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)

We have investigated the expression and regulation of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) by insulin and hyperosmotic stress in L6 rat skeletal muscle cells. NKCC was identified by immunoblotting as a 170 kDa protein in L6 myotubes and mediated 54% of K(+) ((86)Rb(+)) influx based on the sensitivity of ion transport to bumetanide, a NKCC inhibitor. The residual (86)Rb(+) influx occurred via the Na(+),K(+)-ATPase and other transporters not sensitive to bumetanide or ouabain. NKCC-mediated (86)Rb(+) influx was enhanced significantly ( approximately 1.6-fold) by acute cell exposure to insulin, but was inhibited significantly by tyrosine kinase inhibitors, wortmannin and rapamycin, consistent with a role for the insulin receptor tyrosine kinase, phosphoinositide 3 (PI3)-kinase and mTOR, respectively, in cotransporter activation. In contrast, the hormonal activation of NKCC was unaffected by inhibition of the classical Erk-signalling pathway. Subjecting L6 myotubes to an acute hyperosmotic challenge (420 mosmol l(-1)) led to a 40% reduction in cell volume and was accompanied by a rapid stimulation of NKCC activity ( approximately 2-fold). Intracellular volume recovered to normal levels within 60 min, but this regulatory volume increase (RVI) was prevented if bumetanide was present. Unlike insulin, activation of NKCC by hyperosmolarity did not involve PI3-kinase but was suppressed by inhibition of tyrosine kinases and the Erk pathway. While inhibition of tyrosine kinases, using genistein, led to a complete loss in NKCC activation in response to hyperosmotic stress, immunoprecipitation of NKCC revealed that the cotransporter was not regulated directly by tyrosine phosphorylation. Simultaneous exposure of L6 myotubes to insulin and hyperosmotic stress led to an additive increase in NKCC-mediated (86)Rb(+) influx, of which, only the insulin-stimulated component was wortmannin-sensitive. Our findings indicate that L6 myotubes express a functional NKCC that is rapidly activated in response to insulin and hyperosmotic shock by distinct intracellular signalling pathways. Furthermore, activation of NKCC in response to hyperosmotic-induced cell shrinkage represents a critical component of the RVI mechanism that allows L6 muscle cells to volume regulate.
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PMID:Signalling mechanisms underlying the rapid and additive stimulation of NKCC activity by insulin and hypertonicity in rat L6 skeletal muscle cells. 1528 43

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.
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PMID:EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway. 1535 95

3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin-treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.
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PMID:HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage. 1562 77

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.
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PMID:Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication. 1577 87

mTOR is a critical regulator of protein translation, and plays an important role in controlling cellular replication. Recent studies indicate that nutrient and growth factor mediated activation of mTOR is deregulated in human cancer, and therefore represents an attractive tumor target. However, activation of mTOR is a complex process that is not yet fully understood. DNA viruses and tumor cells often perturb similar cellular pathways to facilitate their replication. In a recent study, we used adenovirus as a novel tool to probe the mechanisms underlying the inappropriate activation of mTOR upon virus infection of quiescent primary cells. These studies revealed that adenovirus encodes two viral proteins, E4-ORF1 and E4-ORF4, which activate mTOR, even in the absence of nutrient/growth factor signals, and which play a role in promoting viral replication. E4-ORF1 mimics growth factor signaling to mTOR by activating PI3-kinase, whereas E4-ORF4, which binds and relocalizes PP2A, can substitute for glucose mediated activation of mTOR. We discuss insights from this study, together with the similarities that may exist between viruses and tumor cells with respect to the mechanistic and functional requirements for mTOR activation in driving their aberrant DNA replication.
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PMID:Adenovirus overrides cellular checkpoints for protein translation. 1597 Jun 98

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.
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PMID:Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes. 1600 40

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.
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PMID:Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt. 1603 16

PTEN is a tumor suppressor gene whose loss of function is observed in approximately 40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN.
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PMID:PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2. 1615 32

Ornithine decarboxylase (ODC) overexpression coupled with activated Ras is fully sufficient to oncogenically transform primary keratinocytes. To determine the Ras effector pathways that represent the minimal essential contribution to full oncogenic transformation in this context, we evaluated the cooperativity of different Ras effector mutants with overexpressed ODC in an in vivo tracheal xenotransplantation assay for epithelial cell invasiveness. Primary keratinocytes, isolated from either K6/ODC transgenic mouse skin (expressing increased ODC) or from normal littermate skin were infected with retrovirus producing an activated RasV12 or partial loss-of-function effector mutants of RasV12 that selectively induce only the Raf/ERK, RalGDS, or the PI3-kinase signaling pathway. Whereas keratinocytes expressing a fully activated RasV12 are not invasive in tracheal xenotransplants, ODC-overexpressing keratinocytes acquire an invasive phenotype with additional expression of either RasV12 or activation of the Raf/ERK pathway. Independent of a mutated ras, elevated levels of ODC activate the Akt/mTOR signaling pathway as well as the Rho/Rac pathway in primary keratinocytes. Thus, Raf/ERK signaling is sufficient to cooperate with increased ODC activity in the conversion of normal keratinocytes to invasive cells. In order to promote invasiveness in keratinocytes, elevated levels of ODC may cooperate with Raf/ERK via activation of the Akt and Rho/Rac signaling pathway.
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PMID:Elevated levels of ornithine decarboxylase cooperate with Raf/ERK activation to convert normal keratinocytes into invasive malignant cells. 1627 77

Nutritional excess and/or obesity represent well-known predisposition factors for the development of non-insulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y(632)MPM motif that is implicated in the binding of p85alpha/p110alpha PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.
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PMID:Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation. 1635 80


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