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

Ly Q Hong-Brown1, C Randell Brown, Abid A Kazi

  • 1Department of Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA. lqh10@psu.edu

Journal of Cellular Biochemistry
|February 4, 2010
PubMed

Insights

Alcohol (EtOH) inhibits protein synthesis in myocytes by affecting the mTORC1 complex. This study reveals how EtOH alters mTORC1 component interactions and phosphorylation, mediated by AMPK, to reduce protein production.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • The mechanistic target of rapamycin complex 1 (mTORC1) is crucial for regulating protein synthesis.
  • Previous research indicated mTOR's role in alcohol's (EtOH) effects on protein synthesis.
  • The precise mechanisms by which EtOH influences mTORC1 activity remain unclear.

Purpose of the Study:

  • To investigate the impact of EtOH on the phosphorylation and interactions of mTORC1 components in C2C12 myocytes.
  • To elucidate the specific role of proline-rich Akt substrate 40 kDa (PRAS40) in EtOH-induced mTORC1 regulation.
  • To explore the involvement of AMP-activated protein kinase (AMPK) in these processes.

Main Methods:

  • C2C12 myocytes were treated with EtOH (100 mM, 24 h).
  • Phosphorylation levels and protein-protein interactions of mTORC1 components (mTOR, raptor, PRAS40, GbetaL) were analyzed.
  • Short hairpin RNA (shRNA) knockdown (KD) of PRAS40 was performed.
  • AMPK activity was assessed in vitro using raptor and TSC2 as substrates.

Main Results:

  • EtOH treatment increased raptor and PRAS40 phosphorylation, along with upstream regulators Akt and IRS-1.
  • EtOH enhanced the binding of raptor and PRAS40 to mTOR, and increased raptor's association with 14-3-3.
  • PRAS40 KD mimicked EtOH's inhibitory effect on protein synthesis, altering mTORC1 component interactions.
  • Both EtOH and PRAS40 KD increased AMPK activity towards raptor and TSC2.

Conclusions:

  • EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, potentially inhibiting mTOR kinase activity.
  • Increased interaction of raptor with 14-3-3 contributes to EtOH's inhibitory effects on protein synthesis.
  • These findings suggest a detailed mechanism for EtOH-induced suppression of protein synthesis in myocytes via mTORC1 and AMPK pathways.

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