DETERMINING THE ROLE OF RAP1 IN REGULATING MTORC2 ACTIVITY AND FUNCTION IN CELL MIGRATION

Abstract

Chemotaxis, the directed migration of cells in response to chemical signals, plays a crucial role in physiological processes (McNiven, 2016). Cancer metastasis is due to mis regulation, although the molecular mechanisms are not entirely known. Specifically, the mechanistic Target of Rapamycin Complex 2 (mTORC2) is explored in this study, including its effect on signaling pathways and opportunities for deregulation. Previous studies on Dictyostelium have revealed Rictor and SIN1, mTORC2 components that are unique to this complex. Our hypothesis focuses on Rap1, a small GTPase, and its effect on mTORC2, including membrane localization and downstream AKT activation. Previous investigations indicate that Rap1 positively regulates mTORC2 activation, potentially through an interaction with SIN, which localizes to the plasma membrane. We hypothesize that SIN1 binding to Rap1 and both colocalizing to the plasma membrane will target and stabilize mTORC2 to the plasma membrane, where it can function to activate AKT. This paper sheds light on the involvement of mTORC2 in cell migration, given its downstream regulation of F-actin, suggesting its impact on cell motility and cancer progression. Through maintaining HEK-293 cells, and stimulation followed by immunofluorescence, we investigated the localization of these proteins. Our results show that Rap1 and Rictor colocalize at the plasma membrane when Rap1B is overexpressed or constitutively active, suggesting recruitment of mTORC and subsequent activation. Further, SIN1 and FLAG-Rap1B colocalize under conditions of Rap1B overexpression or constitutive activation, confirmed across various stimulation conditions (IGF-1 and EGF).