Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP1) interacts directly with eIF4E and prevents it from forming initiation factor (eIF4F) complexes required for the initiation of cap-dependent mRNA translation. Insulin and other agents induce the phosphorylation of 4E-BP1 at multiple sites, resulting in its release from eIF4E, and this involves signalling through the mammalian target of rapamycin (mTOR). Here we show that D-glucose promotes the ability of insulin to bring about the phosphorylation of 4E-BP1 and the formation of eIF4F complexes. This appears to involve facilitation of the phosphorylation of at least three phosphorylation sites on 4E-BP1, i.e. Thr-36, Thr-45 and Thr-69. Non-metabolizable glucose analogues cannot substitute for D-glucose, but other hexoses can. This suggests that a product of hexose metabolism mediates the permissive effect of glucose. The effect of glucose was concentration-dependent within the range 1-5 mM. In contrast with the situation for 4E-BP1, glucose does not allow full activation of the 70 kDa ribosomal protein S6 kinase (p70 S6k; another target of mTOR signalling) or phosphorylation, in vivo, of its substrate, ribosomal protein S6. Taken together with earlier data showing that amino acids regulate 4E-BP1 and p70 S6k, the present findings show that 4E-BP1 in particular is regulated in response to the availability of both amino acids and sugars.
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PMID:Glucose exerts a permissive effect on the regulation of the initiation factor 4E binding protein 4E-BP1. 1151 50

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.
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PMID:Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. 1154 73

Inhibition of insulin-induced 3T3-L1 preadipocyte differentiation by rapamycin has been attributed to a blockade of the early critical clonal expansion phase of the adipogenic program. Rapamycin binds to, and inhibits, mTOR (mammalian target of rapamycin), leading to diminution of p70 S6 kinase activity and eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) function. Our objective was to determine if rapamycin-sensitive pathways exist subsequent to the clonal expansion phase. We determined that the mitotic clonal expansion was complete by day 4 of the differentiation protocol, based on the response to Ara-C (cytosine beta-D-arabinofuranoside), which only inhibits differentiation when administered during this phase. Treatment of differentiating 3T3-L1 cells with rapamycin, starting on day 4, exerted potent negative effects on glycerol phosphate dehydrogenase activity, and triacylglycerol accumulation, as well as on the protein expression of adipogenic transcription factors, C/EBPalpha and PPARgamma. Insulin-stimulated p70 S6 kinase activity, and its inhibition by rapamycin, were comparable in preadipocytes at day 0 vs. day 4 post-differentiation. We conclude that a component of the adipogenic program, operating after the completion of clonal expansion, is inhibited by rapamycin, suggesting an ongoing need for mTOR function in this process.
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PMID:Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion. 1157

Adipose tissue is a major site for whole-body glutamine synthesis and we are investigating mechanisms and regulation of glutamine transport across the adipocyte membrane. Glutamine transport in adipocytes includes both high- and low-affinity Na+-dependent components (consistent with observed expression of ASCT2 and ATA2/SAT2 transporter mRNAs respectively) and a Na+-independent transport component (consistent with observed expression of LAT1/2 transporter mRNAs). Hypo-osmotic (235 mosmol/kg) swelling of adipocytes transiently stimulated glutamine uptake (180% increase at 0.05 mM glutamine) within 5 mins. Stimulation was blocked by the tyrosine kinase inhibitor genistein and the MAP kinase pathway inhibitors PD98059 and SB203580, but not by wortmannin (PI 3-kinase inhibitor) or rapamycin (mTOR pathway inhibitor). Cell-swelling also stimulated uptake of glucose but not MeAIB (indicating that ASCT2 rather than ATA2 was stimulated by swelling). Insulin (66 nM) treatment for up to 1 h stimulated Na+-dependent glutamine transport and increased adipocyte water space. Activation of the ERK1-2 MAP kinase pathway by cell swelling or insulin may be important for rapid activation of the ASCT2 glutamine transporter in adipocytes. Insulin may also exert a minor additional stimulatory effect on adipocyte glutamine transport indirectly via cell swelling. The mechanisms regulating glutamine transport in adipose tissue are distinct from those in other major sites of glutamine turnover in the body (notably liver and skeletal muscle).
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PMID:Mechanisms of glutamine transport in rat adipocytes and acute regulation by cell swelling. 1168 15

