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)

Several studies have suggested that activation of p70 ribosomal S6 kinase (p70 S6 kinase) by insulin may be mediated by the phosphatidylinositol 3-kinase (PI 3-kinase)-Akt pathway. However, by temporal analysis of the activation of each kinase in L6 muscle cells, we report that the activation of the two serine/threonine kinases (Akt and p70 S6 kinase) can be dissociated. Insulin stimulated p70 S6 kinase in intact cells in two phases. The first phase (5 min) of stimulation was fully inhibited by wortmannin (IC50 = 20 nM) and LY-294002 (full inhibition at 5 microM). After this early inhibition, p70 S6 kinase was gradually stimulated by insulin in the presence of 100 nM wortmannin. After 30 min, the stimulation was 65% of the maximum attained in the absence of wortmannin. The IC50 of wortmannin for inhibition of this second phase was approximately 150 nM. In contrast, activation of Akt1 by insulin was completely inhibited by 100 nM wortmannin at all time points investigated. Inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase with PD-098059 (10 microM) or treatment with the protein kinase C inhibitor bisindolylmaleimide (10 microM) had no effect on the late phase of insulin stimulation of p70 S6 kinase. We have previously shown that GLUT-1 protein synthesis in these cells is stimulated by insulin via the mTOR-p70 S6 kinase pathway, based on its sensitivity to rapamycin. We therefore investigated whether the signals leading to GLUT-1 synthesis correlated with the early or late phase of stimulation of p70 S6 kinase. GLUT-1 synthesis was not inhibited by wortmannin (100 nM). In summary, insulin activates p70 ribosomal S6 kinase in L6 muscle cells by two mechanisms, one dependent on and one independent of the activation of PI 3-kinase. In addition, activation of Akt1 is fully inhibited by wortmannin, suggesting that Akt1 does not participate in the late activation of p70 S6 kinase. Wortmannin-sensitive PI 3-kinases and Akt1 are not required for insulin stimulation of GLUT-1 protein biosynthesis.
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PMID:Temporal activation of p70 S6 kinase and Akt1 by insulin: PI 3-kinase-dependent and -independent mechanisms. 975 80

We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulin-mimetic bisperoxovanadium 1,10-phenanthroline (bpV(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin- and bpV(phen)-induced activation of the atypical protein kinase C (zeta/lambda) (PKC (zeta/lambda)) was reversed by wortmannin. Inhibition of PKC (zeta/lambda) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (zeta/lambda) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.
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PMID:Regulation of glycogen synthase in rat hepatocytes. Evidence for multiple signaling pathways. 1049 84

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

In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.
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PMID:Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase. 1274 4

We have previously suggested that phosphatidylinositol 3-kinase (PI3K)/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R(+)) and -negative (Trf-R(-)) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in differentiation and proliferation of both cell types, with a particular focus on c-Myc. Similar to p70 S6K, we found that the expression of c-Myc in Trf-R(+) cells is also higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, indicating that the extent of c-Myc inhibition by these inhibitors correlates with a reduction in proliferation, and that c-Myc is downstream from PI3K/p70 S6K. We also determined phosphorylation of the 4E-binding protein 1 (4E-BP1), which is regulated downstream of the mammalian target of rapamycin. The addition of G-CSF failed to enhance the phosphorylation state of 4E-BP1 of HL-60 cells 2 days after DMSO differentiation. An antisense oligonucleotide for c-myc inhibited both G-CSF-dependent enhancement of c-Myc expression and proliferation in Trf-R(+) cells, but did not enhance the differentiation in terms of O(2)(-)-generating ability or fMLP-R expression. In contrast, antisense oligonucleotide for c-myc promoted fMLP-R on non-treated HL-60 cells. We therefore conclude that the PI3K/p70 S6K/c-Myc cascade plays an important role in neutrophilic proliferation in HL-60 cells. Unlike that of rapamycin, however, the antisense oligonucleotide for c-myc could not promote differentiation of Trf-R(+) cells cultured with G-CSF, indicating that another target downstream of p70 S6K may control the differentiation of HL-60 cells in terms of the signal transduction of G-CSF.
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PMID:The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL-60 cells. 1281 73

