Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction

J Cell Biochem. 2010 Apr 15;109(6):1172-84. doi: 10.1002/jcb.22496.

Abstract

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes.

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism*
  • Adaptor Proteins, Signal Transducing
  • Animals
  • Carrier Proteins / metabolism
  • Cell Line
  • Ethanol / pharmacology*
  • Gene Expression Regulation / drug effects*
  • Immunoblotting
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mechanistic Target of Rapamycin Complex 1
  • Mice
  • Multiprotein Complexes
  • Muscle Cells / drug effects
  • Muscle Cells / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • Protein Serine-Threonine Kinases / metabolism*
  • Proteins
  • RNA Interference
  • Regulatory-Associated Protein of mTOR
  • Signal Transduction* / drug effects
  • TOR Serine-Threonine Kinases
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • Multiprotein Complexes
  • Phosphoproteins
  • Proteins
  • Regulatory-Associated Protein of mTOR
  • Rptor protein, mouse
  • Transcription Factors
  • proline-rich Akt substrate, 40 kDa protein, mouse
  • Ethanol
  • mTOR protein, mouse
  • Mechanistic Target of Rapamycin Complex 1
  • Protein Serine-Threonine Kinases
  • TOR Serine-Threonine Kinases
  • AMP-Activated Protein Kinases