ results in significantly enhanced activation of both wild-type S6K1 and S6K1-F5A-DeltaCT. These findings strongly suggest that Rheb induces S6K1 activation via a signaling input that is upstream of mTOR but not PI3K (Tee, 2003).
Overexpression of wild-type Rheb has been shown to lead to a significant increase in its activity and implies that the majority of the overexpressed Rheb must exist in the active GTP bound form. If this is true, then the RhebGAP activity must be a limiting factor. If Tuberin possesses RhebGAP activity, overexpression of Tuberin should switch Rheb from an active GTP bound state to an inactive GDP bound state. To indirectly measure Rheb activity, Rheb-induced S6K1 activation was analyzed within nutrient-deprived HEK293E cells. Coexpression of Hamartin and Tuberin completely block Rheb's ability to activate S6K1, implying that Tuberin may function as a RhebGAP (Tee, 2003).
If the GAP domain of Tuberin is essential for Rheb inactivation, then patient-derived TSC2 GAP domain point mutants should not block Rheb-induced S6K1 activation. To address this, three Tuberin mutants were generated that mimic patient-derived TSC2 mutations that occur within the GAP domain and these Tuberin mutants were coexpressed with Hamartin and S6K1. Under serum-starved conditions, Rheb potently activates S6K1, which was fully blocked by coexpression of wild-type Tuberin with Hamartin. In contrast, the three TSC2 GAP domain point mutants were unable to repress Rheb-induced S6K1 activation, revealing that the GAP domain of Tuberin is critical for Tuberin's ability to repress Rheb-mediated signaling (Tee, 2003).
The in vivo overexpression data strongly suggest that the Tuberin-Hamartin heterodimer inhibits Rheb function. In order to test whether this is a direct inhibition due to the GAP activity of Tuberin, in vitro GAP assays were performed on purified Rheb. Flag-tagged Hamartin and Flag-tagged Tuberin were expressed separately or together in HEK293 cells and the respective protein(s) were immunoprecipitated for use in Rheb-GAP assays. Interestingly, immunoprecipitated Hamartin or Tuberin display nearly identical GAP activity toward Rheb; both enhance the intrinsic GTPase activity of Rheb by approximately 2-fold. This suggests that a complex between Tuberin and Hamartin is essential for Tuberin's GAP activity toward Rheb and that endogenous levels of coimmunoprecipitating Tuberin or Hamartin are limiting in these reactions. In support of this, coexpression and immunoprecipitation of both Tuberin and Hamartin result in immune complexes that enhance Rheb GTPase activity by more than 100-fold over the activity of either alone and approximately 200-fold over intrinsic Rheb activity. This dramatic increase in GAP activity is detected despite no significant difference in the amount of Tuberin immunoprecipitated when expressed alone or with Hamartin. Therefore, Tuberin and Hamartin together form a GTPase-activating protein complex that greatly enhances the intrinsic GTPase activity of Rheb (Tee, 2003).
In order to test if this activity is potentially important in the prevention of the TSC disease, the GAP activity of wild-type Tuberin was compared to that of a patient-derived mutant mapped to the Tuberin GAP domain (N1651S). Compared to wild-type Tuberin, the Tuberin(N1651S) mutant is greatly reduced in its ability to enhance Rheb GTPase activity. This suggests that there is a correlation in the ability of Tuberin to act as a GAP toward Rheb and its ability to suppress the TSC disease (Tee, 2003).
Farnesylation of Rheb is required for cell cycle progression of S. pombe. To investigate whether farnesylation is important for Rheb's ability to activate S6K1, a farnesylation-defective Rheb(C182S) mutant was generated, in which the cysteine within the farnesylation CAAX motif is substituted for a serine. When overexpressed, the Rheb(C182S) mutant is less efficient at enhancing S6K1 activity than wild-type Rheb. The mutant Rheb(C182S) protein migrated as the upper band on SDS-PAGE, which indicates that it is not being prenylated and is consistent with earlier studies showing that prenylated Rheb migrates more quickly on SDS-PAGE. In contrast, the majority of wild-type Rheb resolved as the lower prenylated band. These findings suggest that the membrane localization of Rheb through farnesylation is important for Rheb to efficiently augment mTOR-mediated signaling (Tee, 2003).
Thus, Rheb functions upstream of mTOR within the nutrient signaling pathway. Rheb specifically activates mTOR-mediated signaling rather than cell signaling through MEK/ERK and PI3K, as shown by Rheb-mediated activation of S6K1 but not Akt or RSK1. Therefore, it is unlikely that Rheb activates PI3K and Raf, two downstream effectors of Ras. Rheb has previously been shown to interact with Raf in vitro, but the current data suggest that Raf is not an effector of Rheb in vivo. Additionally, Rheb overexpression does not increase the activity of the rapamycin-resistant S6K1 mutant that is unresponsive to mTOR signaling inputs but is activated in response to PI3K signaling. S6K1 activation is regulated by multiple signaling inputs, one of which is directed by PI3K. Therefore, these findings are important and confirm that Rheb overexpression specifically promotes mTOR rather than PI3K signaling. Furthermore, Rheb-induced 4E-BP1 phosphorylation is completely sensitive to rapamycin but not to wortmannin, which further strengthens the notion that Rheb acts upstream of mTOR rather than PI3K. 4E-BP1 dissociates from eIF4E upon Rheb overexpression, revealing that Rheb-mediated signaling through mTOR promotes cap-dependent translation (Tee, 2003).
