Alcohol and PRAS40 Knockdown Decrease mTOR Activity and Protein Synthesis via AMPK Signaling and Changes in mTORC1 Interaction

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/G beta L and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (Et0H). However, the mechanisms by which Et0H regulates mTORC1 activity have not been established. Here, we investigated the effect of Et0H 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 Et0H (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. Et0H also caused changes in mTORC1 protein-protein interactions. Et0H 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 anti 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to Et0H. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased G beta L-mTOR binding. The effects of Et0H 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 Et0H stabilizes the association of raptor, PRAS40, and G beta L with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of Et0H on mTOR kinase activity and protein synthesis in myocytes. J. Cell. Biochem. 109: 1172-1184, 2010. (C) 2010 Wiley-Liss, Inc.