UNIPROT:P42345 - FACTA Search

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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 (eIF-4E)-binding proteins PHAS-I and PHAS-II were found to have overlapping but different patterns of expression in tissues. Both PHAS proteins were expressed in 3T3-L1 adipocytes, in which insulin stimulated their phosphorylation, promoted dissociation of PHAS.eIF-4E complexes, and decreased the ability of both to bind exogenous eIF-4E. The effects of insulin were attenuated by rapamycin and wortmannin, two agents that block activation of p70(S6K). Unlike PHAS-I, PHAS-II was readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins were attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibited the activation of p70(S6K). In summary, our results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70(S6K) signaling pathway and that in 3T3-L1 adipocytes this pathway is inhibited by increased cAMP.
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PMID:Control of the translational regulators PHAS-I and PHAS-II by insulin and cAMP in 3T3-L1 adipocytes. 893 71

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.
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PMID:Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. 920 8

The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression.
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PMID:Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin. 927 96

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.
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PMID:The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k. 927 19

Treatment of 3T3-L1 adipocytes with insulin (IC50 approximately 200 pM insulin) or insulin-like growth factor-1 (IC50 approximately 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one- and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPalpha. However, the repression of C/EBPalpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.
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PMID:Signaling pathways through which insulin regulates CCAAT/enhancer binding protein alpha (C/EBPalpha) phosphorylation and gene expression in 3T3-L1 adipocytes. Correlation with GLUT4 gene expression. 932 24

The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/RAFT-1/FRAP is the target through which the rapamycin.FKBP12 complex acts to dephosphorylate/deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin.FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivation in situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner.
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PMID:Regulation of eIF-4E BP1 phosphorylation by mTOR. 933 22

PHAS-I and PHAS-II are members of a newly discovered family of proteins that regulate translation initiation. PHAS-I is expressed in a wide variety of cell types, but it is highest in adipocytes, where protein synthesis is markedly increased by insulin. PHAS-II is highest in liver and kidney, where very little PHAS-I is found. PHAS proteins bind to eIF-4E, the mRNA cap-binding protein, and inhibit translation of capped mRNA in vitro and in cells. In rat adipocytes PHAS-I is phosphorylated in at least five sites, all of which conform to the consensus, (Ser/Thr)-Pro. Both PHAS proteins are phosphorylated in response to insulin or growth factors, such as EGF, PDGF and IGF-1. Phosphorylation in the appropriate site(s) promotes dissociation of PHAS/eIF-4E complexes. This allows eIF-4E to bind to eIF-4G (p220), thereby increasing the amount of the eIF-4F complex and the rate of translation initiation. Increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA in vitro, suggesting that regulation of the two proteins differs. However, increasing cAMP in cells also promotes dephosphorylation of PHAS-II. Thus, PHAS proteins appear to be key mediators not only of the stimulatory effects of insulin and growth factors on protein synthesis, but also of the inhibitory effects of cAMP. Moreover, by controlling eIF-4E PHAS proteins may be involved in the control of cell proliferation, as increasing eIF-4E is mitogenic and can even cause malignant transformation of cells. MAP kinase readily phosphorylates both PHAS-I and PHAS-II in vitro, but inhibiting activation of MAP kinase does not attenuate the effects of insulin on increasing phosphorylation of the PHAS proteins in adipocytes or skeletal muscle. MAP kinase phosphorylates neither PHAS-I nor PHAS-II at a significant rate when the proteins are bound to eIF-4E. Therefore, the role of MAP kinase in promoting the dissociation of PHAS/eIF-4E complexes is not clear. Of several protein kinases tested, only casein kinase-II phosphorylated PHAS-I when it was bound eIF-4E. Indeed, the bound form of PHAS-I was phosphorylated more rapidly than the free form. However, it is unlikely that casein kinase II regulates either PHAS protein, as the major site (Ser111) in PHAS-I phosphorylated by casein kinase II in vitro is not phosphorylated in adipocytes, and PHAS-II is not a substrate for casein kinase-II. Pharmacological and genetic evidence indicates that the mTOR/p70S6K pathway is involved in the control of PHAS-I and -II. Thus, PHAS proteins may be mediators of the effects of this pathway on protein synthesis and cell proliferation.
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PMID:PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation. 938 73

The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.
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PMID:Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells. 943 72

Growth factors and hormones activate protein translation by phosphorylation and inactivation of the translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive signaling pathway. The mechanism by which signals emanating from extracellular signals lead to phosphorylation of 4E-BPs is not well understood. Here we demonstrate that the activity of the serine/threonine kinase Akt/PKB is required in a signaling cascade that leads to phosphorylation and inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1. Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1 by Akt requires the activity of FRAP/mTOR. FRAP/mTOR may lie downstream of Akt in this signaling cascade. These results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1.
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PMID:4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. 947 19

The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which become unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p70Delta2-46/DeltaCT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase.
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PMID:Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. 960 62

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