title: Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling.
abstract: Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3, Canada.
authors.forename: Xuemin
authors.initials: X
authors.lastname: Wang
authors.forename: Bruno D
authors.initials: BD
authors.lastname: Fonseca
authors.forename: Hua
authors.initials: H
authors.lastname: Tang
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.forename: Androulla
authors.initials: A
authors.lastname: Elia
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Clemens
authors.forename: Ulrich-Axel
authors.initials: UA
authors.lastname: Bommer
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Proud
mesh_terms: D000596:Amino Acids / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007935:Leucine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011506:Proteins; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D000076205:Ras Homolog Enriched in Brain Protein; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D000090324:Tumor Protein, Translationally-Controlled 1; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D014408:Biomarkers, Tumor; C471205:FKBP8 protein, human; D007004:Hypoglycemic Agents; D007328:Insulin; D046912:Multiprotein Complexes; D009479:Neuropeptides; D011506:Proteins; C490211:RHEB protein, human; D000076205:Ras Homolog Enriched in Brain Protein; C000715647:TPT1 protein, human; D014157:Transcription Factors; D000077005:Tuberous Sclerosis Complex 2 Protein; D000090324:Tumor Protein, Translationally-Controlled 1; D025521:Tumor Suppressor Proteins; D012343:RNA, Transfer; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; C509186:TERT protein, human; D019098:Telomerase; D020559:Monomeric GTP-Binding Proteins; D022021:Tacrolimus Binding Proteins; D007935:Leucine-tRNA Ligase