title: Alcohol-induced modulation of rictor and mTORC2 activity in C2C12 myoblasts.
abstract: BACKGROUND
  The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1.
  
  
  METHODS
  C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by (35) S-methionine/cysteine incorporation into cellular protein.
  
  
  RESULTS
  EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts.
  
  
  CONCLUSIONS
  EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.
authors: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA. lqh10@psu.edu
authors: Ly Q
authors: LQ
authors: Hong-Brown
authors: C Randell
authors: CR
authors: Brown
authors: Maithili
authors: M
authors: Navaratnarajah
authors: Danuta S
authors: DS
authors: Huber
authors: Charles H
authors: CH
authors: Lang
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects*; D002492:Central Nervous System Depressants / Q000378:metabolism / Q000633:toxicity*; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity*; D047468:Immunoprecipitation; D051379:Mice; D032446:Myoblasts / Q000187:drug effects* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects / Q000502:physiology; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D015534:Trans-Activators / Q000187:drug effects / Q000502:physiology*; D014157:Transcription Factors; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D002492:Central Nervous System Depressants; C504923:Crtc2 protein, mouse; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D015534:Trans-Activators; D014157:Transcription Factors; C525637:rictor protein, mouse; D000431:Ethanol; D051057:Proto-Oncogene Proteins c-akt