title: The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses.
abstract: Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.
authors.affiliation: Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Ewan M
authors.initials: EM
authors.lastname: Smith
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Finn
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Tee
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Browne
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Proud
mesh_terms: D055372:AMP-Activated Protein Kinases; D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry; D018797:Cell Cycle Proteins; D002460:Cell Line; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D054736:Elongation Factor 2 Kinase; D039642:Eukaryotic Initiation Factors; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D005947:Glucose / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D000076205:Ras Homolog Enriched in Brain Protein; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D038601:Ribosomal Protein S6 / Q000378:metabolism; D015398:Signal Transduction; D013012:Sorbitol / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013997:Time Factors; D019076:Transgenes; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D009097:Multienzyme Complexes; D009479:Neuropeptides; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D000076205:Ras Homolog Enriched in Brain Protein; D012097:Repressor Proteins; C490213:Rheb protein, mouse; D038601:Ribosomal Protein S6; C000624653:TSC2 protein, human; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D006153:Guanosine Diphosphate; D013012:Sorbitol; D006147:Guanine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; C517689:EEF2K protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517690:Eef2k protein, mouse; D054736:Elongation Factor 2 Kinase; D055372:AMP-Activated Protein Kinases; D020559:Monomeric GTP-Binding Proteins; D005947:Glucose