Characterization of the rictor Thr-1135 phosphorylation. A. The functional study of the rictor T1135 phosphorylation site. Reconstitution of the mTORC2 signaling in rictor null MEFs by expressing the wild type and phospho-rictor mutants (T1135A and T1135D). Following 48 hrs after transfection cells were lysed and cell extracts analyzed by immunoblotting for the indicated proteins and phosphorylation state of Akt on the Ser-473 site. B. The rapamycin-sensitive rictor phosphorylation does not require mTORC2. The wild type and Sin1 KO MEFs were serum starved overnight. As indicated the cells were stimulated by IGFI (50 ng/ml) for 20 min or treated with rapamycin (50 ng/ml) for 15 min prior stimulation of cells with the growth factor. The cells were lysed and analyzed as described in panel A.

Rictor phosphorylation associates with the IGF-I dependent stimulation of the mTORC2 kinase activity. A. The IGFI-dependent rictor phosphorylation. The serum starved MDA-MB-231, MDA-MB-435 and MCF7 cells for 24 hrs were stimulated by IGF-I (40 ng/ml) for 20 min. The lysates were analyzed by immunoblotting for indicated proteins and phosphorylation of Akt. B. The mTORC2 kinase assay. The rictor immunoprecipitates from the serum starved or IGF-I stimulated MDA-MB-435 cells were used in a kinase assay with a full-length wild type Akt1 as a substrate. Immunoblotting was used to detect the phosphorylation of Akt on Ser-473 and the levels of mTOR, Rictor, and Akt1 in the kinase assay. In a first lane of the Akt immunoblots, Akt1 was used in a kinase assay to detect a basal phosphorylation of the substrate. C. Detection of the IGF-I dependent phosphorylation site of rictor. The serum starved MDA-MB-435 cells with or without the IGF-I stimulation were lysed and analyzed by immunoblotting for the indicated proteins and phosphorylation states by the phospo-specific rictor and Akt antibodies (left panel). In parallel, the mTOR associated rictor phosphorylation was analyzed in the mTOR immunoprecipitates as shown in a right panel.

Regulation of the rictor T1135 phosphorylation. A. The phosphorylation of rictor on the Thr-1135 site is mTOR dependant. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and mTOR. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. The cells were lysed and the equal amount of protein lysates were analyzed by western blotting by the indicated antibodies. B. The ILK knockdown inhibits the phosphorylation of rictor on the Thr-1135 site. MDA-MB-435 cells were infected with lentiviruses expressing shRNA targeting luciferase (control) and ILK. Following a serum starvation for 24 hrs and stimulation by IGFI (40 ng/ml) for 30 min. C. The growth factor-dependent phosphorylation of rictor is prevented by inhibition of PI3K. The MDA-MB-435, A549 and Hela cells were serum starved for 24h. Where indicated, cells were pre-incubated or not with the PI3-kinase inhibitor LY294002 at 50 mM concetration for 1 hour and stimulated with IGF-I (40 ng/ml) for 30 min. D. The rictor phosphorylation is rapamycin dependent. The MDA-MB-435, A549 and Hela cell lines were grown in the growth medium containing 10% serum with or without rapamycin at 100 nM concentration for the indicated periods of time.

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.