Evidence has also been provided that Rheb functions within the nutrient signaling cascade upstream of mTOR, as shown by Rheb's ability to potently stimulate S6K1 activity during amino acid insufficiency. During amino acid withdrawal, acute insulin stimulation is still able to elicit high levels of Akt phosphorylation but poorly activates S6K1, showing that the nutrient-mediated mTOR signaling input is essential for optimal S6K1 activation. Therefore, Rheb overexpression supersedes the dependency of the nutrient input to mTOR, suggesting that Rheb is an activator of mTOR within the nutrient-signaling pathway. Interestingly, resupplying cells with amino acids further enhances the activity of S6K1 when Rheb is overexpressed, suggesting that amino acids may promote the activation of Rheb. This research has revealed that the Tuberin-Hamartin heterodimer functions as an inhibitor of nutrient signaling through mTOR. The Tuberin-Hamartin heterodimer inhibits Rheb-induced S6K1 activation during conditions of amino acid withdrawal. This work, therefore, extends these earlier studies, revealing that inhibition of Rheb is the mechanism by which the Tuberin-Hamartin heterodimer inhibits nutrient-mediated signaling. Importantly, the Rheb-inhibitory function of Tuberin-Hamartin heterodimers depends on an intact Tuberin GAP domain; patient-derived point mutations within the GAP domain of TSC2 prevented the Tuberin-Hamartin heterodimer from blocking Rheb-induced S6K1 activation. These data indicate that the GAP activity of Tuberin promotes inactivation of Rheb in vivo, presumably through increasing the intrinsic GTPase activity of Rheb. Confirming this hypothesis, in vitro Rheb GTPase activity assays have revealed that Tuberin enhances the intrinsic GTPase activity of Rheb. Interestingly, coexpression of Hamartin with Tuberin markedly enhances the GTPase activity of Rheb, implying that Hamartin promotes the GAP function of Tuberin toward Rheb. A model is proposed whereby Rheb promotes mTOR signaling when it is in an active GTP bound form, whereas the Tuberin-Hamartin heterodimer inhibits Rheb by converting it to an inactive GDP bound state. These findings reveal that the Tuberin-Hamartin heterodimer and Rheb respectively inhibit and activate the nutrient-signaling input to mTOR. Small G proteins are additionally regulated by guanine nucleotide exchange factors (GEFs). In this model it is proposed that a RhebGEF becomes activated during conditions of nutrient sufficiency, and its activation switches Rheb to an active GTP bound form. Therefore, identifying this Rheb-GEF will be of great importance and may provide new insights into how mTOR senses intracellular amino acids. However, at this point the possibility that Rheb- and nutrient-mediated signaling may function in parallel pathways upstream of mTOR cannot be ruled out. Further experiments will be carried out to investigate this possibility (Tee, 2003).
Inorganic polyphosphate (poly P: chains of hundreds of phosphate residues linked by 'high-energy' bonds as in ATP) has been conserved from prebiotic times in all cells. Poly P is essential for a wide variety of functions in bacteria, including virulence in pathogens. In this study, the unique and many-fold stimulation by poly P in vitro is observed of the protein kinase mTOR (mammalian target of rapamycin). To explore the role of poly P in mammalian cells, a yeast polyphosphatase, PPX1, was inserted into the chromosomes of MCF-7 mammary cancer cells. The transfected cells are markedly deficient in their response to mitogens, such as insulin and amino acids, as seen in their failure to activate mTOR to phosphorylate one of its substrates, PHAS-I (the initiation factor 4E-binding protein). In addition, the transfected cells are severely reduced in their growth in a serum-free medium. On the basis of these findings, it is suggested that poly P (and/or PPX1) serves as a regulatory factor in the activation of mTOR in the proliferative signaling pathways of animal cells (Wang, 2003).
Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. This study shows that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801 (Drosophila homologs: scylla and charybde). Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia (Brugarolas, 2004).
The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. The stress response REDD1 gene has been identified as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, because regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway (Sofer, 2005).
The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila a Ste20 family member (MAP4K3; Drosophila homolog happyhour) was identified that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. These results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3 (Findley, 2007).
In metazoans both nutrient amino acids and mitogens are required to promote mTOR signalling and cell growth. Activation of mTOR signalling by mitogens involves activation of protein kinases such as PKB, p90RSK and ERK that have been reported to inhibit the function of the TSC1-2 complex which is then thought to lead to increased GTP loading of the Ras-like GTPase Rheb. In contrast, amino acids do not appear to regulate Rheb GTP loading, and, although a class III PI3K has been implicated upstream of mTORC1, no analogous protein kinases have been identified that respond to amino acids and activate mTOR signalling. This study found, both in Drosophila and in mammalian cells, that suppression of MAP4K3 inhibited mTOR signalling to S6K in the context of activation of the pathway induced by deficiency in TSC1-2. Since a variety of oncogenic signalling pathways inhibit TSC1-2, and TSC1-2 is predicted also to be inactivated by loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) via activation of PKB, MAP4K3 may represent a promising new candidate for inhibition of the pathway in diseases of TSC1-2 or PTEN loss-of-function. Although the mechanism of MAP4K3 action on mTOR signalling has not been defined, the data on the positive regulation of both S6K1 activity and phosphorylation of 4E-BP1 by MAP4K3 overexpression points to the mTORC1 complex as the likely target of MAP4K3 action. Interestingly, addition of excess amino acids (relative to the normal concentrations found in DMEM) has been reported to fully activate S6K in the absence of growth factors, which is similar to current findings with overexpressed MAP4K3, suggesting that MAP4K3 may be activated further by amino acid excess. Future studies will be required to clarify these points. However, having defined the action of a new protein kinase in the mTOR pathway, delineating the signals downstream of amino acids that activate MAP4K3, and determining its mechanism of action will likely shed further light on nutrient regulation of cell growth (Findley, 2007).
