Figure 5. PI3K regulates the level of active RAS in RAS wild type cells.

(A) SK-BR-3 cells (left) or BT-474 cells (right) were treated with BAY 80-6946 (50nM) and collected at indicated times. RAS-GTP level was assessed by RAF1-RBD pull down (PD) and lysates from the same samples were immunoblotted. A control for the PD (Neg. Ctrl.) was performed in the same manner as the other samples but without the addition of RAF1-RBD. Immunoblots demonstrate that PI3K inhibition blocks AKT phosphorylation and causes a loss of RAS-GTP level (total and individual isoforms) corresponding with changes in RAF-MEK-ERK activity.

(B) SK-BR-3 cells (left) or BT-474 cells (right) were treated with BAY 80-6946 (50nM) or MK2206 (2μM) for 30 minutes, EGF (100ng/mL) for 5 minutes, Lapatinib (2μM) for 1h, or left untreated, and collected for RAS-GTP analysis (as in (A)) or immunoblotting. Treatment with EGF activates RAS and RAF/MEK/ERK, while RAS-ERK signaling is inhibited by RTK or PI3K inhibition but not AKT inhibition.

(C) SK-BR-3 cells stably transfected with TTIGp-MLUEX-NRAS Q61K were pretreated for 24h with doxycycline (1μg/mL) to induce the expression of exogenous NRAS Q61K (right) or not pretreated (left) before the addition of the PI3K inhibitor BAY 80-6946 (50nM) for indicated times, EGF (100ng/mL) for 5 minutes, or Lapatinib (2μM) for 1h. RAS-GTP analysis was performed as in (A), and lysates were immunoblotted. PI3K inhibition decreases the GTP-bound level of the endogenous wild type, but not the exogenous mutant, RAS protein. Accordingly, P-ERK was only decreased in cells without the induction of NRAS Q61K expression.

N.S. denotes non-specific band.