Insulin regulates the expression of several hepatic genes. Although the general definition of insulin signaling has progressed dramatically, the elucidation of the complete signaling pathway from insulin receptor to transcription factors involved in the regulation of a specific gene remains to be established. In fact, recent works suggest that multiple divergent insulin signaling pathways regulate the expression of distinct genes. 5-Aminolevulinate synthase (ALAS) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. It has been reported that insulin caused the rapid inhibition of housekeeping ALAS transcription, but the mechanism involved in this repression has not been explored. The present study investigates the role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase pathways in insulin signaling relevant to ALAS inhibition. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in rat hepatocytes and HepG2 cells. Wortmannin and LY294002, PI3-kinase inhibitors, as well as lovastatin and PD152440, Ras farnesylation inhibitors, and MEK inhibitor PD98059 abolished the insulin repression of ALAS transcription. The inhibitor of mTOR/p70(S6K) rapamycin had no effect whatsoever upon hormone action. The overexpression of vectors encoding constitutively active Ras, MEK, or p90(RSK) mimicked the inhibitory action of insulin. Conversely, negative mutants of PKB, Ras, or MEK impaired insulin inhibition of ALAS promoter activity. Furthermore, inhibition of one of the pathways blocks the inhibitory effect produced by the activation of the other. Our findings suggest that factors involved in two signaling pathways that are often considered to be functionally separate during insulin action, the Ras/ERK/p90(RSK) pathway and the PI3K/PKB pathway, are jointly required for insulin-mediated inhibition of ALAS gene expression in rat hepatocytes and human hepatoma cells.
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PMID:Phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase signaling pathways are required for the regulation of 5-aminolevulinate synthase gene expression by insulin. 1171 32

The "metabolic cocktail" comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by >/=45% versus control hearts (P<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy.
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PMID:Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. 1173 85

Insulin inhibits the expression of the hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) and glucose-6-phosphatase (G6Pase) genes. The signaling pathway that mediates these events requires the activation of phosphatidylinositol 3-kinase, whereas transfection studies have suggested an involvement of Akt (protein kinase B) and FKHR, a transcription factor regulated by Akt. We now demonstrate that insulin repression of endogenous IGFBP-1 gene transcription was blocked by rapamycin or by amino acid starvation. Rapamycin inhibited the mammalian target of rapamycin (mTOR) and the subsequent activation of p70/p85 S6 protein kinase-1 (S6K1) by insulin, whereas amino acid depletion prevented insulin induction of these signaling molecules. Importantly, we demonstrate that insulin regulation of the thymine-rich insulin response element of the IGFBP-1 promoter was also inhibited by rapamycin. However, sustained activation of S6K1 did not repress this promoter. In addition, rapamycin did not affect insulin regulation of G6Pase expression or Akt activation. We propose that these observations indicate that an mTOR-dependent, but S6K-independent mechanism regulates the suppression of IGFBP-1 (but not G6Pase) gene expression by insulin. Therefore, although the insulin-responsive sequence of the G6Pase gene promoter is related to that of the IGFBP-1 promoter, the signaling pathways that mediate suppression of these genes are distinct.
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PMID:Insulin regulation of insulin-like growth factor-binding protein-1 gene expression is dependent on the mammalian target of rapamycin, but independent of ribosomal S6 kinase activity. 1178 21

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent stimulator of Erk, leads to the phosphorylation of 4E-BP1 and its dissociation from eIF4E. In contrast to agonists such as insulin, this occurs independently of PKB activation. In this report, we investigate the mechanism by which TPA regulates 4E-BP1 phosphorylation. Treatment of HEK293 cells with TPA was found to result in the phosphorylation of 4E-BP1 at Ser(64), Thr(69), and Thr(36/45). The TPA-stimulated phosphorylation of all these sites is sensitive to inhibitors of MEK and to the inhibitor of mTOR, rapamycin, indicating that inputs from both mTOR and MEK are required for the regulation of 4E-BP1 phosphorylation by TPA. Indeed, evidence is presented that mTOR may initially be required for the phosphorylation of Thr(45) in a priming step, which is necessary for the subsequent phosphorylation of Ser(64) and Thr(69) through an Erk-dependent pathway. Overexpression of constitutively active MEK in HEK293 cells resulted both in the phosphorylation of 4E-BP1 at Ser(64) and Thr(36/45) and its release from eIF4E. In this case, the phosphorylation of these sites was also blocked by inhibitors of MEK or by rapamycin. In conclusion, the Erk pathway, via mechanisms also requiring mTOR, regulates the phosphorylation of multiple sites in 4E-BP1 in vivo and this is sufficient for the release of 4E-BP1 from eIF4E.
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PMID:The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. 1179 19

Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway.
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PMID:Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1. 1180 94

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.
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PMID:Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle. 1183 61


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