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)

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.
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PMID:Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha ). Functions of lipid kinase-deficient PI3Kalpha in signaling. 1127 89

Mammalian target of rapamycin (mTOR) is the central element of a signaling pathway involved in the control of mRNA translation and cell growth. The actions of mTOR are mediated in part through the phosphorylation of the eukaryotic initiation factor 4E-binding protein, PHAS-I. In vitro mTOR phosphorylates PHAS-I in sites that control PHAS-I binding to eukaryotic initiation factor 4E; however, whether mTOR directly phosphorylates PHAS-I in cells has been a point of debate. The Arg-Ala-Ile-Pro (RAIP motif) and Phe-Glu-Met-Asp-Ile (tor signaling motif) sequences found in the NH2- and COOH-terminal regions of PHAS-I, respectively, are required for the efficient phosphorylation of PHAS-I in cells. Here we show that mutations in either motif markedly decreased the phosphorylation of recombinant PHAS-I by mTOR in vitro. Wild-type PHAS-I, but none of the mutant proteins, was coimmunoprecipitated with hemagglutinin-tagged raptor, an mTOR-associated protein, after extracts of cells overexpressing raptor had been supplemented with recombinant PHAS-I proteins. Moreover, raptor overexpression enhanced the phosphorylation of wild-type PHAS-I by mTOR but not the phosphorylation of the mutant proteins. The results not only provide direct evidence that both the RAIP and tor signaling motifs are important for the phosphorylation by mTOR, possibly by allowing PHAS-I binding to raptor, but also support the view that mTOR phosphorylates PHAS-I in cells.
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PMID:Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor. 1266 11

The enzyme p70S6 kinase (S6K1) is critical for cell growth, and we have reported its activation during cardiac hypertrophy. Because cardiac hypertrophy also involves integrin activation, we analyzed whether integrins could contribute to S6K1 activation. Using adult feline cardiomyocytes, here we report that integrin-interacting Arg-Gly-Asp (RGD) peptides activate S6K1 as observed by band shifting, kinase activity and phosphorylation at Thr-389 and Thr-421/Ser-424 of S6K1, and S6 protein phosphorylation. Perturbation of specific integrin function with blocking antibodies and by overexpressing the beta1A cytoplasmic tail revealed that beta3 but not beta1 integrin mediates the RGD-induced S6K1 activation. This activation is focal adhesion complex-independent and is accompanied by the activation of extracellular signal-regulated kinases 1/2 (ERK) and mammalian target of rapamycin (mTOR). Studies using specific inhibitors and dominant negative c-Raf expression in cardiomyocytes indicate that the S6K1 activation involves mTOR, MEK/ERK, and phosphatidylinositol 3-kinase pathways and is independent of protein kinase C and c-Raf. Finally, addition of fluorescent-labeled RGD peptide to cardiomyocytes exhibits its internalization and localization to the endocytic vesicles, and pretreatment of cardiomyocytes with endocytic inhibitors reduced the S6K1 activation. These data suggest that RGD interaction with beta3 integrin and its subsequent endocytosis trigger specific signaling pathway(s) for S6K1 activation in cardiomyocytes and that this process may contribute to hypertrophic growth and remodeling of myocardium.
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PMID:RGD-containing peptides activate S6K1 through beta3 integrin in adult cardiac muscle cells. 1290 16

Amino acids, especially branched-chain amino acids such as l-Leucine, have been revealed to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through mTOR signaling pathway. In this study, we showed that a cationic amino acid, l-Arginine, also activated this signaling pathway in a rapamycin-sensitive manner in rat intestinal epithelial cells, and this l-Arginine-induced amino acid signal transduction involved the cationic amino acid transport system. The manner of l-Arginine- and l-Leucine-induced activation of p70 S6 kinase depended on the stimulation time and the concentration of each amino acid, which suggested that the mechanism of this amino acid signal acceptance might be saturable. l-Arginine and l-Leucine induced activation of p70 S6 kinase and phosphorylation of 4E-BP1 in a rapamycin-sensitive manner, which suggested the involvement of mTOR signaling pathway in these effects. l-Arginine-induced activation of p70 S6 kinase was inhibited by NG-Methyl-L-Arginine (NMMA) and L-N5-(1-Iminoethyl) Ornithine (NIO), inhibitors of nitric oxide synthase (NOS) which also block cationic amino acid transporters, system y(+). However, l-Leucine-induced activation of p70 S6 kinase was not affected with treatment of NOS inhibitors. In conclusion, l-Arginine regulates p70 S6 kinase activity and phosphorylation of 4E-BP1 through mTOR signaling pathway, which involves system y(+), cationic amino acid transporters.
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PMID:Arginine and Leucine regulate p70 S6 kinase and 4E-BP1 in intestinal epithelial cells. 1501 Aug 53

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.
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PMID:Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity. 1534 59

