Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42345 (mTOR)
 26,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nutrient overload induces constitutive S6K1 (S6 kinase 1) activation, which leads to insulin resistance by suppressing insulin-induced class I PI3K (phosphoinositide 3-kinase) signalling [Um, Frigerio, Watanabe, Picard, Joaquin, Sticker, Fumagalli, Allegrini, Kozma, Auwerx and Thomas (2004) Nature 431, 200-205]. This finding gave rise to the question of the mechanism by which nutrients, such as AAs (amino acids), enter the mTOR (mammalian target of rapamycin)/S6K1 signalling pathway. Counter to the prevailing view, our recent studies have shown that the AA input into the mTOR/S6K1 signalling pathway is not mediated by the tumour suppressor TSC1 (tuberous sclerosis complex 1)/TSC2 or its target, the proto-oncogene Rheb (Ras homologue enriched in brain). Instead, we found that the AA input was mediated by class 3 PI3K, or hVps34 (human vacuolar protein sorting 34). In brief, ectopic expression of hVps34 drives S6K1 activation, but only in the presence of AAs, and this effect is blocked by small interfering RNAs directed against hVps34. Moreover, stimulation of cells with AAs increases hVps34 activity, as indicated by the production of PI3P (phosphatidylinositol 3-phosphate). PI3P mediates the recruitment of proteins containing FYVE (Fab1p, YOTB, Vac1p and EEA1) or PX (Phox homology) domains to endosomal membranes, with PI3P-rich micro-domains acting as signalling platforms. Additional evidence indicating hVps34 as the mediator of AA input to S6K1 came from experiments in which S6K1 activation was attenuated by ectopic expression of a cDNA containing two FYVE domains, which bind to PI3P, preventing binding of proteins containing either FYVE or PX domains [Nobukuni, Joaquin, Roccio, Dann, Kim, Gulati, Byfield, Backer, Natt, Bos, Zwartkruis and Thomas (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14238-14243].
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PMID:Nutrient sensing in the mTOR/S6K1 signalling pathway. 1737 Dec 47

Autophagy is a lysosomal pathway involved in the turnover of cellular macromolecules and organelles. Starvation and various other stresses increase autophagic activity above the low basal levels observed in unstressed cells, where it is kept down by mammalian target of rapamycin complex 1 (mTORC1). In starved cells, LKB1 activates AMP-activated protein kinase (AMPK) that inhibits mTORC1 activity via a pathway involving tuberous sclerosis complex 1 and 2 (TSC1/2) and its substrate Rheb. The present study suggests hat AMPK inhibits mTORC1 and autophagy also in nonstarved cells. Various Ca(2+) mobilizing agents (vitamin D compounds, thapsigargin, ATP and ionomycin) activate MPK via activation of Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta), and his pathway is required for Ca(2+)-induced autophagy. Thus, we propose that an increase in free cytosolic Ca(2+) ([Ca(2+)](c)) induces autophagy via the CaMKK/beta-AMPK-TSC1/2-Rheb-mTORC1 signaling pathway and that AMPK is a more general regulator of autophagy than previously expected.
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PMID:AMP-activated protein kinase: a universal regulator of autophagy? 1724 28

The small GTPase Rheb is a positive upstream regulator of the target of rapamycin (TOR) complex 1 in mammalian cells and can bind directly to TOR complex 1. To identify the regions of the Rheb surface most critical for signaling to TOR complex 1, we created a set of 26 mutants wherein clusters of 1-5 putative solvent-exposed residues were changed to alanine, ultimately changing 65 residues distributed over the entire Rheb surface. The signaling function of these mutants was assessed by their ability, in comparison to wild type Rheb, to restore the phosphorylation of S6K1(Thr389) when expressed transiently in amino acid-deprived 293T cells. The major finding is that two mutants situated in the Rheb switch 2 segment, Y67A/I69A and I76A/D77A, exhibit a near total loss of function, whereas extensive replacement of the switch 1 segment and other surface residues with alanines causes relatively little disturbance of Rheb rescue of S6K1 from amino acid withdrawal. This is surprising in view of the minimal impact of guanyl nucleotide on Rheb switch 2 configuration. The loss of function Rheb switch 2 mutants are well expressed and exhibit partial agonist function in amino acid-replete cells. They are unimpaired in their ability to bind GTP or mammalian (m)TOR in vivo or in vitro, and the mTOR polypeptides retrieved with these inactive Rheb mutants exhibit kinase activity in vitro comparable with mTOR bound to wild type Rheb. We conclude that Rheb signaling to mTOR in vivo requires a Rheb switch 2-dependent interaction with an element other than the three known polypeptide components of TOR complex 1.
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PMID:The Rheb switch 2 segment is critical for signaling to target of rapamycin complex 1. 1747 Apr 30

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.
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PMID:The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. 1751 83

Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-tuberous sclerosis complex (TSC), non-lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.
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PMID:Activation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasms. 1752 3

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.
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PMID:The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe. 1755 68

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.
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PMID:PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. 1760 71

The TSC/Rheb/TOR signaling pathway plays important roles in growth and cell cycle regulation. The main player TOR belongs to the PI3K-related protein kinase family. Recent studies utilizing fission yeast Tor2 have led to the identification of a number of amino acid changes that lead to inactivation as well as activation of TOR kinase. Also, constitutive active mutations in its upstream regulator, Rheb, have been identified. Isolation and characterization of temperature sensitive Tor2 mutants have established that this kinase functions as a key switch that determines cell fate between growth and sexual development. Introduction of Tor2 activating mutations into mTOR conferred nutrient independent activation of mTOR. Interestingly, these studies point to regions of TOR kinase important for its function.
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PMID:The TSC/Rheb/TOR signaling pathway in fission yeast and mammalian cells: temperature sensitive and constitutive active mutants of TOR. 1763 64

TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.
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PMID:Generation and characterization of monoclonal antibodies against tuberous sclerosis complex 2. 1772 89

Insulin and amino acids act independently to stimulate protein synthesis in skeletal muscle of neonatal pigs, and the responses decrease with development. The purpose of this study was to compare the separate effects of fed levels of INS and AA on the activation of signaling components leading to translation initiation and how these responses change with development. Overnight-fasted 6- (n = 4/group) and 26-day-old (n = 6/ group) pigs were studied during 1) euinsulinemic-euglycemiceuaminoacidemic conditions (controls), 2) euinsulinemic-euglycemichyperaminoacidemic clamps (AA), and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA, increased the phosphorylation of protein kinase B (PKB) and tuberous sclerosis 2 (TSC2). Both INS and AA increased protein synthesis and the phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase-1, and eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), and these responses were higher in 6-day-old compared with 26-day-old pigs. Both INS and AA decreased the binding of 4E-BP1 to eIF4E and increased eIF4E binding to eIF4G; these effects were greater in 6-day-old than in 26-day-old pigs. Neither INS nor AA altered the composition of mTORC1 (raptor, mTOR, and GbetaL) or mTORC2 (rictor, mTOR, and GbetaL) complexes. Furthermore, neither INS, AA, nor age had any effect on the abundance of Rheb and the phosphorylation of AMP-activated protein kinase and eukaryotic elongation factor 2. Our results suggest that the activation by insulin and amino acids of signaling components leading to translation initiation is developmentally regulated and parallels the developmental decline in protein synthesis in skeletal muscle of neonatal pigs.
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PMID:Activation by insulin and amino acids of signaling components leading to translation initiation in skeletal muscle of neonatal pigs is developmentally regulated. 1787 22


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