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Activation of AMPK / TSC 2/ PLD by Alcohol Regulates m TORC 1 and m TORC 2 Assembly in C 2 C 12 Myocytes
Author(s) -
HongBrown Ly Q.,
Brown C. Randell,
Navaratnarajah Maithili,
Lang Charles H.
Publication year - 2013
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/acer.12174
Subject(s) - mtorc1 , mtorc2 , phosphorylation , ampk , p70 s6 kinase 1 , protein kinase b , pi3k/akt/mtor pathway , phosphatidic acid , phospholipase d , chemistry , wortmannin , nox4 , microbiology and biotechnology , signal transduction , protein kinase a , biology , biochemistry , nadph oxidase , reactive oxygen species , phospholipid , membrane
Background Ethanol ( E t OH ) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (m TORC )1 and increased m TORC 2 activities. In contrast, phospholipase D ( PLD ) and its metabolite phosphatidic acid ( PA ) positively regulate m TORC 1 signaling, whereas their role in m TORC 2 function is less well defined. Herein, we examine the role that PLD and PA play in E t OH ‐mediated m TOR signaling. Methods C2C12 myoblasts were incubated with E t OH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50‐minute incubation with PA in the presence or absence of E t OH . The phosphorylation state of various proteins was assessed by immunoblotting. Protein–protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit. Results PA levels and PLD activity increased in C2C12 myocytes exposed to E t OH (100 mM). Increased PLD activity was blocked by inhibitors of AMP ‐activated protein kinase ( AMPK ) (compound C) and phosphoinositide 3‐kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented E t OH ‐induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14‐3‐3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in m TORC 2 activity were not associated with altered binding of complex members and 14‐3‐3. PA increased S6K1 phosphorylation, with the associated increase in m TORC 1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with m TOR , as well as suppression of m TORC 2. Conclusions E t OH ‐induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. E t OH and PA regulate m TORC 1 via a PI3K/AMPK/TSC2/PLD signaling cascade. PA stimulates m TORC 1 function and suppresses activation of m TORC 2 as part of an m TORC 1/2 feedback loop.