Enhanced Akt-rpS6 activation and <i>in vitro</i> inhibition of rpS6 activation in Oo<i>Pten</i>^−/− oocytes by rapamycin.

Abstract

<p>(<b>A</b>) Comparison of Akt-rpS6 signaling in Oo<i>Pten</i>^−/− and Oo<i>Pten</i>^+/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in <i>Materials and Methods</i>. Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo<i>Pten</i>^−/− oocytes compared with Oo<i>Pten</i>^+/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. (<b>B</b>) Activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of Oo<i>Pten</i>^−/− mice at PD 12–14 as described in <i>Materials and Methods</i>. Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo<i>Pten</i>^−/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.</p