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Query: UNIPROT:P42345 (mTOR)
 26,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we have investigated the effects of insulin, chemical and hyperthermic stresses upon the activity of the System A amino acid transporter in L6 rat muscle cells. Uptake of alpha-methyl-aminoisobutyric acid (Me-AIB), a non-metabolisable System A substrate, was increased by between 50% and 80% when muscle cells were exposed to a maximally effective concentration of insulin (100 nM), sodium arsenite (ARS, 0.5 mM) or a 42 degrees C heat shock (HS). The observed activation in System A in response to all three stimuli was maximal within 20 min and in the case of insulin and ARS primarily involved an increase in the Vmax of System A transport. In contrast, HS induced significant increases in both Vmax and Km of System A transport suggesting that hyperthermic stress may activate System A by a mechanism distinct from that mediating the effects of insulin and ARS. The hormonal stimulation of System A was blocked by the phosphoinositide 3-kinase (PI3k) inhibitor, wortmannin, but not by rapamycin or PD 98059 which respectively inhibit the mTOR and classical MAP kinase pathways. Exposure of L6 cells to ARS, but not HS, caused a 4.7-fold stimulation in MAPKAP-K2 activity that was blocked by SB 203580, a specific inhibitor of the stress activated protein kinase SAPK2/p38. However, neither SB 203580, rapamycin nor wortmannin were able to suppress the ARS- or HS-induced stimulation in System A transport. In summary, our results demonstrate that activity of the System A transporter can be rapidly upregulated in response to hormonal and stress stimuli through changes in the transport kinetics of the System A carrier. Our data show that whilst the hormonal response is PI3k dependent, the signalling mechanisms which instigate changes in System A activity in response to chemical or hyperthermic stress do not appear to involve PI3k or components of the mTOR, p42/p44 MAP kinase or SAPK2/p38 signalling pathways.
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PMID:Regulation of System A amino acid transport in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress. 987 56

Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3, and this is likely to have important regulatory implications for translation initiation.
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PMID:L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport. 1094 49

Rapamycin and its analogues have shown promising anticancer activities in preclinical and clinical studies. However, the mechanism whereby rapamycin inhibits signaling through the mammalian target of rapamycin (mTOR) remains poorly understood. Here, we show that the FKBP12/rapamycin complex is an essentially irreversible inhibitor of mTOR kinase activity in vitro. However, we observe no suppression of mTOR catalytic activity after immunoprecipitation from rapamycin-treated cells. These results suggest either that rapamycin acts as a reversible kinase inhibitor in intact cells or that the cellular effects of rapamycin are not mediated through global suppression in mTOR kinase activity. To better understand the cellular pharmacology of rapamycin, we compared the individual and combined effects of rapamycin and kinase-inactive mTOR expression on a panel of mTOR-dependent cellular responses. These studies identified glycolytic activity, amino acid transporter trafficking, and Akt kinase activity as novel, mTOR-modulated functions in mammalian cells. Whereas kinase-inactive mTOR did not enhance the decreases in cell size and glycolysis induced by rapamycin, expression of this mTOR mutant significantly enhanced the inhibitory effects of rapamycin on cell proliferation, 4EBP1 phosphorylation, and Akt activity. Unexpectedly, amino acid transporter trafficking was perturbed by kinase-inactive mTOR but not by rapamycin, indicating that this process is rapamycin insensitive. These results indicate that rapamycin exerts variable inhibitory actions on mTOR signaling functions and suggest that direct inhibitors of the mTOR kinase domain will display substantially broader anticancer activities than rapamycin.
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PMID:Differential effects of rapamycin on mammalian target of rapamycin signaling functions in mammalian cells. 1467 9

Skeletal muscle is a major insulin target tissue and has a prominent role in the control of body amino acid economy, being the principal store of free and protein-bound amino acids and a dominant locus for amino acid metabolism. Interplay between diverse stimuli (e.g., hormonal/nutritional/mechanical) modulates muscle insulin action to serve physiological need through the action of factors such as intramuscular signaling molecules. Ceramide, a product of sphingolipid metabolism and cytokine signaling, has a potent contra-insulin action with respect to the transport and deposition of glucose in skeletal muscle, although ceramide effects on muscle amino acid turnover have not previously been documented. Here, membrane permeant C2-ceramide is shown to attenuate the basal and insulin-stimulated activity of the Na+-dependent System A amino acid transporter in rat muscle cells (L6 myotubes) by depletion of the plasma membrane abundance of SNAT2 (a System A isoform). Concomitant with transporter down-regulation, ceramide diminished both intramyocellular amino acid abundance and the phosphorylation of translation regulators lying downstream of mTOR. The physiological outcome of ceramide signaling in this instance is a marked reduction in cellular protein synthesis, a result that is likely to represent an important component of the processes leading to muscle wasting in catabolic conditions.
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PMID:Ceramide down-regulates System A amino acid transport and protein synthesis in rat skeletal muscle cells. 1561 Nov 52