Ischaemic preconditioning (IPC) protects the heart against myocardial infarction acutely as well as several hours later (e.g. 24-48 h). The mechanism of the profound cardioprotection is not completely explored. We hypothesized that PI3K/PDK1/Akt/mTOR/p70S6K-mediated pro-survival pathway is involved in delayed cardioprotection induced by IPC. Under Hypnorm-Diazepam anaesthesia, male New Zealand White rabbits were either sham-operated (SC) or preconditioned by four cycles of 5-min ischaemia and 10-min reperfusion on day 1. Twenty-four hours after recovery, the animals were anaesthetized with sodium pentobarbitone and subjected to 30-min ischaemia followed by 180-min reperfusion. Wortmannin (0.6 mg/kg, i.v.), an irreversible PI3 kinase (PI3K) inhibitor, rapamycin (0.25 mg/kg, i.v.), which prevents the phosphorylation of p70S6 kinase (p70S6K), or DMSO (control vehicle) was given 15 min prior to IPC. IPC significantly reduced infarct size compared to the control group (SC) (31.9 +/- 5.8% (n = 7) vs. 54.9 +/- 2.9% (n = 6), P < 0.05). Wortmannin and rapamycin alone had no effect on infarct size (56.3 +/- 1.6% (n = 6) and 54.7 +/- 3.8% (n = 6), respectively). However, when wortmannin or rapamycin were given prior to IPC the protection was completely abolished (49.9 +/- 2.8% (n = 6), 45.1 +/- 4.6% (n = 7), P < 0.05 vs. IPC). Western blot analysis showed that wortmannin, at a dose of 0.6 mg/kg, and rapamycin, at a dose of 0.25 mg/kg, were sufficient to prevent phosphorylation of Akt and p70S6K, respectively, when the inhibitors were given prior to IPC. We conclude that PI3K/PDK1/Akt/mTOR/p70S6K-signalling pathway plays an essential role in the development of the cardioprotection against infarction in rabbits.
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PMID:Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase. 1296 24

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.
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PMID:Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling. 1462 99

Adequate extravillous trophoblast (EVT) invasion is an essential step for placental formation. The aim of this study was to examine the possible role of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling in epidermal growth factor (EGF)-induced EVT migration and to determine if the 70 kDa ribosomal S6 kinase (p70S6K) is involved in this process. In this study, EGF significantly stimulated HTR8/SVneo cell migration and the phosphorylation of AKT, ERK1/2 and p70S6K in a concentration-dependent manner. The MAPK inhibitor U0126 decreased cell migration and ERK phosphorylation, but it did not influence p70S6K phosphorylation in response to EGF. In the presence of PI3K inhibitors (Wortmannin), EGF-stimulated trophoblast migration and phosphorylation of AKT and P70S6K (Thr(389) and Thr(421)/Ser(424)) were decreased, while EGF-induced ERK phosphorylation was not affected. Expression of an activated AKT (Myr-AKT2) increased basal phospho-p70S6K (Thr(389) and Thr(421)/Ser(424)) content, but failed to stimulate cell migration. However, it induced cell migration in the presence of EGF and Wortmannin, in which both AKT and MAPK pathways were activated. In addition, there was a concentration-dependent inhibition of cell migration and p70S6K phosphorylation (Thr(389) and Thr(421)/Ser(424)) in the presence of Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR, a downstream of AKT). Taken together, our data suggest that EGF-induced trophoblast migration involves the coordinated regulation of both PI3K/AKT and MAPK signalling pathways. mTOR/p70S6K is important in PI3K- but not MAPK-mediated trophoblast migration in response to EGF.
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PMID:Both mitogen-activated protein kinase and phosphatidylinositol 3-kinase signalling are required in epidermal growth factor-induced human trophoblast migration. 1523 5

To determine whether the lipoxygenase metabolites of arachidonic acid, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids [5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, respectively] are angiogenic, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All three HETEs stimulated HDMVEC tube formation and migration. Because 15(S)-HETE was found to be more potent than 5(S)-HETE and 12(S)-HETE in HDMVEC tube formation, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 15(S)-HETE stimulated Akt and S6K1 phosphorylation in HDMVEC in a time-dependent manner. Wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and S6K1 phosphorylation, whereas rapamycin, a specific inhibitor of Akt downstream effector, mammalian target of rapamycin (mTOR), suppressed only S6K1 phosphorylation induced by 15(S)-HETE suggesting that this eicosanoid activates the PI3K-Akt-mTOR-S6K1 signaling in HDMVEC. Wortmannin, LY294002, and rapamycin also inhibited 15(S)-HETE-induced HDMVEC tube formation and migration. In addition, all three HETEs stimulated angiogenesis as measured by in vivo Matrigel plug assay with 15(S)-HETE being more potent. Pharmacologic inhibition of PI3K-Akt-mTOR-S6K1 signaling completely suppressed 15(S)-HETE-induced in vivo angiogenesis. Consistent with these observations, adenoviral-mediated expression of dominant-negative Akt also blocked 15(S)-HETE-induced HDMVEC tube formation and migration and in vivo angiogenesis. Together, these results show for the first time that 15(S)-HETE stimulates angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling.
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PMID:15(S)-hydroxyeicosatetraenoic acid induces angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling. 1610 79

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser473) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71-101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT-induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71-101 dose-dependently inhibited 5-HT-induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT-induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species-dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT-induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.
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PMID:Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1. 1619 41


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