Figure 4

PTEN inhibits mTORC2 formation by hyperphosphorylation of Rictor at Threonine 1135 position. (a) PTEN^mu and PTEN^wt cells were lysed for co-immunoprecipitation (co-IP) study. Protein was incubated with anti-mTOR antibody overnight at 4^oC followed by incubation with protein A-Sepharose 4B, washed with ice-cold phosphate-buffered saline (PBS). The immunocomplex was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently identified using the respective antibodies. Co-IP revealed that Raptor is associated with mTOR in both the cells, whereas Rictor is associated with mTOR only in PTEN^mu cells. (b) PTEN^wt cells were transfected with siPTEN. After 24 h of transfection, cell lysates were subjected to co-IP analysis. Cells transfected with siPTEN showed increased association of Rictor with mTOR. (c) Untreated PTEN^wt and PTEN^mu cell lysates were subjected for western blot analysis. Expression of phosphorylated Rictor at Thr1135 was detected higher in PTEN^wt than PTEN^mu cells. (d, e) PTEN^wt cells were transfected with siPTEN. After 24 h of transfection cell lysates were subjected to western blot analysis. Status of Rictor phosphorylation at Thr1135 position was determined. Densitometric analysis showed that there was about 35% reduction of phosphorylation of Rictor (Thr1135) after PTEN knockdown from PTEN^wt cells. Each value is the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. (f) Other PTEN^mu (T98G, U373MG) and PTEN^wt (LN18) GBM cell lysates were prepared and subjected for western blot analysis. Status of Rictor Thr1135 phosphorylation was compared. (g, h) PTEN^wt cells (8 × 10⁵) were seeded and incubated for 24 h. Next, cells were transfected with wild-type pRK-5/Rictor (myc-tag) or the T1135A mutant of pRK-5/Rictor (myc-tag, 2 μg/well) or empty vector alone. After 36 h of transfection, cell lysates were prepared and subjected to co-IP (g) and western blot analysis (h). (g) Co-IP showed Rictor was associated with mTOR in cells transfected with pRK-5/Rictor T1135A. (h) Immunoblot analysis showed the status of myc-tagged Rictor, p-AKT Ser473 and p-GSK3β Ser9 in those transfected cells. (i) Schematic representation: PTEN-mediated regulation of mTORC2 formation and downstream signaling. (j, k) PTEN^wt cells were transfected with siRaptor or co-transfected with siRaptor and siPTEN consecutively. After 24 h of transfection, cells were lysed and subjected to western blot analysis. (j) Western blot showed the relative status of mTOR, p-mTOR, p-S6K1, Rictor, p-Rictor Thr1135, AKT, p-AKT Ser473, Raptor and PTEN. (k) Densitometry analysis showing the fold change of p-mTOR Ser2481 and p-Rictor Thr1135. Each value is the mean±s.d. of three independent experiments. *P<0.05, significant difference between two test groups. In all the western blot experiments, β-actin served as loading control.