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)

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.
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PMID:Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells. 1259 28

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
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PMID:Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1. 1271 46

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

The molecular interplay between the phosphoinositide 3-kinase (PI3K) pathway and mammalian target of rapamycin (mTOR) signalling in the control of cell growth and proliferation has been the subject of much interest and debate amongst cell biologists. A recent escalation of research in this area has come from the discovery of the tuberous sclerosis complex gene products, tuberin and hamartin, as central regulators of mTOR activation. The PI3K effector Akt/protein kinase B has been found to directly phosphorylate tuberin and is thereby thought to activate mTOR through inhibition of the tuberin-hamartin complex. The many recent studies aimed at defining the molecular nature of this revamped PI3K/Akt/mTOR pathway are reviewed here. The collective data discussed have laid the groundwork for important new insights into the many cancers caused by aberrant PI3K activation and the clinically challenging tuberous sclerosis complex disease and have suggested a possible means of treatment for both.
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PMID:United at last: the tuberous sclerosis complex gene products connect the phosphoinositide 3-kinase/Akt pathway to mammalian target of rapamycin (mTOR) signalling. 1277 58

In this study, we investigate the extracellular and intracellular signals that drive cell cycle progression of activated B cells in the absence of T cell help. We find that brief engagement of the B cell receptor is sufficient to induce a single cell division in a fraction of cells, but that survival during successive cell divisions requires sustained receptor stimulation. In contrast, T cells have been shown previously to commit to multiple cell divisions following brief TCR engagement. Both early and late B cell receptor signals are blocked by inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin and are associated with S6 kinase activation and increased cell size. The requirement for ongoing Ag receptor signaling can be overcome by engagement of CD40 but only partially by IL-4. Proliferation driven by LPS also requires sustained exposure to the stimulus. These findings reveal checkpoints that may limit T-independent B cell responses when Ag exposure is transient.
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PMID:Proliferation and survival of activated B cells requires sustained antigen receptor engagement and phosphoinositide 3-kinase activation. 1279 10

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo.
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PMID:Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin. 1286 26

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.
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PMID:WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. 1461 43

mRNA abundance for a number of genes is increased by amino acid limitation. From an array screening study in HepG2 human hepatoma cells, it was established that one set of genes affected by amino acid availability is the set associated with cell-cycle control. The present study describes the increased expression of both mRNA and protein for the cyclin-dependent kinase inhibitors p21 and p27 in response to deprivation of HepG2 cells for a single essential amino acid, histidine. The increase in p21 and p27 mRNA content depended on de novo protein synthesis and involved a post-transcriptional mRNA stabilization component. For p21, increase in mRNA by histidine depletion appeared to be independent of p53 transactivation, and the absolute level of p53 protein was unaffected by this treatment. Histidine limitation caused an increase in the phosphorylation of ERK1/ERK2 (extracellular-signal-regulated kinase), and inhibition of the ERK signal transduction pathway resulted in a reduction in the starvation-dependent increase in p21 mRNA. Blockade of the phosphoinositide 3-kinase and mTOR (mammalian target of rapamycin) pathways also blunted the increase in p21 mRNA content. These results document the amino acid-dependent control of the synthesis of specific cell-cycle regulators and help to explain the block at G1 phase after amino acid limitation.
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PMID:Induction of p21 and p27 expression by amino acid deprivation of HepG2 human hepatoma cells involves mRNA stabilization. 1471 82

Mitogens activate protein translation through phosphorylation of p7S6 kinase (p70(S6K)) and eIF4E binding protein 1 (4E-BP1) mediated by the mammalian target of rapamycin (mTOR) or phosphoinositide 3-kinase (PI3K). A recent report (Science 294, 1942, 2001) has implicated phospholipase D (PLD) in mTOR signaling. We studied the role of PLD in the phosphorylation of p70(S6K) and 4E-BP1 induced by lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) using fibroblasts deficient in PLD activity and also 1-butanol, which inhibits phosphatidic acid production by PLD. The reduction in PLD activity in both situations impaired the effect of LPA on mTOR signaling but did not inhibit the effect of PDGF. PDGF induced marked phosphorylation of Akt (a PI3K target) but this was not affected by PLD deficiency. LPA caused much less phosphorylation of Akt and this was dependent on PLD activity. Toxin B, which inactivates Rho GTPases, markedly impaired PLD1 activation and phosphorylation of Akt, p70(S6K), and 4E-BP1 induced by LPA but had a minimal or no effect on the actions of PDGF. These results support the hypothesis that LPA activates protein translation through the action of PLD1-generated PA on mTOR and the PI3K/Akt pathway whereas PDGF acts through P13K/Akt independent of PLD1.
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PMID:Role of phospholipase D1 in the regulation of mTOR activity by lysophosphatidic acid. 1476 25

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1-/- mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.
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PMID:Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. 1503 Mar 12


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