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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
Summary
This summary is machine-generated.

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.

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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.