Pathological fetal growth is associated with perinatal morbidity and the development of diabetes and cardiovascular disease later in life. Placental nutrient transport is a primary determinant of fetal growth. In human intrauterine growth restriction (IUGR) the activity of key placental amino acid transporters, such as systems A and L, is decreased. However the mechanisms regulating placental nutrient transporters are poorly understood. We tested the hypothesis that the mammalian target of rapamycin (mTOR) signalling pathway regulates amino acid transport in the human placenta and that the activity of the placental mTOR pathway is reduced in IUGR. Using immunohistochemistry and culture of trophoblast cells, we show for the first time that the mTOR protein is expressed in the transporting epithelium of the human placenta. We further demonstrate that placental mTOR regulates activity of the l-amino acid transporter, but not system A or taurine transporters, by determining the mediated uptake of isotope-labelled leucine, methylaminoisobutyric acid and taurine in primary villous fragments after inhibition of mTOR using rapamycin. The protein expression of placental phospho-S6K1 (Thr-389), a measure of the activity of the mTOR signalling pathway, was markedly reduced in placentas obtained from pregnancies complicated by IUGR. These data identify mTOR as an important regulator of placental amino acid transport, and provide a mechanism for the changes in placental leucine transport in IUGR previously demonstrated in humans. We propose that mTOR functions as a placental nutrient sensor, matching fetal growth with maternal nutrient availability by regulating placental nutrient transport.
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PMID:Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth. 1746 46

p70S6 kinase is a multipotent kinase that phosphorylates substrates in response to extracellular stimuli. This kinase activity inhibits apoptosis, regulates cell size and controls translation. In the CNS, p70S6K also participates in synaptic plasticity. In this study, we report that leucine, a branched-chain amino acid, induces phosphorylation and activation of p70S6 kinase in cortical neurons. Leucine also induces phosphorylation of S6 protein, a substrate of p70S6K. These effects of leucine are completely inhibited by rapamycin, consistent with mammalian target of rapamycin mediating p70S6 phosphorylation. Finally, we demonstrate that the action of leucine on cortical neurons is mediated by the system L amino acid transporter. Neurons express components of system L amino acid transporter LAT1, LAT2, and CD98. Leucine uptake and its effect on p70S6 kinase are both inhibited by a specific inhibitor of system L amino acid transporter. We propose that leucine plays important roles in regulating signaling by p70S6 kinase by acting as an intercellular communicator in the CNS.
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PMID:Leucine induces phosphorylation and activation of p70S6K in cortical neurons via the system L amino acid transporter. 1843 29

The activity of placental amino acid transporters is decreased in intrauterine growth restriction (IUGR), but the underlying regulatory mechanisms have not been established. Inhibition of the mammalian target of rapamycin (mTOR) signaling pathway has been shown to decrease the activity of the system L amino acid transporter in human placental villous fragments, and placental mTOR activity is decreased in IUGR. In the present study, we used cultured primary trophoblast cells to study mTOR regulation of placental amino acid transporters in more detail and to test the hypothesis that mTOR alters amino acid transport activity by changes in transporter expression. Inhibition of mTOR by rapamycin significantly reduced the activity of system A (-17%), system L (-28%), and taurine (-40%) amino acid transporters. mRNA expression of isoforms of the three amino acid transporter systems in response to mTOR inhibition was measured using quantitative real-time PCR. mRNA expression of l-type amino acid transporter 1 (LAT1; a system L isoform) and taurine transporter was reduced by 13% and 50%, respectively; however, mTOR inhibition did not alter the mRNA expression of system A isoforms (sodium-coupled neutral amino acid transporter-1, -2, and -4), LAT2, or 4F2hc. Rapamycin treatment did not significantly affect the protein expression of any of the transporter isoforms. We conclude that mTOR signaling regulates the activity of key placental amino acid transporters and that this effect is not due to a decrease in total protein expression. These data suggest that mTOR regulates placental amino acid transporters by posttranslational modifications or by affecting transporter translocation to the plasma membrane.
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PMID:Regulation of placental amino acid transporter activity by mammalian target of rapamycin. 1898 52

The mTOR (mammalian target of rapamycin) signalling pathway functions as a nutrient sensor, both in individual cells and, more globally, in organs such as the fat body in Drosophila and the hypothalamus in the rat. The activity of placental amino acid transporters is decreased in IUGR (intrauterine growth restriction), and recent experimental evidence suggests that these changes contribute directly to the restricted fetal growth. We have shown that mTOR regulates the activity of the placental L-type amino acid transporter system and that placental mTOR activity is decreased in IUGR. The present review summarizes the emerging evidence implicating placental mTOR signalling as a key mechanism linking maternal nutrient and growth factor concentrations to amino acid transport in the human placenta. Since fetal growth is critically dependent on placental nutrient transport, placental mTOR signalling plays an important role in the regulation of fetal growth.
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PMID:Placental mTOR links maternal nutrient availability to fetal growth. 1914 50

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC(50) value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.
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PMID:Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: a possible target for combination therapy with anti-proliferative aminopeptidase inhibitors. 2051 Jun 78

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a multicomponent, nutrient-sensitive protein that is implicated in a wide range of major human diseases. mTORC1 responds to both growth factors and changes in local amino acid levels. Until recently, the intracellular amino acid-sensing mechanism that regulates mTORC1 had remained unexplored. However, studies in human cells in culture have demonstrated that in response to amino acid stimulation, mTOR (a conserved member of the PI3K superfamily) is shuttled to late endosomal and lysosomal compartments, where it binds the Ragulator-Rag complex and is assembled into active mTORC1. Members of the proton-assisted amino acid transporter (PAT/SLC36) family have been identified as critical components of the amino acid-sensing system that regulates mTORC1 present in endosomal and lysosomal membranes. These discoveries not only highlight several new potential drug targets that could impact selectively on mTORC1 activity in cancer cells, but also provide novel insights into the strategies used by such cells to outcompete their neighbors in growth factor- and nutrient-depleted conditions. In this review, recent mechanistic insights into how mTORC1 activity is controlled by amino acids and the potential for the selective targeting this regulatory input are discussed.
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PMID:Intracellular amino acid sensing and mTORC1-regulated growth: new ways to block an old target? 2115 18


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