FIG. 3.
TSC inhibits and Rheb promotes mTOR P-S1261. (A) TSC1−/− MEFs exhibit high levels of mTORC1-associated mTOR P-S1261 compared to WT MEFs. Littermate-matched, 3T3 immortalized mouse embryonic fibroblasts derived from TSC1+/+ or TSC−/− animals were serum deprived (24 h) and lysed in CHAPS buffer. Triplicate lysates were immunoprecipitated with raptor antibodies and immunoblotted as indicated. Note: as we found that TSC1−/− fibroblasts express higher levels of total raptor and mTOR protein, two-thirds of the immunoprecipitate from TSC1−/− cells was loaded relative to that for TSC1+/+ cells in order to normalize the amounts of raptor and mTOR between the two cell lines. WCL were loaded similarly. (B) Amino acids are required to maintain mTORC1-associated mTOR P-S1261 in TSC1−/− MEFs. Littermate-matched, TSC1+/+, and TSC1−/− MEFs were cultured under steady-state conditions. TSC1−/− MEFs were also amino acid deprived by incubation in D-PBS/Glc for 60 min. Raptor was immunoprecipitated, and the level of P-S1261 on raptor-associated mTOR was assessed by immunoblotting (IB). WCL were immunoblotted as indicated. (C) Knockdown of Rheb in TSC1−/− MEFs reduces mTORC1-associated mTOR P-S1261. Parental TSC1−/− MEFs (Par) were infected with a control scrambled shRNA lentivirus (Scr) or with shRNA lentiviruses targeting two distinct regions of Rheb (Rheb-1 and Rheb-3). Infected cells were selected in puromycin and serum deprived. Raptor was immunoprecipitated, and the level of P-S1261 on raptor-associated mTOR was assessed by immunoblotting. WCL were immunoblotted to confirm Rheb knockdown and the expected decrease in mTORC1 signaling as well as to observe the level of P-S1261 on total mTOR.