Thioredoxin inhibits MPK38-induced ASK1, TGF-β, and p53 function in a phosphorylation-dependent manner

Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family. The factors that regulate MPK38 activity and function are not yet elucidated. Here, thioredoxin (Trx) was shown to be a negative regulator of MPK38. The redox-dependent association of MPK38 and Trx was mediated through the C-terminal domain of MPK38. Single. and double amino acid substitution mutagenesis of MPK38 (C286S, C339S, C377S, and C339S/C3775) and Trx (C32S, C35S, and C32S/C35S) demonstrated that Cys(339) and Cys(377) of MPK38 and Cys(32) and Cys(35) of Trx are required for MPK38-Trx complex formation. MPK38 directly interacted with and phosphorylated Trx at Thr(76). Expression of wild-type Trx, but not the Trx mutants C32S/C35S and T76A, inhibited MPK38-induced ASK1, TGF-beta, and p53 function by destabilizing MPK38. The E3 ubiquitin-protein ligase Mdm2 played a critical role in the regulation of MPK38 stability by Trx. Treatment of cells with 1-chloro-2,4-dinitrobenzene, a specific inhibitor of Trx reductase, decreased MPK38-Trx complex formation and subsequently increased MPK38 stability and activity, indicating that Trx negatively regulates MPK38 activity in vivo. Finally, we used ASK1-, Smad3-, and p53-null mouse embryonic fibroblasts to demonstrate that ASK1, Smad3, and p53 play important roles in the activity and function of MPK38, suggesting a functional link between MPK38 and ASK1, TGF-beta, and p53 signaling pathways. These results indicate that Trx functions as a physiological inhibitor of MPK38, which plays an important role in inducing ASK1-, TGF-beta, and p53-mediated activity. (C) 2013 Published by Elsevier Inc.