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. Author manuscript; available in PMC: 2012 Jan 1.

Published in final edited form as: Mol Cancer Res. 2010 May 25;8(6):896–906. doi: 10.1158/1541-7786.MCR-09-0409

The mTORC2 signaling up-regulation by the ras or PI3K oncogenes is associated with the rictor T1135 phosphorylation. A loss of the growth factor-dependent rictor phosphorylation caused by the oncogenic form of H-ras. The oncogenic form of H-ras and empty vector as a control have been constitutively expressed in the non-tumorigenic MCF10A cells (A) or in A549, Hela, and MDA-MB-435 cells (B) based on the retroviral expression system. The transduced cells were selected and following the indicated treatments were lysed and analyzed by immunoblotting for the indicated proteins and phosphorylation states by the phospo-specific rictor, mTOR and Akt antibodies. As indicated, cells were growing in 10% serum with or without Wortmaninn at 100 nM concentration for 1 hour. In parallel, cells were serum starved for 24 hr or stimulated by IGFI (40 ng/ml) for 30 min. C. Expression of the oncogenic PI3K is sufficient in stimulation of the rictor Thr-1135 phosphorylation. The constitutively active form of the PI3K catalytic unit p110 (H1047R) has been constitutively expressed in A549, Hela and MDA-MB-435 cells based on the retroviral pBabe expression vector. The pBabe empty vector was used as control. The transduced and selected cells were starved for 24h and stimulated or not with IGF for 30 min at the concentration of 40 ng/ml.

The mTORC2 signaling up-regulation by the ras or PI3K oncogenes is associated with the rictor T1135 phosphorylation. A loss of the growth factor-dependent rictor phosphorylation caused by the oncogenic form of H-ras. The oncogenic form of H-ras and empty vector as a control have been constitutively expressed in the non-tumorigenic MCF10A cells (A) or in A549, Hela, and MDA-MB-435 cells (B) based on the retroviral expression system. The transduced cells were selected and following the indicated treatments were lysed and analyzed by immunoblotting for the indicated proteins and phosphorylation states by the phospo-specific rictor, mTOR and Akt antibodies. As indicated, cells were growing in 10% serum with or without Wortmaninn at 100 nM concentration for 1 hour. In parallel, cells were serum starved for 24 hr or stimulated by IGFI (40 ng/ml) for 30 min. C. Expression of the oncogenic PI3K is sufficient in stimulation of the rictor Thr-1135 phosphorylation. The constitutively active form of the PI3K catalytic unit p110 (H1047R) has been constitutively expressed in A549, Hela and MDA-MB-435 cells based on the retroviral pBabe expression vector. The pBabe empty vector was used as control. The transduced and selected cells were starved for 24h and stimulated or not with IGF for 30 min at the concentration of 40 ng/ml.