The mTOR kinase is a Trx1 substrate.
A, schematic of how the Trx1 substrate-trapping mutant (Trx1C35S) works. B, Trx1C35S traps mTOR in the heart. After 3 h of ischemia, FLAG-Trx1C35S was immunoprecipitated (IP) with anti-FLAG antibody. C, Trx1C35S traps mTOR in cultured cardiomyocytes. FLAG-Trx1C35S-HA was expressed via an adenovirus vector. Cardiomyocytes were incubated with 100 μm H2O2 for 30 min, and then FLAG-Trx1C35S-HA was immunoprecipitated with anti-FLAG antibody with or without 1 mm DTT. D, stable interaction between endogenous mTOR and Trx1 is not observed in cultured cardiomyocytes. Cardiomyocytes were treated with 100 μm H2O2 for 30 min, and endogenous mTOR was immunoprecipitated. E, mTOR intermolecular disulfide formation in primary cultured cardiomyocytes. Cardiomyocytes were incubated with 100 μm H2O2 for 30 min (left panel) or 500 μm H2O2 for 10 min (right panel). F, mTOR forms disulfide bonds with either mTOR itself or other proteins. FLAG-mTOR and Myc-mTOR were expressed in HEK293 cells. After H2O2 treatment (3 mm, 30 min), FLAG-mTOR was immunoprecipitated with anti-FLAG antibody. G, data interpretation of the results shown in F. E and F, Western blot analyses were performed after SDS-PAGE under non-reducing (without 2ME) conditions. Ox, oxidized form; Red, reduced form. Western blot results were verified by two (B–D and F) and three (E) additional independent experiments.