Fig. 2.
Activation of mTORC1 leads to defective AKT phosphorylation in MYC-induced lymphomagenesis. (A) Tumors of the indicated genotype were cultured ex vivo and stimulated with 100 nM insulin, 10 μg/ml LPS, or 10 μg/ml IgM. Extracts were prepared and probed for AKT (total and p-S473) and ACTIN. (B) The distribution of mTOR among mTORC1 and mTORC2 is altered in Tsc2LOHEμ-myc tumors. Tsc2+/+Eμ-myc and Tsc2LOHEμ-myc tumors were analyzed for total levels of mTOR, RICTOR, and RAPTOR. MTOR-containing complexes were immunoprecipitated from Eμ-myc tumors of the indicated genotypes, and RICTOR and RAPTOR levels were assessed by Western blotting. (Top) Total levels of mTOR, RICTOR, and RAPTOR in whole-cell extracts. (Bottom) Amounts of RICTOR and RAPTOR coprecipitating (Co-IP) with mTOR. The quantitation of mTORC2:mTORC1 ratios in three tumors of the indicated genotype is shown and represents the average of three experiments ±SD. (C) Tsc2+/+Eμ-myc and Tsc2LOHEμ-myc tumors were analyzed for levels of RICTOR-associated mTOR. (D) Amounts of RICTOR and RAPTOR associating with mTOR in Eμ-myc, Tsc2LOH, myr-Akt, and Pten+/−Eμ-myc tumors. (E) AKT (S473) phosphorylation is decreased in Tsc2LOHEμ-myc tumors. Extracts prepared from MSCV/myr-Akt-infected Tsc2+/+Eμ-myc or Tsc2LOHEμ-myc tumors were analyzed by Western blotting for AKT (total and p-S473), S6 (total and p-S235/236), and tubulin. AKT and myr-AKT are denoted by an arrow and arrowhead, respectively.