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)

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KDeltaCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (Km) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4+/-0.29 and 0.9+/-0.48 microM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors.
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PMID:Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay. 1621 57

The objective of this work was to evaluate the possible role of PI3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity. E47 cells (HepG2 cells transfected with human CYP2E1 cDNA) exposed to 25 microM iron-nitrilotriacetate+5 microM arachidonic acid (AA+Fe) developed higher toxicity than C34 cells (HepG2 cells transfected with empty plasmid). Toxicity was associated with increased oxidative stress and activation of calcium-dependent hydrolases calpain and phospholipase A2. Treatment of E47, but not C34 cells, with arachidonic acid and iron (AA+Fe) led to a decrease in the phosphorylation state of AKT. 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific inhibitor of PI3-kinase, produced a further decrease of phosphorylated AKT in AA+Fe-treated E47 cells. LY294002 and down-regulation of endogenous AKT with small interference RNAs increased the toxicity of AA+Fe in E47 cells. Toxicity of AA+Fe in rat hepatocytes was also increased by LY294002. LY294002 did not affect phospholipase A2 or calpain activation, CYP2E1 activity, or lipid peroxidation elicited by AA+Fe. alpha-Tocopherol prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of phosphorylated AKT. LY294002 potentiated AA+Fe-induced loss of mitochondrial membrane potential and ATP, whereas overexpression of constitutively active AKT partially prevented mitochondrial impairment and toxicity. Mitochondrial permeability transition inhibitors prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of mitochondrial membrane potential. These results suggest that: i) AA+Fe+CYP2E1-induced oxidative stress decreases AKT activation; ii) AKT inactivation induces mitochondrial impairment associated with opening of the permeability transition pore but is not dependent on the activation state of bad, glycogen synthase kinase-3beta, mammalian target of rapamycin, or bcl-xL; and iii) PI3-kinase/AKT may serve as a survival pathway against CYP2E1-dependent toxicity.
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PMID:Role of phosphatidylinositol 3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity. 1662 72

Beginning with phenylketonuria, dietary therapy for inborn errors has focused primarily on the restriction of the precursor to an affected catabolic pathway in an attempt to limit the production of potential toxins. Anaplerotic therapy is based on the concept that there may exist an energy deficit in these diseases that might be improved by providing alternative substrate for both the citric acid cycle (CAC) and the electron transport chain for enhanced ATP production. This article focuses on this basic problem, as it may relate to most catabolic disorders, and provides our current experience involving inherited diseases of mitochondrial fat oxidation, glycogen storage, and pyruvate metabolism using the anaplerotic compound triheptanoin. The observations have led to a realization that 'inter-organ' signalling and 'nutrient sensors' such as adenylate monophosphate mediated-protein kinase (AMPK) and mTOR (mammalian target of rapamycin) appear to play a significant role in the intermediary metabolism of these diseases. Activated AMPK turns on catabolic pathways to augment ATP production while turning off synthetic pathways that consume ATP. Information is provided regarding the inter-organ requirements for more normal metabolic function during crisis and how anaplerotic therapy using triheptanoin, as a direct source of substrate to the CAC for energy production, appears to be a more successful approach to an improved quality of life for these patients.
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PMID:Anaplerotic diet therapy in inherited metabolic disease: therapeutic potential. 1676 96

We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-beta independent. High glucose reduced AMPK alpha-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1beta-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.
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PMID:A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy. 1701 41

Hyperthyroidism increases metabolic rate, mitochondrial ATP production, and protein synthesis, but it remains to be determined whether all tissues and synthesis of specific protein pools are equally affected by hyperthyroidism. Previous studies showed that mitochondrial function was less responsive to elevated triiodothyronine (T(3)) levels in the low-oxidative plantaris muscle compared with other tissues in rats. We tested the hypothesis that in T(3)-treated animals mitochondrial protein synthesis would increase in oxidative but not glycolytic tissues. Male rats received either T(3) (200 mug/day, n = 10) or saline (controls, n = 9) by subcutaneous pump for 14 days, and then in vivo protein synthesis rates were measured using [(15)N]phenylalanine in liver, heart, plantaris, and red gastrocnemius (Red Gast). Mitochondrial protein synthesis rate in T(3)-treated rats was higher than in controls by 62% in Red Gast and plantaris and 89 and 115% in liver and heart, respectively (P < 0.01). Cytoplasmic protein synthesis rates in the T(3) group were 107-176% higher than control values (P < 0.01). There was also indirect evidence that protein breakdown was increased in all tissues of the T(3)-treated rats. Phosphorylation of selected regulators of protein synthesis in plantaris and Red Gast (mTOR, p70 S6 kinase, 4E-BP1), however, were not significantly affected by T(3). We conclude that T(3) infusion stimulates a general increase in mitochondrial and cytoplasmic protein synthesis rate among tissues and that this does not appear to explain the tissue-specific responses in mitochondrial oxidative capacity.
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PMID:Effect of T(3)-induced hyperthyroidism on mitochondrial and cytoplasmic protein synthesis rates in oxidative and glycolytic tissues in rats. 1704 59