Amino acids, especially branched-chain amino acids such as l-leucine, have been shown to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through the mTOR signaling pathway. In our recent study, l-arginine was also shown to activate the mTOR signaling pathway in rat intestinal epithelial cells. l-Glutamine is an amino acid that is required for culturing of numerous cell types, including rat intestinal epithelial cells. In this study, we showed that l-glutamine inhibited the activation of p70 S6 kinase and phosphorylation of 4E-BP1 induced by arginine or leucine in rat intestinal epithelial cells. Although the molecular mechanism of l-glutamine-induced inhibition of the mTOR signaling pathway is still unknown, the presence of this novel signal pathway may indicate that individual amino acids play specific roles for cellular proliferation and growth.
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PMID:Glutamine is a key regulator for amino acid-controlled cell growth through the mTOR signaling pathway in rat intestinal epithelial cells. 1556 68

Fetal growth retardation, a common end point for a variety of conditions affecting mother and fetus, is associated with reduced liver mass. We have performed studies to determine the mechanism for decreased liver mass in a maternal starvation model of fetal growth restriction in the rat. Pregnant dams were deprived of food for 48 h before delivery on embryonic day 19 (E19). Fetal body weight was not affected. However, fetal liver weight was reduced by approximately 15%. Immunostaining of fetal liver for proliferating cell nuclear antigen and flow cytometry on isolated fetal hepatocytes showed G1 cell cycle arrest in samples from starved dams. Based on our prior studies showing attenuated hepatic insulin signaling in the late gestation fetal rat, we tested the hypothesis that G1 arrest in our model might be due to altered nutrient signaling. Fetal plasma amino acid analyses showed no decrease in branched-chain amino acids, but arginine concentrations were decreased in fetuses of fasted mothers. Reduced arginine in E19 fetal hepatocyte culture media was associated with decreased DNA synthesis. Whereas levels of cyclins D and E were unchanged in fetal hepatocytes exposed to low arginine, cyclin E-dependent kinase activity was reduced. Low arginine also induced changes in the translational machinery, indicative of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin. Our results are consistent with the hypothesis that restricted nutrient availability signals to the hepatocyte cell cycle in fetuses of fasted mothers, thereby accounting for decreased hepatocyte proliferation and liver mass.
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PMID:Effects of maternal starvation on hepatocyte proliferation in the late gestation fetal rat. 1561 45

The small GTPase Rheb displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human Rheb in complexes with GDP, GTP, and GppNHp (5'-(beta,gamma-imide)triphosphate), which reveal novel structural features of Rheb and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of Rheb undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the alpha-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of Rheb. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in Rheb is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of Rheb as the catalytic residue.
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PMID:Structural basis for the unique biological function of small GTPase RHEB. 1572 74

There is much interest in precise functions of amino acids on mammalian growth and development. Some of amino acids play important roles in the control of gene expression by controlling the initiation phase of mRNA translation. The signal induced by leucine or arginine may stimulate cell growth. On the other hand, the other signal induced by glutamine may stimulate cellular proliferation and increase cell number, but inhibit the growth of cell size. However, there was no clear evidence that an individual amino acid specifically works as a signaling molecule. In our recent study, not only leucine, but also arginine is shown to activate the mTOR signaling pathway in rat intestinal epithelial cells. Furthermore, regarding L-Glutamine, an important amino acid that is required for culturing of numerous cell types, including rat intestinal epithelial cells, we have shown that it had an inhibitory effect on leucine- or arginine-induced activation of the mTOR signaling pathway. We have demonstrated that L-Glutamine inhibited the activation of p70 S6 kinase and phosphorylation of 4E-BP1 induced by arginine or leucine in rat intestinal epithelial cells. Based on these results, we are planning to confirm the effect of each amino acid including glutamine in an in vivo model using new born mice.
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PMID:Rational role of amino acids in intestinal epithelial cells (Review). 1601 50

An early signaling event activated by amino acids and growth factors in many cell types is the phosphorylation of the mammalian target of rapamycin (mTOR; FRAP), which is functionally linked to ribosomal protein s6 kinase (p70(s6k)), a kinase that plays a critical regulatory role in the translation of mRNAs and protein synthesis. We previously showed that intestinal cell migration, the initial event in epithelial restitution, is enhanced by l-arginine (ARG). In this study, we used amino acids as prototypic activators of mTOR and ARG, IGF-1, or serum as recognized stimulators of intestinal cell migration. We found that 1) protein synthesis is required for intestinal cell migration, 2) mTOR/p70(s6k) pathway inhibitors (rapamycin, wortmannin, and intracellular Ca(2+) chelation) inhibit cell migration, 3) ARG activates migration and mTOR/p70(s6k) (but not ERK-2) in migrating enterocytes, and 4) immunocytochemistry reveals abundant p70(s6k) staining in cytoplasm, whereas phospho-p70(s6k) is virtually all intranuclear in resting cells but redistributes to the periphery on activation by ARG. We conclude that mTOR/p70(s6k) signaling is essential to intestinal cell migration, is activated by ARG, involves both nuclear and cytoplasmic events, and may play a role in intestinal repair.
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PMID:Role of mTOR signaling in intestinal cell migration. 1671 51


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