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

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.
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PMID:Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. 1036 59

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282% of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292%, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336%, P = 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647%, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292%, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2 alpha phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2 alpha. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.
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PMID:Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle. 1296 87

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.
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PMID:Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids. 1468 79

Arsenite is ubiquitous in the environment, particularly in the form of contaminated water. Although this metal is a known human carcinogen, its exact mechanism of action remains unclear. P70S6K1 phosphorylates the ribosomal 40S protein leading to increased protein translation, and is an important regulator of cell growth and proliferation. Hypoxia inducible factor-1 (HIF-1) is a basic helix-loop-helix transcription factor composed of two subunits, HIF-1alpha and HIF-1beta. HIF-1 activates the transcription of a number of genes that mediate angiogenesis and tumor formation. In this study we demonstrated that arsenite treatment increased levels of p70S6K1 phosphorylation and p70S6K1 activity in a PI3K and mTOR sensitive manner. We have also shown that arsenite specifically induces HIF-1alpha, but not HIF-1beta, protein levels in prostate cancer cells in a mTOR-dependent manner.
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PMID:Arsenite induces p70S6K1 activation and HIF-1alpha expression in prostate cancer cells. 1497 42

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.
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PMID:Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein. 1505

Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNX-induced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.
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PMID:Role of mammalian target of rapamycin signaling in compensatory renal hypertrophy. 1578 77

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.
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PMID:Acetoacetate induces CYP2E1 protein and suppresses CYP2E1 mRNA in primary cultured rat hepatocytes. 1598 59

Insulin stimulates protein synthesis by increasing translation initiation. This response is mediated by mTOR and is believed to result from 4EBP1 phosphorylation, which allows eIF4E to bind eIF4G. Here, we present evidence that mTOR interacts directly with eIF3 and that mTOR controls the association of eIF3 and eIF4G. Activating mTOR signaling with insulin increased by as much as five-fold the amount of eIF4G bound to eIF3. This novel effect was blocked by rapamycin and other inhibitors of mTOR, and it required neither eIF4E binding to eIF4G nor eIF3 binding to the 40S ribosomal subunit. The increase in eIF4G associated with eIF3 occurred rapidly and at physiological concentrations of insulin. Moreover, the magnitude of the response was similar to the increase in eIF4E binding to eIF4G produced by insulin. Thus, increasing eIF4G association with eIF3 represents a potentially important mechanism by which insulin, as well as amino acids and growth factors that activate mTOR, stimulate translation.
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PMID:mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin. 1654 Nov 3

UVA is the major portion (90-99%) of solar radiation reaching the surface of the earth and has been described to lead to formation of benign and malignant tumors. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. Pomegranate fruit extract (PFE) possesses strong antioxidant and anti-inflammatory properties. Our recent studies have shown that PFE treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated activation of MAPK and NF-kappaB pathways. Signal transducers and activators of transcription 3 (STAT3), Protein Kinase B/AKT and Map Kinases (MAPKs), which are activated by a variety of factors, modulate cell proliferation, apoptosis and other biological activities. The goal of this study was to determine whether PFE affords protection against UVA-mediated activation of STAT3, AKT and extracellular signal-regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/cm2 of UVA exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. Pretreatment of NHEK with PFE (60-100 microg/mL) for 24 h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. mTOR, structurally related to PI3K, is involved in the regulation of p70S6K, which in turn phosphorylates the S6 protein of the 40S ribosomal subunit. We found that UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. PFE pretreatment resulted in a dose-dependent inhibition in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. Our data further demonstrate that PFE pretreatment of NHEK resulted in significant inhibition of UVA exposure-mediated increases in Ki-67 and PCNA. PFE pretreatment of NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of the cell cycle and the expression of Bax and Bad (proapoptotic proteins), with downregulation of Bcl-X(L) expression (antiapoptotic protein). Our data suggest that PFE is an effective agent for ameliorating UVA-mediated damages by modulating cellular pathways and merits further evaluation as a photochemopreventive agent.
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PMID:Photochemopreventive effect of pomegranate fruit extract on UVA-mediated activation of cellular pathways in normal human epidermal keratinocytes. 1661 91

More than 10 years ago, Rheb (Ras homolog enriched in brain) was identified as a highly conserved protein that is a member of the Ras superfamily of small GTPases, which play critical roles in cell growth and proliferation. Recently, a convergence of genetic and biochemical evidence from yeast, Drosophila, and mammalian cells has placed Rheb upstream of the mammalian target of rapamycin (mTOR) and immediately downstream of the tumor suppressors TSC1 (hamartin) and TSC2 (tuberin). Rheb plays a key role in the regulation of cell growth in response to growth factors, nutrients, and amino acids linking PI3K and TOR signaling. Rheb activation of the nutrient and energy-sensitive TOR pathway leads to the direct phosphorylation of two known downstream translational control targets by mTOR, the 40S ribosomal S6 kinase 1 (S6K1) and the eukaryotic translation initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1). Appropriate regulation of this pathway is crucial for the proper control of cell growth, proliferation, survival, and differentiation. Inappropriate regulation of these signaling molecules, therefore, can lead to a variety of human diseases. In this chapter, we describe cell biological and biochemical methods commonly used to study Rheb activation and dissect its role in the mTOR-signaling pathway.
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PMID:Rheb activation of mTOR and S6K1 signaling. 1675 52


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