In conclusion, these data indicate that amino acids stimulate the activity of a Ste20-family kinase, MAP4K3, with maximal kinase activation occurring concordant with phosphorylation of the Thr389 site of S6K1. In HeLa cells MAP4K3 activity is required for amino acid-induced activation of S6K1 and mediates rapamycin-senstive signalling to two effectors of mTORC1, but is not itself stimulated by insulin or inhibited by rapamycin. Lastly, MAP4K3 promotes cell growth in human HeLa cells in culture in a similar manner to Rheb and mTORC1 (Findley, 2007).
FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. This study unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis (Chen, 2010; see graphical abstract).
The mTOR pathway is the central regulator of cell size. External signals from growth factors and nutrients converge on the mTORC1 multi-protein complex to modulate downstream targets, but how the different inputs are integrated and translated into specific cellular responses is incompletely understood. Deregulation of the mTOR pathway occurs in polycystic kidney disease (PKD), where cilia (filiform sensory organelles) fail to sense urine flow because of inherited mutations in ciliary proteins. It was therefore investigated if cilia have a role in mTOR regulation. This study shows that ablation of cilia in transgenic mice results in enlarged cells when compared with control animals. In vitro analysis demonstrated that bending of the cilia by flow is required for mTOR downregulation and cell-size control. Surprisingly, regulation of cell size by cilia is independent of flow-induced calcium transients, or Akt. However, the tumour-suppressor protein Lkb1 localises in the cilium, and flow results in increased AMPK phosphorylation at the basal body. Conversely, knockdown of Lkb1 prevents normal cell-size regulation under flow conditions. These results demonstrate that the cilium regulates mTOR signalling and cell size, and identify the cilium-basal body compartment as a spatially restricted activation site for Lkb1 signalling (2010).
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. This study demonstrates that the products of two p53 target genes, Sestrin1 and Sestrin2 (see Drosophila Sestrin), 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 (Budanov, 2008).
The mTOR signaling pathway is a central regulator of cell growth and survival. It is therefore not surprising that adverse environmental conditions negatively regulate cell growth by inhibiting mTOR. In addition to nutrient limitation, mTOR activity is negatively regulated by genotoxic stress and hypoxia, conditions that activate tumor suppressor p53. The ability of p53 to inhibit mTOR signaling is in line with its function as a negative regulator of cell growth and proliferation. The results described above strongly suggest that the ability of p53 to inhibit mTOR signaling depends on two of its target genes: Sesn1 and Sesn2 (Budanov, 2008).
The Sestrins belong to a small and evolutionary conserved family composed of 3 members in mammals, of which Sesn1 and 2 are stress inducible and p53 regulated. The ability of Sesn1/2 to inhibit cell growth and proliferation was attributed to their redox activity. The present work, however, demonstrates that Sesn1/2 are potent inhibitors of mTOR signaling, acting in a manner that does not depend on their redox activity, which only makes a partial contribution to their growth inhibitory activity. Sesn1 and 2 inhibit TORC1 activity towards p70S6K and 4E-BP1 in a variety of human and mouse cell lines, as well as in mouse liver. Notably, the ability of the hepatocarcinogen DEN to inhibit S6 phosphorylation is restricted to zone 3 hepatocytes, which are the main site in which it undergoes metabolic activation to become a potent alkylating agent, and this inhibitory activity is Sesn2-dependent. By inhibiting 4E-BP1 phosphorylation, Sesn2 enhances its interaction with eIF-4E and inhibits expression of growth regulatory proteins, such as cyclin D1 and c-Myc, whose translation is eIF-4E-dependent and sensitive to 4E-BP1 phosphorylation (Budanov, 2008).
The Sestrins impact TORC1 activity through the TSC1:TSC2 complex. Being a GAP for Rheb, the direct activator of TORC1, the TSC1:TSC2 complex is a central regulator of mTOR signaling. Sesn2 expression decreases Rheb GTP loading and the ability of both Sesn1 and Sesn2 to inhibit mTOR signaling is TSC2-dependent. One way to regulate TSC1:TSC2 GAP activity is through TSC2 phosphorylation, but other modes of regulation may also exist. Although the Sestrins have no effect on ERK and its target RSK or GSK3β, which can all serve as TSC2 kinases, they stimulate the activity of AMPK, a major TSC2 kinase. Furthermore, Sestrin expression enhanced TSC2 phosphorylation in live cells and this effect required the N-terminus of Sesn2, which mediates AMPKα binding. Sesn2 did not stimulate TSC1 phosphorylation and Sesn2-activated AMPK did not phosphorylate TSC1 (Budanov, 2008).
