The Mechanism Of Rb Inhibits MTORC2 Function

Objective: To study whether tumor suppressor Rb inhibits mTORC2 function and its underlying mechanism(s).Methods: Western blot was used to investigate the mTOR signaling in established Rb-/-MEFs, several Rb shRNA stable cancer cell lines and tet-on cancer cell line.Co-immunoprecipitate, GST pull-down and gel filtration chromatography were used to further confirm that Sinl is the only protein in mTOR complex that Rb interacts with.Through truncation of Sin into 4 fragments, we further investigated the fragment of Sinl that associates with Rb by Co-IP and GST pull-down.Results: Compared with Rbloxp/loxp MEFs, Rb-/- MEFs show increased mTORC2-Akt activity,while the mTORC1 activity, which is designated by the phosphorylation of S6K Thr389 remains unaffected. This phenotype has been confirmed by HeLa, OVCAR5 and MDA-MB-231Rb shRNA stable cell lines. The tet-on OVCAR5 stable cell line exhibits with attenuated Akt-pS473 after induction of Rb overexpression by doxycycline. Overexpression,semi-endogeous and endogeous IP indicates Sinl is the only component in mTOR complex that Rb interacts with. Co-IP and GST pull down indicates the PH domain of Sinl associates with Rb.Conclusion: Through interacts with Sin1-PH domain, Rb directly inhibits mTORC2 activity,but not mTORC1 activity.