The AMP-activated protein kinase (AMPK) has been referred to as an "energy sensor" because it binds to and is regulated by both AMP and ATP. The binding of AMP to AMPK allows it to be phosphorylated by upstream kinases, resulting in its activation. In contrast, the binding of ATP prevents its activation. AMPK regulates a multitude of metabolic processes that cumulatively function to maintain cellular energy homeostasis through repression of a number of energy-consuming processes with simultaneous enhancement of energy-producing processes. One downstream AMPK target that has been recently identified is the mammalian target of rapamycin (mTOR), a positive effector of cell growth and division. The focus of the present review is to briefly summarize current knowledge concerning the regulation of mTOR signaling by AMPK.
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PMID:Interaction between the AMP-activated protein kinase and mTOR signaling pathways. 1709 30

Identification of the key roles of protein kinases in signaling pathways leading to development of cancer has caused pharmacological interest to concentrate extensively on targeted therapies as a more specific and effective way for blockade of cancer progression. This review will mainly focus on inhibitors targeting these key components of cellular signaling by employing a technology-based point of view with respect to ATP- and non-ATP-competitive small molecule inhibitors and monoclonal antibodies of selected protein kinases, particularly, mammalian target of rapamycin (mTOR), BCR-ABL, MEK, p38 MAPK, EGFR PDGFR, VEGFR, HER2 and Raf. Inhibitors of the heat shock protein Hsp90 are also included in a separate section, as this protein plays an essential role for the maturation/proper activation of cancer-related protein kinases. In the following review, the molecular details of the mode of action of these inhibitors as well as the emergence of drug resistance encountered in several cases are discussed in light of the structural, molecular and clinical studies conducted so far.
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PMID:Protein kinases as drug targets in cancer. 1710 May 68

Muscle mass is determined by the difference between the rate of protein synthesis and degradation. If synthesis is greater than degradation, muscle mass will increase (hypertrophy) and when the reverse is true muscle mass will decrease (atrophy). Following resistance exercise/increased loading there is a transient increase in protein synthesis within muscle. This change in protein synthesis correlates with an increase in the activity of protein kinase B/Akt and mTOR (mammalian target of rapamycin). mTOR increases protein synthesis by increasing translation initiation and by inducing ribosomal biogenesis. By contrast, unloading or inactivity results in a decrease in protein synthesis and a significant increase in muscle protein breakdown. The decrease in synthesis is due in part to the inactivation of mTOR and therefore a decrease in translation initiation, but also to a decrease in the rate of translation elongation. The increase in degradation is the result of a co-ordinated response of the calpains, lysosomal proteases and the ATP-dependent ubiquitin-proteosome. Caspase 3 and the calpains act upstream of the ubiquitin-proteosome system to assist in the complete breakdown of the myofibrillar proteins. Two muscle specific E3 ubiquitin ligases, MuRF1 and MAFbx/atrogen-1, have been identified as key regulators of muscle atrophy. In this chapter, these pathways and how the balance between anabolism and catabolism is affected by loading and unloading will be discussed.
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PMID:Resistance exercise, muscle loading/unloading and the control of muscle mass. 1714 80

Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis. Leucine treatment upregulates mTOR signaling, which enhances protein synthesis; however, the mechanisms are not well understood. Herein, treatment of C2C12 myoblast cells with leucine enhanced the phosphorylation of mTOR and ribosomal protein S6 kinase. Leucine treatment also decreased the adenosine monophosphate/ATP ratio in myoblasts by 36.4 +/- 9.1% (P < 0.05) and reduced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) alpha subunit at Thr172 (28.6 +/- 4.9% reduction, P < 0.05) and inhibited AMPK activity (43.6 +/- 3.5% reduction, P < 0.05). In addition, leucine increased the phosphorylation of mTOR at Ser2448 by 63.5 +/- 10.0% (P < 0.05) and protein synthesis by 30.6 +/- 6.1% (P < 0.05). Applying 5-aminoimidazole-4-carbox-amide 1-beta-d-ribonucleoside, an activator of AMPK, abolished the stimulation of mTOR signaling by leucine, showing that AMPK negatively controls mTOR signaling. To further show the role of AMPK in mTOR signaling, myoblasts expressing a dominant negative AMPKalpha subunit were employed. Negative myoblasts had very low AMPK activity. The activation of mTOR induced by leucine in these cells was abated, showing that AMPK contributed to mTOR activation. In conclusion, leucine stimulates mTOR signaling in part through AMPK inhibition. This study implicates AMPK as an important target for nutritional management to enhance mTOR signaling and protein synthesis in muscle cells, thereby increasing muscle growth.
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PMID:Leucine stimulates mammalian target of rapamycin signaling in C2C12 myoblasts in part through inhibition of adenosine monophosphate-activated protein kinase. 1717 7

Leptin is a hormone primarily secreted by adipocytes and participating in the regulation of food intake and energy expenditure. Its blood levels usually correlate with adiposity. The secretion of this hormone is affected, among others, by food consumption, insulin, fasting and cold exposure. Regulation of leptin secretion depends on many intracellular events. It is known that the activation of mTOR (the mammalian target of rapamycin) as well as increase in ATP and malonyl-CoA content in adipocytes enhance secretion of leptin. The rise in intracellular cAMP and fatty acids is thought to evoke the opposite effect. Moreover, the undisturbed action of endogenous adenosine in adipocytes and the proper intracellular Ca(2+) concentration in these cells were also found to have an important function in leptin release. The role of mTOR, ATP, cAMP, fatty acids, malonyl-CoA, adenosine and Ca(2+) in the regulation of leptin secretion from adipocytes is discussed.
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PMID:Intracellular mediators in regulation of leptin secretion from adipocytes. 1718 48


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