Importantly, the mTOR inhibitory activity of Sesn1/2 depends on AMPKα, whose phosphorylation at the activation loop was enhanced upon Sestrin expression. Inhibition of AMPK using compound C as well as shRNA silencing of AMPKα1 attenuated the ability of Sesn2 to inhibit mTOR signaling. Co-immunoprecipitation and gel filtration analyses revealed an interaction between Sesn2 and AMPKα, suggesting that Sestrins are engaged in formation of a large protein complex containing AMPK and TSC1:TSC2. It is proposed that Sesn1/2 induction in response to genotoxic stress results in binding of Sestrins, most likely as dimers, to AMPK and TSC1:TSC2, as well as auto-activation of AMPK through a mechanism based on induced proximity. In addition to activation of AMPK the Sestrins recruit it to phosphorylate TSC2. Phosphorylation of TSC2 correlates with enhancement of its GAP activity that leads to inhibition of Rheb and mTOR (Budanov, 2008).
Importantly, ample and clear evidence was obtained that Sesn1/2 are critical mediators of p53's ability to inhibit mTOR signaling. Using shRNA-mediated silencing it was found that both Sesn1 and Sesn2 participate in mTOR inhibition upon p53 activation in human cancer cells. Furthermore, disruption of the Sesn2 gene in mice attenuated the inhibition of p70S6K activity by the DNA-damaging agents: camptothecin in fibroblasts and DEN in hepatocytes. In both cases inhibition of p70S6K was p53-mediated, but unlike the p53 deficiency, the absence of Sesn2 has no effect on induction of p21Waf1, another p53 target gene. Thus, Sesn2 (and presumably Sesn1) seems to mediate only one aspect of p53 signaling -- inhibition of mTOR. Correspondingly, the growth-inhibitory activity of Sesn2 is not as strong as that of p53, which has additional targets with anti-proliferative activity, such as p21Waf1 (Budanov, 2008).
p53 deficiency and activation of mTOR signaling are hallmarks of human cancer. Several mechanisms account for mTOR activation in cancer, including activation of Ras, PI3K and AKT and inactivation of tumor suppressors that negatively regulate these molecules: PTEN, TSC1, TSC2 and LKB1. Although p53 can induce expression of several negative regulators of mTOR, including PTEN, TSC2, AMPKβ1 and IGF-BP3 in a cell type-dependent manner, the results demonstrate that p53-mediated inhibition of mTOR depends mainly on Sesn1 and 2 in mouse fibroblasts and certain human cancer cell lines and on Sesn2 in mouse liver (Budanov, 2008).
Inhibition of mTOR suppresses cell growth and proliferation. Sesn2 was known to inhibit cell proliferation, but its mechanism of action was heretofore unknown. The results strongly suggest that Sesn1 and Sesn2 exert their growth inhibitory effect via mTOR and may cooperate with other anti-proliferative p53 targets, such as p21Waf1. Interestingly, the SESN1 (6q21) and SESN2 (1p35) loci are frequently deleted in a variety of human cancers, suggesting they harbor one or more tumor-suppressors. Sesn2 deficiency was found to render murine fibroblasts more susceptible to oncogenic transformation and this effect may depend on mTOR inhibition. Hence, SESN1 and SESN2 may indeed be important components of the tumor suppressor network activated by p53 (Budanov, 2008).
In summary, while more remains to be learned about Sestrin biology and mechanism of action, the results establish these proteins as critical links between p53 and mTOR that enable p53 to inhibit cell growth (Budanov, 2008).
The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. This study identified DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. The gene for DEPDC6 is found only in vertebrates, and encodes a protein with tandem N-terminal DEP (dishevelled, egl-10, pleckstrin) domains and a C-terminal PDZ (postsynaptic density 95, discs large, zonula occludens-1) domain. 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, this study identified a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival (Peterson, 2009).
Loss-of-function data indicate that DEPTOR inhibits both the mTORC1 and mTORC2 pathways. However, by inhibiting mTORC1, DEPTOR overexpression relieves mTORC1-mediated inhibition of PI3K, causing an activation of PI3K and, paradoxically, of mTORC2-dependent outputs, like Akt (Peterson, 2009).
mTOR interacts with DEPTOR via its PDZ domain, and so far there is no information about the function of the tandem DEP domains the protein also contains. In other proteins, DEP domains mediate protein-protein interactions, but in numerous DEPTOR purifications additional DEPTOR-interacting proteins were not identified, besides the known components of mTORC1 and mTORC2. Therefore, based on current evidence, DEPTOR appears dedicated to mTOR regulation, and it is proposed that in vertebrates it is likely to be involved in regulating other outputs of the mTOR signaling network besides the growth and survival pathways examined in this study. The mTOR complexes and DEPTOR negatively regulate each other, suggesting the existence of a feedforward loop in which the loss of DEPTOR leads to an increase in mTOR activity, which then further reduces DEPTOR expression. This type of regulatory circuit should result in DEPTOR expression being tightly coupled to mTOR activity, and, interestingly, it was noted that DEPTOR mRNA levels strongly anticorrelate with cell size, a readout of mTORC1 activity (Peterson, 2009).
