Fig. 7. Rac1 associates with and promotes plasma membrane localization of mTOR and anchorage-independent growth of MET-mutant cells through its C-terminal RKR motif.

(A) Wild-type and M1268T MET-expressing cells were transiently transfected with GFP-Rac1-T17N or GFP-Rac1-T17N-AAA constructs. GFP-Rac1 was immunoprecipipated (IP) with GFP-Trap beads (GFP). Pull-down with control beads (Ctl) was performed. Western blots were performed to detect the GFP immunoprecipitates (IP) and the co-immunoprecipitated (co-IP) proteins mTOR and Tiam1. Lysates for each condition were also blotted and are indicated with “+”. Numbers below were obtained following densitometry of the blots. Thy represent the level of mTOR co-immunoprecipitated with GFP in M1268T MET-expressing cells. Thus, for each construct, mTOR level was normalised to GFP level. The obtained value was normalised to mTOR levels in lysate. The value was set as 1 for GFP-Rac1-T17N construct. Data are mean +/- SEM, N=3 independent biological replicates. (B and C) M1268T MET-expressing cells were transiently transfected with GFP-Rac1-T17N (B) or GFP-Rac1-T17N-AAA (B and C) constructs. After flow cytometry separation, the cells expressing GFP or the GFP negative cells (No GFP) were (B) immunostained for mTOR and the percentage of cells with mTOR at the plasma membrane was evaluated, and (C) grown in soft agar and treated with DMSO or PHA-665752 (PHA, 100 nM). Data are mean +/- SEM, N=3 independent biological replicates. P values were obtained with the Student’s t test. NS: non-significant, **P<0.01, ***P<0.005. (D) Model suggested for the role of Rac1 downstream of oncogenic MET to induce cell migration, and anchorage-independent growth.