PMID- 15772076 OWN - NLM STAT- MEDLINE DCOM- 20050712 LR - 20211203 IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 280 IP - 19 DP - 2005 May 13 TI - The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. PG - 18717-27 AB - 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. FAU - Smith, Ewan M AU - Smith EM AD - Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom. FAU - Finn, Stephen G AU - Finn SG FAU - Tee, Andrew R AU - Tee AR FAU - Browne, Gareth J AU - Browne GJ FAU - Proud, Christopher G AU - Proud CG LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20050316 PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Adaptor Proteins, Signal Transducing) RN - 0 (Amino Acids) RN - 0 (Carrier Proteins) RN - 0 (Cell Cycle Proteins) RN - 0 (EIF4EBP1 protein, human) RN - 0 (Eif4ebp1 protein, mouse) RN - 0 (Eukaryotic Initiation Factors) RN - 0 (Multienzyme Complexes) RN - 0 (Neuropeptides) RN - 0 (Phosphoproteins) RN - 0 (Protein Synthesis Inhibitors) RN - 0 (Ras Homolog Enriched in Brain Protein) RN - 0 (Repressor Proteins) RN - 0 (Rheb protein, mouse) RN - 0 (Ribosomal Protein S6) RN - 0 (TSC2 protein, human) RN - 0 (Tsc2 protein, mouse) RN - 0 (Tuberous Sclerosis Complex 2 Protein) RN - 0 (Tumor Suppressor Proteins) RN - 146-91-8 (Guanosine Diphosphate) RN - 506T60A25R (Sorbitol) RN - 5Z93L87A1R (Guanine) RN - 86-01-1 (Guanosine Triphosphate) RN - 8L70Q75FXE (Adenosine Triphosphate) RN - 98600C0908 (Cycloheximide) RN - BBX060AN9V (Hydrogen Peroxide) RN - EC 2.7.- (Protein Kinases) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.1.1 (mTOR protein, mouse) RN - EC 2.7.1.17 (EEF2K protein, human) RN - EC 2.7.11.1 (Protein Serine-Threonine Kinases) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 2.7.11.17 (Calcium-Calmodulin-Dependent Protein Kinases) RN - EC 2.7.11.20 (Eef2k protein, mouse) RN - EC 2.7.11.20 (Elongation Factor 2 Kinase) RN - EC 2.7.11.31 (AMP-Activated Protein Kinases) RN - EC 3.6.5.2 (Monomeric GTP-Binding Proteins) RN - IY9XDZ35W2 (Glucose) SB - IM MH - AMP-Activated Protein Kinases MH - Adaptor Proteins, Signal Transducing MH - Adenosine Triphosphate/chemistry MH - Amino Acids/chemistry/*metabolism MH - Animals MH - Calcium-Calmodulin-Dependent Protein Kinases/metabolism MH - Carrier Proteins/chemistry MH - Cell Cycle Proteins MH - Cell Line MH - Cells, Cultured MH - Cycloheximide/pharmacology MH - Dose-Response Relationship, Drug MH - Elongation Factor 2 Kinase MH - Eukaryotic Initiation Factors MH - Fibroblasts/metabolism MH - Gene Expression Regulation MH - Glucose/chemistry MH - Guanine/chemistry MH - Guanosine Diphosphate/chemistry MH - Guanosine Triphosphate/chemistry MH - Humans MH - Hydrogen Peroxide/pharmacology MH - Hydrolysis MH - Immunoblotting MH - Immunoprecipitation MH - Mice MH - Models, Biological MH - Monomeric GTP-Binding Proteins/chemistry/metabolism/*physiology MH - Multienzyme Complexes/metabolism MH - Mutation MH - Neuropeptides/chemistry/metabolism/*physiology MH - Phosphoproteins/chemistry MH - Phosphorylation MH - Protein Kinases/*metabolism MH - Protein Serine-Threonine Kinases/metabolism MH - Protein Structure, Tertiary MH - Protein Synthesis Inhibitors/pharmacology MH - Ras Homolog Enriched in Brain Protein MH - Repressor Proteins/metabolism/*physiology MH - Ribosomal Protein S6/metabolism MH - Signal Transduction MH - Sorbitol/pharmacology MH - TOR Serine-Threonine Kinases MH - Time Factors MH - Transgenes MH - Tuberous Sclerosis Complex 2 Protein MH - Tumor Suppressor Proteins/metabolism/*physiology EDAT- 2005/03/18 09:00 MHDA- 2005/07/13 09:00 CRDT- 2005/03/18 09:00 PHST- 2005/03/18 09:00 [pubmed] PHST- 2005/07/13 09:00 [medline] PHST- 2005/03/18 09:00 [entrez] AID - S0021-9258(20)67516-0 [pii] AID - 10.1074/jbc.M414499200 [doi] PST - ppublish SO - J Biol Chem. 2005 May 13;280(19):18717-27. doi: 10.1074/jbc.M414499200. Epub 2005 Mar 16.