About 28% of human multiple myelomas (MMs) overexpress DEPTOR. These results are consistent with a published survey of 67 MM tumors and 43 MM cell lines, in which 21% were shown to possess copy number gains and associated expression increases of the genes within a 6 Mb region of chromosome 8q24 that contains DEPTOR. Furthermore, it appears that deregulated overexpression of c-MAF and MAFB is an additional, perhaps even more prevalent, mechanism for increasing DEPTOR expression in MMs. The related c-MAF and MAFB transcription factors are expressed (frequently as the result of chromosomal translocations) in a large fraction of MMs, but not in the plasma cells from which they are derived. Consistent with c-MAF playing a key role in promoting DEPTOR expression, a knockdown of c-MAF in a MM cell line having a c-MAF translocation decreases the expression of DEPTOR and mimics the effects of a DEPTOR knockdown on mTOR and PI3K signaling. The levels of the DEPTOR and c-MAF or MAFB mRNAs highly correlate with each other and, importantly, DEPTOR expression correlates with poor survival in patients with multiple myeloma (Peterson, 2009).
In many multiple myeloma cell lines, DEPTOR is massively overexpressed compared to the levels found in other cancer cell lines, such as HeLa cells. In these cells, the great overexpression of DEPTOR inhibits mTORC1 growth signaling and drives outputs dependent on PI3K. Interestingly, a reduction in DEPTOR expression to the lower levels seen in non-multiple myeloma cell lines causes cell death via apoptosis. This suggests that a pharmacologically induced reduction in DEPTOR expression or disruption of the DEPTOR-mTOR interaction could have therapeutic benefits for the treatment of multiple myeloma. There has been progress in developing small-molecule inhibitors of protein-protein interactions mediated by PDZ domains, so it is conceivable that blockers of the DEPTOR-mTOR interaction could be made (Peterson, 2009).
Although a number of other cancer cell lines have high levels of DEPTOR, as a class only multiple myelomas appear to consistently overexpress it. Besides activating PI3K/Akt signaling, DEPTOR overexpression in MM cells may provide these cells with benefits that are not relevant in other cancer types or perhaps even detrimental. For example, the high demand that MM cells place on the protein synthesis machinery to produce large amounts of immunoglobulins, causes a significant ER stress, which renders these cells susceptible to apoptosis induction via agents that induce further ER stress, such as proteasome inhibitors. DEPTOR overexpression, by partial inhibition of protein synthesis through the suppression of mTORC1, may reduce the levels of ER stress below the threshold that triggers apoptosis. In contrast, in other cancer cells in which ER stress is not a significant factor, DEPTOR overexpression may be selected against because reduced rates of protein synthesis may not be tolerated. That mTORC1-stimulated protein synthesis leads to ER stress is already appreciated as TSC1 or TSC2 null cells have increased sensitivity to ER stress-induced death (Peterson, 2009).
It is curious that DEPTOR is overexpressed mostly in MMs characterized by chromosomal translocations instead of those that are hyperdiploid because of aneuploidy. Elevated DEPTOR expression might be tolerated better in the nonhyperdiploid MMs because aneuploidy itself increases sensitivity to conditions, like mTORC1 inhibition, that interfere with protein synthesis. Moreover, the state of high mTORC2 and low mTORC1 signaling that this work indicates that some MM cells prefer cannot be achieved by mutations that activate PI3K signaling, perhaps explaining why multiple myelomas exhibit low rates of PTEN-inactivating or PI3K-activating mutations (Peterson, 2009).
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. This study shows that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation (Nicklin, 2009).
p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or in response to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. A mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro results in a synergistic stimulation of p70alpha activity to levels similar to those attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined (Isotani, 2000).
PDK1-catalyzed phosphorylation of Thr-252 on the p70alpha activation loop is a critical and probably final step in the physiologic activation of p70alpha in vivo. The ability of PDK1 to phosphorylate Thr-252 is regulated primarily by the accessibility of the p70alpha activation loop to PDK1, which in turn is controlled by a series of prior p70alpha phosphorylations. Phosphorylation of the multiple Ser/Thr-Pro motifs clustered in the pseudosubstrate autoinhibitory segment of the p70alpha carboxyl-terminal tail serves to disocclude the catalytic domain, greatly enhancing access to PDK1. A similar effect can be achieved by deletion of the p70alpha carboxyl-terminal tail (to give p70alpha-DeltaCT104). At any level of PDK1 activity, the extent of Thr-252 phosphorylation of p70alpha-DeltaCT104 is substantially greater than with a similar amount of full-length p70alpha polypeptide. In addition, the S6 kinase activity generated by any extent of PDK1-catalyzed Thr-252 phosphorylation is significantly higher for p70alpha-DeltaCT104 as compared with full-length p70alpha. Displacement of the p70alpha carboxyl-terminal tail is also necessary for the phosphorylation of Thr-412 in vivo, and modification of Thr-412 itself significantly enhances the ability of PDK1 to phosphorylate Thr-252. In addition, the simultaneous phosphorylation of Thr-412 and Thr-252 appears to generate a synergistic activation of p70alpha. Thus, the substitution of Thr-412 by Glu in p70alpha-DeltaCT104 alone gives a 6-fold increase in S6 kinase activity, and the PDK1 catalyzed phosphorylation of p70alpha-DeltaCT104 Thr-252 alone gives a 15-fold increase, but the two modifications together give at least a 240-fold increase in S6 kinase activity over the unmodified p70alpha-DeltaCT104 polypeptide. The importance of the strong positively cooperative effect of Thr-252 and Thr-412 phosphorylation for the physiologic activation of p70alpha is illustrated by the response of p70alpha to the inhibitors rapamycin and wortmannin; these agents each cause a rapid dephosphorylation of Thr-412 but a slower and lesser dephosphorylation of Thr-252. Despite the preservation of Thr-252 phosphorylation, S6 kinase activity in the presence of rapamycin or wortmannin declines in parallel to Thr-412 dephosphorylation. In view of the ability of mTOR to catalyze the in vitro phosphorylation of p70alpha Thr-412 as well as sites within the autoinhibitory segment in the p70alpha carboxyl-terminal tail and the potential effects of such phosphorylations on the response of p70alpha to PDK1, the p70alpha activation achieved in vitro by mTOR or PDK1 alone have been compared to that achieved by sequential phosphorylation by mTOR and PDK1 and to that achieved in vivo by stimulation of the cells with 10% serum. mTOR alone increases the S6 kinase activity of p70alpha in vitro by more than 10-fold, whereas PDK1 alone hardly activates p70alpha, presumably reflecting the relatively poor access of Thr-252 to PDK1 in full-length, inactive p70alpha. In contrast, phosphorylation of p70alpha by PDK1 after a prior phosphorylation by mTOR increases the p70alpha activity by 10-fold over that engendered by mTOR alone, to a level roughly 70-fold greater than that generated by PDK1 acting alone. Moreover, the S6 kinase activity generated in vitro by the sequential action of mTOR and PDK1 is indistinguishable from that achieved in vivo by stimulation of cells with 10% serum (Isotani, 2000).
