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)

Excessive supply of fatty acids to the liver might be a contributing factor to hepatic insulin resistance associated with obesity and type 2 diabetes mellitus. The aim of this study was to investigate direct effects of palmitate on insulin signaling in hepatocytes. The ability of metformin to reverse changes induced by palmitate was also studied. Rat hepatocytes in primary culture exhibited a rightward shift of the insulin dose-response curve for PKB phosphorylation during culture with palmitate. The insulin-stimulated phosphorylation of GSK-3beta, a metabolic substrate of PKB, was diminished in palmitate hepatocytes. By contrast, the mTOR protein kinase was overstimulated in cells incubated with palmitate. Hepatocytes cultured with palmitate displayed hyperphosphorylation of IRS-1 at Ser residues 632/635, known to be phosphorylated by mTOR. Metformin treatment of the hepatocytes resulted in activation of the AMP-activated kinase, attenuation of the mTOR/S6K1 pathway, reduction of IRS-1 phosphorylation, and a leftward shift in the insulin dose-response curve for PKB activation. These data suggest a link between an oversupply of fatty acid to hepatocytes, a disproportionate stimulation of mTOR/S6K1, and resistance to insulin.
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PMID:Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes. 1769 34

Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS-1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB-alpha (HepG2-CA-Akt/PKB). IRS-1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS-1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2-CA-Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS-1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2-CA-Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS-1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2-CA-Akt/PKB cells. These results suggest that knockdown of IRS-1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS-1 knockdown in cells overexpressing constitutively active Akt1/PKB-alpha either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells.
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PMID:Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS-1 knockdown HepG2 cells. 1772 85

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.
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PMID:The selectivity of protein kinase inhibitors: a further update. 1785 Feb 14

We hypothesized that rates of myofibrillar and patellar tendon collagen synthesis would fall over time during disuse, the changes being accompanied in muscle by decreases in focal adhesion kinase (FAK) phosphorylation and in gene expression for proteolytic enzymes. We studied nine men (22 +/- 4 years, BMI 24 +/- 3 kg m(-2) (means +/- s.d.) who underwent unilateral lower leg suspension for 23 days; five were studied between 0 and 10 days and four between 10 and 21 days. Muscle and tendon biopsies were taken in the postabsorptive state at days 0, 10 and 21 for measurement of protein synthesis, gene expression and protein phosphorylation. Muscle cross-sectional area decreased by 5.2% at 14 days and 10.0% (both P < 0.001), at 23 days, i.e. 0.5% day(-1), whereas tendon dimensions were constant. Rates of myofibrillar protein synthesis fell (P < 0.01) from 0.047% h(-1) at day 0 to 0.022% h(-1) at 10 days without further changes. Tendon collagen synthetic rates also fell (P < 0.01), from 0.052 to 0.023% h(-1) at 10 days and then to 0.010% h(-1) at 21 days. FAK phosphorylation decreased 30% (P < 0.01) at 10 days. No changes occurred in the amounts/phosphorylation of PKB-P70s6k-mTOR pathway components. Expression of mRNA for MuRF-1 increased approximately 3-fold at 10 days without changes in MAFbx or tripeptidyl peptidase II mRNA, but all decreased between 10 and 21 days. Thus, both myofibrillar and tendon protein synthetic rates show progressive decreases during 21 days of disuse; in muscle, this is accompanied by decreased phosphorylation of FAK, with no marked increases in genes for proteolytic enzymes.
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PMID:The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse. 1790 Nov 16

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.
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PMID:Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor. 1796 Nov 25

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC(50), 0.149 +/- 0.045 micromol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors.
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PMID:Discovery of lactoquinomycin and related pyranonaphthoquinones as potent and allosteric inhibitors of AKT/PKB: mechanistic involvement of AKT catalytic activation loop cysteines. 1798 20

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.
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PMID:Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes. 1821 34

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3beta (GSK-3beta) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.
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PMID:Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity. 1826 25

A chronic increase in physical activity and (or) endurance training can improve insulin sensitivity in insulin-resistant skeletal muscle. Cellular mechanisms responsible for the development of insulin resistance are unclear, though one proposed mechanism is that nutrient overload chronically increases available energy, over-activating the mammalian target of rapamycin (mTOR) and ribosomal S6 kinase 1 (S6K1) signaling pathway leading to increased phosphorylation of serine residues on insulin receptor substrate-1 (IRS-1). The objective of this study was to determine if increased physical activity would inhibit mTOR/S6K1 signaling and reduce IRS-1 serine phosphorylation in rat skeletal muscle. Soleus muscle was collected from fed male Sprague-Dawley sedentary rats (Inactive) and rats with free access to running wheels for 9 weeks (Active). Immunoblotting methods were used to measure phosphorylation status of mTOR, S6K1, IRS-1, and PKB/Akt (protein kinase B/AKT), and total abundance of proteins associated with the mTOR pathway. Muscle citrate synthase activity and plasma insulin and glucose concentrations were measured. Phosphorylation of mTOR (Ser2448), S6K1 (Thr389), and IRS-1 (Ser636-639) was reduced in Active rats (p<0.05). Total protein abundance of mTOR, S6K1, IRS-1, 4E-BP1, eEF2, PKB/Akt and AMPKalpha, and phosphorylation of PKB/Akt were unaffected (p>0.05). Total SKAR protein, a downstream target of S6K1, and citrate synthase activity increased in Active rats (p<0.05), though plasma insulin and glucose levels were unchanged (p>0.05). Reduced mTOR/S6K1 signaling during chronic increases in physical activity may play an important regulatory role in the serine phosphorylation of IRS-1, which should be examined as a potential mechanism for attenuation of insulin resistance associated with increased IRS-1 serine phosphorylation.
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PMID:A chronic increase in physical activity inhibits fed-state mTOR/S6K1 signaling and reduces IRS-1 serine phosphorylation in rat skeletal muscle. 1834 58

In mammals, feeding promotes protein accretion in skeletal muscle through a stimulation of the insulin- and amino acid- sensitive mammalian target of rapamycin (mTOR) signaling pathway, leading to the induction of mRNA translation. The purpose of the present study was to characterize both in vivo and in vitro the activation of several major kinases involved in the mTOR pathway in the muscle of the carnivorous rainbow trout. Our results showed that meal feeding enhanced the phosphorylation of the target of rapamycin (TOR), PKB, p70 S6 kinase, and eIF4E-binding protein-1, suggesting that the mechanisms involved in the regulation of mRNA translation are well conserved between lower and higher vertebrates. Our in vitro studies on primary culture of trout muscle cells indicate that insulin and amino acids regulate TOR signaling and thus may be involved in meal feeding effect in this species as in mammals. In conclusion, we report here for the first time in a fish species, the existence and the nutritional regulation of several major kinases involved in the TOR pathway, opening a new area of research on the molecular bases of amino acid utilization in teleosts.
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PMID:An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss). 1843 42


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