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)

IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.
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PMID:PKCdelta and mTOR interact to regulate stress and IGF-I induced IRS-1 Ser312 phosphorylation in breast cancer cells. 1595 59

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.
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PMID:Acetoacetate induces CYP2E1 protein and suppresses CYP2E1 mRNA in primary cultured rat hepatocytes. 1598 59

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses.
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PMID:PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle. 1601 56

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), we detected only Ser-318, but not Ser-307 phosphorylation, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. As we have observed recently that the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, we studied the role of Shp2 in this context. We found that phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depended on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 were blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1.
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PMID:Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1. 1605 40

Increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is associated with cellular insulin resistance. We have recently identified serine 318 (Ser318) as a novel protein kinase C-zeta (PKC-zeta)-dependent phosphorylation site within IRS-1. As other kinases may phosphorylate at this serine residue as well, we aimed to identify such kinases in the present study. In C2C12 myotubes, exposure to insulin or phorbol ester markedly increased Ser318 phosphorylation. In contrast, high glucose, tumor necrosis factor-alpha, and free fatty acids did not provoke Ser318 phosphorylation. JNK and the PI 3-kinase/mTOR pathway were found to be implicated in insulin-induced Ser318 phosphorylation, but not in TPA-stimulated phosphorylation that was, at least partly, mediated by classical or novel PKC. In conclusion, with JNK and the PI 3-kinase/mTOR pathway as mediators of insulin-induced Ser318 phosphorylation, we have identified kinases that have previously been reported to play key roles in phosphorylation of other serine residues in IRS-1.
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PMID:Insulin-induced stimulation of JNK and the PI 3-kinase/mTOR pathway leads to phosphorylation of serine 318 of IRS-1 in C2C12 myotubes. 1609 31

In skeletal muscle, amino acids, together with hormones, are key regulators of protein metabolism. Leucine, in particular, has inhibitory effects of protein degradation in skeletal muscles, but the mechanisms are poorly understood. The present study addressed the role of leucine as a regulator of myofibrillar proteolysis in cultured chick myotubes and chick skeletal muscles, and aimed to determine which cellular responses regulate the process. In chick myotubes, leucine suppressed myofibrillar proteolysis (as measured by N(tau)-methylhistidine release), while also decreasing ubiquitin and proteasome C2 subunit mRNA. Oral administration of leucine also suppressed myofibrillar proteolysis (as measured by plasma N(tau)-methylhistidine concentration), while also decreasing proteasome C2 subunit mRNA in chick skeletal muscle. Leucine activated the phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) (but not the mammalian target of rapamycin) inhibition of these pathways and increased myofibrillar proteolysis, ubiquitin and proteasome C2 subunit mRNA. Thus, an important component of muscle proteolysis inhibition by leucine, through the PI3K and PKC, is its ability to suppress transcription of the ubiquitin and proteasome C2 subunit, and degradation of myofibrillar protein.
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PMID:Leucine suppresses myofibrillar proteolysis by down-regulating ubiquitin-proteasome pathway in chick skeletal muscles. 1615 8

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KDeltaCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (Km) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4+/-0.29 and 0.9+/-0.48 microM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors.
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PMID:Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay. 1621 57

Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase beta (LPAAT-beta), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-beta converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-beta was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-beta using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-beta also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-beta was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-alpha. These findings identify LPAAT-beta as a potential prognostic and therapeutic target in ovarian and endometrial cancer.
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PMID:Lysophosphatidic acid acyltransferase-beta is a prognostic marker and therapeutic target in gynecologic malignancies. 1623 Apr 5

We reported previously that insulin elevated alpha-class glutathione S-transferase (GSTs) protein levels in primary cultured rat hepatocytes (Kim et al., 2003b). In contrast, glucagon down-regulated alpha- and pi-class GST expression, and mechanistic research implicated cAMP and protein kinase A in this process (Kim et al., 2003b). The present study examines the signaling pathways involved in the regulation of alpha-class GST in response to insulin in primary cultured rat hepatocytes. Protein levels of GSTA1/2 and GSTA3/5 and activity of GST toward 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were increased in an insulin concentration-dependent manner. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or rapamycin, an inhibitor of mammalian target of rapamycin and ribosomal p70 S6 kinase (p70S6K) phosphorylation, or with an adenovirus containing green fluorescent protein and a dominant-negative and kinase-dead Akt, effectively inhibited the insulin-mediated increase in alpha-class GST expression and GST activity toward NBD. In contrast, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of c-Jun N-terminal kinase, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imadazole], an inhibitor of p38 mitogen-activated protein kinase, or bisindolylmaleimide, a broad spectrum inhibitor of protein kinase C, did not inhibit the insulin-mediated increase in alpha-class GST protein levels in hepatocytes. These results show that PI3K/Akt/p70S6K signaling is active in the insulin-mediated up-regulation of the antioxidant defense system and that low insulin levels, as encountered in diabetes, potentially increase the susceptibility of hepatocytes to xenobiotic-mediated and/or oxidative stress-mediated damage.
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PMID:Identification of the insulin signaling cascade in the regulation of alpha-class glutathione S-transferase expression in primary cultured rat hepatocytes. 1629 13

Thrombopoietin (TPO) and its receptor (c-Mpl) are the major regulators of megakaryocyte and platelet production and serve a critical and non-redundant role in hematopoietic stem cell (HSC) biology. TPO signals through the Jak-STAT, Ras-Raf-MAPK, and PI3K pathways, and promotes survival, proliferation, and polyploidization in megakaryocytes. The proto-oncogene c-myc also plays an important role in many of these same processes. In this work we studied the regulated expression of c-myc in megakaryocytic cell lines and primary cells by quantitative real-time RT-PCR. We found that TPO induced expression of c-myc in 1 h in both hematopoietic cell lines (UT-7 and BaF3/Mpl) and mature murine megakaryocytes. The TPO-induced expression of c-myc was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting that TPO stimulated c-myc expression through a PI3K-dependent pathway. Of interest, our study showed that overexpression of active Akt did not rescue the effect of PI3K blockade on c-myc expression, rather, enhanced it. In addition, inhibitors of protein kinase C (PKC)zeta and the target of rapamycin (mTOR) also failed to affect c-myc mRNA expression, while c-myc mRNA expression was reduced by inhibition of the mitogen activated protein kinase (MAPK) pathway. Therefore, we conclude that TPO stimulates c-myc expression in primary megakaryocytes through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR.
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PMID:Thrombopoietin (TPO) induces c-myc expression through a PI3K- and MAPK-dependent pathway that is not mediated by Akt, PKCzeta or mTOR in TPO-dependent cell lines and primary megakaryocytes. 1638 Feb 30


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