The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. A conserved TOR signaling (TOS) motif has been identified in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs; the TOS motif is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, since mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. It is concluded that S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1 (Schalm, 2002).
Neuropoletic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)-signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamycin (mTOR)-p70 S6 kinase. Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context. This study investigates the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. CNTF-induced phosphorylation of Ser727 is inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide is efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreases activation of the reporter to the same degree as a STAT3 Ser727Ala mutant. The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR in mammalian cells to include transcriptional regulation (Yokogami, 2000).
Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21Cip1 arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21Cip1 independent of cell cycle arrest. Rapamycin induces p21Cip1 binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21Cip1 (Huang, 2003).
In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. The mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Ser 44 (S44) and hyperphosphorylation of Ser 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target for mTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of rRNA synthesis (Mayer, 2004).
An important question is whether mTOR itself, or a downstream kinase, phosphorylates TIF-IA. S6K1 is known to act downstream of mTOR, raising the possibility that S6K1 directly phosphorylates TIF-IA. However, TIF-IA contains no consensus recognition motif for S6K1, and neither immunopurified mTOR nor S6K1 were capable of phosphorylating TIF-IA in vitro. Ser 44 resides within a recognition motif for Cdks (SPxR). This raises the possibility that Cdk-mediated phosphorylation of S44 links nutrient-sensing pathways to cellcycle control. Pol I transcription oscillates during the cell cycle, being highest in S2 and G2 phase, shutting down at mitosis and recovering during G1-phase progression. It is not yet known whether or not TIF-IA is inactivated during mitosis and is reactivated by phosphorylation by G1-specific Cdks. The finding that immunopurified Cdk2/cyclin E phosphorylates S44 and phosphorylation at this site is required for TIF-IA function, supports the view that phosphorylation by Cdks is essential for regulation of TIF-IA activity and, hence, cell cycle-dependent fluctuations of Pol I transcription. In contrast, although rapamycin inhibits Cdk activation and compromises cell-cycle progression, inhibition of S44 phosphorylation by G1-phase Cdks is unlikely to be responsible for the rapid switch-off of pre-rRNA synthesis, because TIF-IA inactivation was observed as early as 1 h after rapamycin administration. Inactivation of TIF-IA by rapamycin has not been observed in the presence of protein phosphatase inhibitors, indicating that activation of phosphatase(s) rather than inhibition of Cdks induces rapamycin-dependent hypophosphorylation of S44. In support of this, PP2A is very rapidly activated by rapamycin, and the regulated activity of PP2A has been proposed as a master switch to regulate the activity of downstream effectors of mTOR, such as S6K1 and 4E-BP. Like TIF-IA, S6K1 and 4E-BP are phosphorylated at multiple sites and are dephosphorylated within 30 min after rapamycin treatment (Mayer, 2004).
While NF-kappaB is considered to play key roles in the development and progression of many cancers, the mechanisms whereby this transcription factor is activated in cancer are poorly understood. A key oncoprotein in a variety of cancers is the serine- threonine kinase Akt, which can be activated by mutations in PI3K, by loss of expression/activity of PTEN, or through signaling induced by growth factors and their receptors. A key effector of Akt-induced signaling is the regulatory protein mTOR (mammalian target of rapamycin). This study shows that mTOR downstream from Akt controls NF-kappaB activity in PTEN-null/inactive prostate cancer cells via interaction with and stimulation of IKK. The mTOR-associated protein Raptor is required for the ability of Akt to induce NF-kappaB activity. Correspondingly, the mTOR inhibitor rapamycin is shown to suppress IKK activity in PTEN-deficient prostate cancer cells through a mechanism that may involve dissociation of Raptor from mTOR. The results provide insight into the effects of Akt/mTOR-dependent signaling on gene expression and into the therapeutic action of rapamycin (Dan, 2008).
