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)

Thymidine phosphorylase (TP) has emerged as a promising target for antiangiogenesis treatment of cancer. Angiogenesis, the formation of blood vessels, is essential for tumors to grow in order to be supplied with nutrients and oxygen. The association of TP with angiogenesis was demonstrated in several clinical studies in various tissue types. It has been postulated that the angiogenic effect of TP is related to its enzymatic activity, which catalyzes the breakdown of thymidine to thymine and deoxyribose-1-phosphate (dR-1-P). The latter, in its parent form or in its sugar form, deoxyribose, may play a role in angiogenesis. It may interfere in cellular energy metabolism or be substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose and a specific TP inhibitor, TPI, can reverse these effects, supporting the role of the enzymatic reaction and that of the sugar. Although TP is usually high in the tumor, we also observed a high expression in tumor-associated stromal cells and macrophages. In order to elucidate the mechanism of TP induced angiogenesis we have investigated the association of TP with angiogenesis, the effect of thymidine and its metabolites on angiogenic parameters (e.g. invasion), the modulation by TPI, the formation and retention of the sugar metabolites of thymidine, and the potential signalling pathways involved in the angiogenic process. We used cell lines without/low TP expression (Colo320 and RT112) and TP transfected variants (Colo320TP1 and RT112/TP). Intrinsic TP expression in cancer cells did not stimulate these cells to invade more. On the other hand, Colo320 and Colo320TP1 cells could attract endothelial cells to a high extent, but Colo320TP1 did not attract them to a higher extent. RT112/TP cells attracted more endothelial cells than RT112 (2 fold). The difference between the RT112's and Colo320's may be related to different formation of sugars. Exposure of tumor cells to thymidine resulted in a rapid formation of dR-1-P, which was rapidly degraded to deoxyribose and further metabolized to other sugar derivatives. Of the possible sugars that can be produced by the conversion of TdR, dR-5-P seems to accumulate the most. dR accumulated 3 fold higher extent in RT112/TP than in Colo320/TP1 cells. dR could be converted to advanced glycation endproducts (AGE), however this was to a lower extent than ribose. Thymidine also induced several signalling pathways in the cells, involved in migration and invasion, such as the Focal adhesion kinase (FAK), which subsequently stimulated p70/S6 phosphorylation. The latter is a downstream kinase of rapamycin and its phosphorylation is inhibited by rapamycin, an mTOR inhibitor. The association between rapamycin and TP was shown by the protection by thymidine of rapamycin induced cytotoxicity, while TPI inhibited the effect of thymidine addition. These studies clearly show a mechanistic link between TP, signalling pathways, and cell migration.
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PMID:Thymidine phoshorylase as a target for antiangiogenesis treatment. 1877 37

The signaling pathways that are regulated by sphingosine-1-phosphate (S1P) and mammalian target of rapamycin (mTOR) modulate cell growth, mitogenesis and apoptosis in various cell types and are of major interest for the development of new cancer therapeutics. Previous reports show that S1P can cross-activate the mTOR pathway although the mechanisms that connect both pathways are still unknown. We found that S1P-treatment activates mTOR in several cancer cell lines and primary cells. The activation was independent of ERK, Akt and PI3-kinase, but instead was mediated by the E3 ubiquitin ligase Protein Associated with Myc (PAM). Increased intracellular PAM concentrations facilitated S1P- and insulin-induced mTOR activation as well as p70S6K and 4EBP1 phosphorylation while genetic deletion of PAM decreased S1P- and insulin-induced mTOR activation. PAM activated by facilitating the GDP/GTP-exchange of Rheb which is an activator of mTOR. In conclusion we show that PAM is a novel regulator of the mTOR pathway and that PAM may directly activate Rheb as a guanosine exchange factor (GEF).
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PMID:Sphingosine-1-phosphate induced mTOR-activation is mediated by the E3-ubiquitin ligase PAM. 1900 Jul 55

Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mammalian target of rapamycin suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization toward the vacuole of structures labeled by the phosphatidylinositol 3-phosphate indicator YFP-2xFYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels, parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.
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PMID:Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth. 1902 80

Safingol, the synthetic L-threo-stereoisomer of endogenous (D-erythro-) sphinganine, is an inhibitor of protein kinase C and sphingosine kinase in vitro, and in some cell types has been implicated in ceramide generation and induction of apoptosis. Utilizing electron microscopy, acridine orange staining, and immunoblot and fluorescent localization studies of the myosin light chain-associated protein (LC3), we determined that safingol induces cell death of an exclusively autophagic character and lacking any of the hallmarks of apoptosis. Safingol inhibited PKCbeta-I, PKC delta and PKC epsilon, and inhibited phosphorylation of critical components of the PI3k/Akt/mTOR pathway (Akt, p70S6k and rS6) and the MAPk pathway (ERK). Inhibition of PI3k with LY294002 or suppression of PKC delta and PKC epsilon with siRNA in HCT-116 cells induced autophagy, though not to the extent caused by safingol. Conversely, activation of PKCs with phorbol 12,13-dibutyrate (PDBu) or transient transfection of a constitutively active form of Akt each reduced safingol's autophagic induction, but not completely, indicating that Akt- and PKC-dependent pathways both contribute partially and independently to safingol-induced autophagy. Accordingly, combining siRNA depletion of PKC epsilon with LY294002 inhibition of PI3k induced autophagy to a degree comparable to safingol. Liquid chromatography, electrospray tandem mass spectrometry analysis indicated that safingol did not elevate levels of any endogenous sphingolipids previously shown to induce autophagy (ceramide, sphingosine-1-phosphate and dihydroceramide); therefore, these effects may be due to safingol per se or another metabolite. Thus, our studies establish that safingol induces autophagy through inhibition of PKCs and PI3k by safingol directly rather than via changes in endogenous sphingolipids.
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PMID:Safingol (L-threo-sphinganine) induces autophagy in solid tumor cells through inhibition of PKC and the PI3-kinase pathway. 1909 47

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR's access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.
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PMID:Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. 1945 Dec 32

Regulatory T cells (T(reg) cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that sphingosine 1-phosphate (S1P) receptor type 1 (S1P1) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T(reg) cells. Combining loss- and gain-of-function genetic approaches, we found that S1P1 blocked the differentiation of thymic T(reg) precursors and function of mature T(reg) cells and affected T(reg) cell-mediated immune tolerance. S1P1 induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T(reg) cells. Dynamic regulation of S1P1 contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T(reg) cell-mediated immune suppression, the lipid-activated S1P1-Akt-mTOR pathway orchestrates adaptive immune responses.
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PMID:The receptor S1P1 overrides regulatory T cell-mediated immune suppression through Akt-mTOR. 1948 17

We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).
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PMID:Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose. 1965 3

The p53-inducible TIGAR protein functions as a fructose-2,6-bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy--a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.
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PMID:Modulation of intracellular ROS levels by TIGAR controls autophagy. 1971 38

PiT1 is a Na(+)-phosphate (P(i)) cotransporter located at the plasma membrane that enables P(i) entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely related family member PiT2, PiT1 is the ubiquitous supplier of P(i) to the cell. Moreover, the role of P(i) in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that P(i) availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either P(i) or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na(+)-P(i) transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a P(i) transporter in cell proliferation, tumor growth, and cell signaling.
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PMID:Identification of a novel function of PiT1 critical for cell proliferation and independent of its phosphate transport activity. 1972 92

We provide a standard phosphate-affinity SDS-PAGE (Mn(2+)-Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins ( approximately 150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn(2+)-Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol.
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PMID:Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. 1979 84


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