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.