The mammalian target of rapamycin, mTOR, is a central regulator of cell growth. Its activity is regulated by Rheb, a Ras-like small guanosine triphosphatase (GTPase), in response to growth factor stimulation and nutrient availability. Rheb regulates mTOR through FKBP38, a member of the FK506-binding protein (FKBP) family that is structurally related to FKBP12. FKBP38 binds to mTOR and inhibits its activity in a manner similar to that of the FKBP12-rapamycin complex. Rheb interacts directly with FKBP38 and prevents its association with mTOR in a guanosine 5'-triphosphate (GTP)-dependent manner. These findings suggest that FKBP38 is an endogenous inhibitor of mTOR, whose inhibitory activity is antagonized by Rheb in response to growth factor stimulation and nutrient availability (Bai, 2007).
mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo (Hara, 2002).
mTOR/RAFT1/FRAP is the target of the immunosuppressive drug rapamycin and the central component of a nutrient- and hormone-sensitive signaling pathway that regulates cell growth. mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway. Raptor has a positive role in nutrient-stimulated signaling to the downstream effector S6K1, maintenance of cell size, and mTOR protein expression. The association of raptor with mTOR also negatively regulates the mTOR kinase activity. Conditions that repress the pathway, such as nutrient deprivation and mitochondrial uncoupling, stabilize the mTOR-raptor association and inhibit mTOR kinase activity. It is proposed that raptor is a missing component of the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels (Kim, 2002).
mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. A member of this pathway, a protein named GbetaL, has been identified that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in the control of cell size and in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, it is proposed that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway (Kim, 2003).
The translational repressor protein eIF4E-binding protein 1 (4E-BP1, also termed PHAS-I) is regulated by phosphorylation through the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. Recent studies have identified two regulatory motifs in 4E-BP1, an mTOR-signaling motif (TOS) in the C-terminus of 4E-BP1 and a RAIP motif (after its sequence) in the N-terminus. Other work has shown that the protein raptor binds to mTOR and 4E-BP1. Raptor binds to full-length 4E-BP1 or a C-terminal fragment containing the TOS motif but not to an N-terminal fragment containing the RAIP motif. Mutation of several residues within the TOS motif abrogates binding to raptor, indicating that the TOS motif is required for this interaction. 4E-BP1 undergoes phosphorylation at multiple sites in intact cells. The effects of removal or mutation of the RAIP and TOS motifs differ. The RAIP motif is absolutely required for phosphorylation of sites in the N- and C-termini of 4E-BP1, while the TOS motif primarily affects phosphorylation of Ser64/65 and Thr69/70, and also the rapamycin-insensitive site Ser101. Phosphorylation of N-terminal sites that are dependent upon the RAIP motif are sensitive to rapamycin. The RAIP motif thus promotes the mTOR-dependent phosphorylation of multiple sites in 4E-BP1 independently of the 4E-BP1/raptor interaction (Beugnet, 2003).
Mammalian target of rapamycin (mTOR) is the central element of a signaling pathway involved in the control of mRNA translation and cell growth. The actions of mTOR are mediated in part through the phosphorylation of the eukaryotic initiation factor 4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eukaryotic initiation factor 4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The Arg-Ala-Ile-Pro (RAIP motif) and Phe-Glu-Met-Asp-Ile (tor signaling motif) sequences found in the NH2- and COOH-terminal regions of PHAS-I, respectively, are required for the efficient phosphorylation of PHAS-I in cells. Mutations in either motif markedly decrease the phosphorylation of recombinant PHAS-I by mTOR in vitro. Wild-type PHAS-I, but none of the mutant proteins, is coimmunoprecipitated with hemagglutinin-tagged raptor, an mTOR-associated protein, after extracts of cells overexpressing raptor have been supplemented with recombinant PHAS-I proteins. Moreover, raptor overexpression enhances the phosphorylation of wild-type PHAS-I by mTOR but not the phosphorylation of the mutant proteins. The results not only provide direct evidence that both the RAIP and tor signaling motifs are important for the phosphorylation by mTOR, possibly by allowing PHAS-I binding to raptor, but also support the view that mTOR phosphorylates PHAS-I in cells (Choi, 2003).
The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1; see Drosophila Thor). Raptor (regulatory associated protein of mTOR) also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs shown to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring S6K and 4E-BP1 sensitivity to rapamycin and amino acid sufficiency (Nojima, 2003).
The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). A TOR signaling (TOS) motif is present in the N terminus of S6K1 and the C terminus of 4E-BP1. In S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it has been unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. A functional TOS motif is shown to be required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. These data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth (Schalm, 2003).
The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GΔL proteins to form a complex that is the target of rapamycin. mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GΔL but not raptor, and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. The rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCa) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals (Sarbassov, 2004).
Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005a).
The raptor-mTOR protein complex is a key component of a nutrient-sensitive signaling pathway that regulates cell size by controlling the accumulation of cellular mass. How nutrients regulate signaling through the raptor-mTOR complex is not well known. This study shows that a redox-sensitive mechanism regulates the phosphorylation of the raptor-mTOR effector S6K1, the interaction between raptor and mTOR, and the kinase activity of the raptor-mTOR complex. In cells treated with the oxidizing agents diamide or phenylarsine oxide, S6K1 phosphorylation increased and became insensitive to nutrient deprivation. Conversely, the reducing reagent BAL (British anti-Lewisite, also known as 2,3-dimercapto-1-propanol) inhibits S6K1 phosphorylation and stabilizes the interaction of mTOR and raptor to mimic the state of the complex under nutrient-deprived conditions. These findings suggest that a redox-based signaling mechanism may participate in regulating the nutrient-sensitive raptor-mTOR complex and pathway (Sarbassov, 2005b).
