Figure 5.

Studies of mTORC1 and PI3K signaling in OoTsc1^−/− and OoTsc1^+/+ oocytes. (A) Comparison of Tsc1/mTORC1 signaling in OoTsc1^−/− and OoTsc1^+/+ oocytes. Oocytes were isolated from ovaries of mice at PD12–14 and western blot was performed as described in Materials and Methods. Loss of Tsc1 led to complete absence of Tsc2 protein in OoTsc1^−/− oocytes. mTORC1 signaling in OoTsc1^−/− oocytes was enhanced, as indicated by elevated levels of phosphorylated S6K1 (p-S6K1, T389) and phosphorylated rpS6 (p-rpS6, S235/6 and S240/4). Increased phosphorylation of 4E-BP1 (p-4E-BP1, S65) and eIF4B (p-eIF4B, S422), and elevated total protein levels of eIF4B were seen in OoTsc1^−/− oocytes. Levels of total S6K1, rpS6, 4E-BP1 and β-actin were used as internal controls. (B) Enhanced cap-dependent translation initiation in OoTsc1^−/− oocytes. Oocytes were isolated from ovaries of OoTsc1^−/− and OoTsc1^+/+ mice at PD12–14 and cap-binding assays were performed as described in Materials and Methods. The ability of rpS6 to bind to m7GTP cap beads was found to be elevated in OoTsc1^−/− oocytes compared with that in OoTsc1^+/+ oocytes. Equivalent amounts of protein between precipitates were monitored by the levels of eIF4E, which also binds to the m7GTP beads. Equal amounts of rpS6 in oocyte lysates were also shown. The experiments were repeated three times. For each experiment, material from 15–20 mice was used per lane. Representative images are shown. (C) Constitutively activated mTORC1 signaling, but unaltered PI3K–Akt signaling in OoTsc1^−/− oocytes. Phosphorylation of S6K1 (p-S6K1, T389) was constitutively higher in OoTsc1^−/− oocytes, which could not be elevated further by KL treatment. However, phosphorylation of Akt (p-Akt, S473) and S6K1 (p-S6K1, T229), which are activated by PI3K signaling, was similar in OoTsc1^−/− and OoTsc1^+/+ oocytes both at the basal level and upon stimulation by KL (100 ng/ml, 2 min). Levels of total S6K1, Akt, rpS6 and β-actin were used as internal controls.