title: Activation of AMPK/TSC2/PLD by alcohol regulates mTORC1 and mTORC2 assembly in C2C12 myocytes.
abstract: BACKGROUND
  Ethanol (EtOH) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (mTORC)1 and increased mTORC2 activities. In contrast, phospholipase D (PLD) and its metabolite phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less well defined. Herein, we examine the role that PLD and PA play in EtOH-mediated mTOR signaling.
  
  
  METHODS
  C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50-minute incubation with PA in the presence or absence of EtOH. The phosphorylation state of various proteins was assessed by immunoblotting. Protein-protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit.
  
  
  RESULTS
  PA levels and PLD activity increased in C2C12 myocytes exposed to EtOH (100 mM). Increased PLD activity was blocked by inhibitors of AMP-activated protein kinase (AMPK) (compound C) and phosphoinositide 3-kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented EtOH-induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14-3-3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in mTORC2 activity were not associated with altered binding of complex members and 14-3-3. PA increased S6K1 phosphorylation, with the associated increase in mTORC1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with mTOR, as well as suppression of mTORC2.
  
  
  CONCLUSIONS
  EtOH-induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. EtOH and PA regulate mTORC1 via a PI3K/AMPK/TSC2/PLD signaling cascade. PA stimulates mTORC1 function and suppresses activation of mTORC2 as part of an mTORC1/2 feedback loop.
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.
authors.forename: Ly Q
authors.initials: LQ
authors.lastname: Hong-Brown
authors.forename: C Randell
authors.initials: CR
authors.lastname: Brown
authors.forename: Maithili
authors.initials: M
authors.lastname: Navaratnarajah
authors.forename: Charles H
authors.initials: CH
authors.lastname: Lang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002492:Central Nervous System Depressants / Q000378:metabolism / Q000494:pharmacology*; D000431:Ethanol / Q000378:metabolism / Q000494:pharmacology*; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D032342:Muscle Cells; D010712:Phosphatidic Acids / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D002492:Central Nervous System Depressants; D046912:Multiprotein Complexes; D010712:Phosphatidic Acids; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D000431:Ethanol; C546843:mTOR protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D010739:Phospholipase D
keywords: 14-3-3 Proteins; Ethanol; Phosphatidic Acid; Protein Synthesis; Rag GTPases
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