The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. This study shows that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. This work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types (Sarbassov, 2006).
The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473. This study investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in AML cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation and glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematological malignancies who received RDs in clinical studies. This study provides first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias (Zeng, 2006).
The enzyme mTOR (mammalian target of rapamycin) is a major target for therapeutic intervention to treat many human diseases, including cancer, but very little is known about the processes that control levels of mTOR protein. This study shows that mTOR is targeted for ubiquitination and consequent degradation by binding to the tumor suppressor protein FBXW7. Human breast cancer cell lines and primary tumors showed a reciprocal relation between loss of FBXW7 and deletion or mutation of PTEN (phosphatase and tensin homolog), which also activates mTOR. Tumor cell lines harboring deletions or mutations in FBXW7 are particularly sensitive to rapamycin treatment, which suggests that loss of FBXW7 may be a biomarker for human cancers susceptible to treatment with inhibitors of the mTOR pathway (Mao, 2008).
The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, mice deficient for raptor, rictor, or mLST8 were generated. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCα, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3β, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCα pathways (Guertin, 2007).
The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of at least two distinct signaling complexes, referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Studies with cultured cell lines indicate that the complexes participate in different pathways and recognize distinct substrates, the specificity of which is determined by unique mTOR-interacting proteins. mTORC1 controls cell growth in part by phosphorylating S6 Kinase 1 (S6K1) and the eIF-4E-binding protein 1 (4E-BP1), known regulators of protein synthesis. It has been proposed that mTORC2 phosphorylates and activates Akt/PKB, which regulates cell proliferation, growth, survival, and metabolism. Full activation of Akt/PKB requires phosphorylation of S473 of the hydrophobic motif, the site proposed to depend on mTORC2, as well as phosphorylation of T308 of the activation loop by PDK1. The clinically valuable drug rapamycin specifically inhibits mTORC1 activity, although recent studies indicate that prolonged rapamycin treatment can also inhibit mTORC2 assembly and function in some cell types. Both complexes participate in signaling pathways associated with human diseases, including tuberous sclerosis complex (TSC), lymphangioleiomyomatosis (LAM), Cowden disease, Peutz-Jeghers syndrome (PJS), neurofibromatosis, familial cardiac hypertrophy, and cancers characterized by hyperactivation of PI3K/Akt (Guertin, 2007).
The roles of mTORC1 and mTORC2 during mammalian development are not well understood. In addition to mTOR, mTORC1 contains raptor (regulatory-associated protein of mTOR) and mLST8 (also called GβL). mTORC2 also contains mTOR and mLST8, but instead of raptor, this complex contains rictor (rapamycin-insensitive companion of mTOR). Germline disruption of mTOR in mice causes embryonic lethality at or around implantation. However, when these mice were engineered, it was not known that mTOR is part of two distinct complexes and pathways. With this new information, it becomes difficult to interpret the phenotypes of the mTOR null mice because these animals lack both mTORC1 and mTORC2 function. Thus, to determine the in vivo role of each branch of the mTOR signaling network, mice lacking the expression of raptor, rictor, and mLST8 were generated and characterized. It is conclude that during mammalian development, mTORC1 and mTORC2 have different, but essential, roles; that mLST8 is required only for mTORC2 function; and that mTORC2 is a crucial component of the Akt/PKB-FOXO and PKCα signaling networks (Guertin, 2007).
Many forms of long-lasting behavioral and synaptic plasticity require the synthesis of new proteins. For example, long-term potentiation (LTP) that endures for more than an hour requires both transcription and translation. The signal-transduction mechanisms that couple synaptic events to protein translational machinery during long-lasting synaptic plasticity, however, are not well understood. One signaling pathway that is stimulated by growth factors and results in the translation of specific mRNAs includes the rapamycin-sensitive kinase mammalian target of rapamycin (mTOR, also known as FRAP and RAFT-1). Several components of this translational signaling pathway, including mTOR, eukaryotic initiation factor-4E-binding proteins 1 and 2, and eukaryotic initiation factor-4E, are present in the rat hippocampus as shown by Western blot analysis, and these proteins are detected in the cell bodies and dendrites in the hippocampal slices by immunostaining studies. In cultured hippocampal neurons, these proteins are present in dendrites and are often found near the presynaptic protein, synapsin I. At synaptic sites, their distribution completely overlaps with a postsynaptic protein, PSD-95. These observations suggest the postsynaptic localization of these proteins. Disruption of mTOR signaling by rapamycin results in a reduction of late-phase LTP expression induced by high-frequency stimulation; the early phase of LTP is unaffected. Rapamycin also blocks the synaptic potentiation induced by brain-derived neurotrophic factor in hippocampal slices. These results demonstrate an essential role for rapamycin-sensitive signaling in the expression of two forms of synaptic plasticity that require new protein synthesis. The localization of this translational signaling pathway at postsynaptic sites may provide a mechanism that controls local protein synthesis at potentiated synapses (Tang, 2002).
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