Figure 1.
Treatment with MEK inhibitor RG7167 or rapamycin activates AKT pathway in NIH3T3 vector control clones but not in BRAFV600E clones.
(A) Western blot analysis of AKT, MEK, and ERK phosphorylation in the isogenic pair of NIH3T3 clones 4 hours post treatment with MEK inhibitor RG7167 at indicated concentration. (B) Western blot analysis of AKT substrates (FOXO1, GSK3α/β, PRAS40) phosphorylation in the isogenic pair of NIH3T3 cells 4 hours post treatment with either MEK inhibitor RG7167 or rapamycin at indicated concentrations. (C) Western blot analysis of MEK and AKT phosphorylation in the isogenic pair of NIH3T3 cells 24 hours after CRAF or BRAF knock-down and 4 hours post RG7167 treatment. (D) Schematic presentation of a model of the cross-talk between MAPK and AKT pathways in engineered NIH3T3 clones. The left panel - MEK inhibitor induces pAKT by suppressing ERK-dependent negative feedback loop in control NIH3T3 cells. The right panel – although MEK inhibitor should induce pAKT through alleviating ERK-dependent feedback loop, the presence of BRAFV600E imparts a negative impact on pAKT induction.
Figure 2.
Melanoma cell lines with different genetic backgrounds respond differently to treatment-induced AKT phosphorylation.
Western blot analysis of the phosphorylation of AKT, ERK and S6 ribosome proteins in melanoma cell lines 4 hours after treatment with MEK inhibitor RG7167 or rapamycin.
Figure 3.
BRAFV600E is required and sufficient to suppress AKT phosphorylation in a subset of melanoma cells.
(A) Western blot analysis of AKT phosphorylation 24 hours after BRAF or CRAF knock-down in A375 melanoma cell line 4 hour post RG7167 treatment. (B) Western blot analysis of AKT substrates (FOXO1, GSK3α/β, PRAS40) phosphorylation 24 hours after BRAF knock-down in A375 melanoma cell line. (C) Western blot analysis of AKT phosphorylation 24 hours after BRAF knock-down in LOX melanoma cell line. (D) Western blot analysis of AKT, MEK, and ERK phosphorylation in CHL1 melanoma cell line 24 hours after transient transfection (wild-type BRAF or BRAFV600E) and treated with RG7167 for 4 hours.
Figure 4.
BRAFV600E suppresses AKT phosphorylation through rictor, but independent of MEK, ERK and its kinase activity.
(A) Western blot analysis of AKT phosphorylation and PTEN in A375 melanoma cell line 24 hours after knock-down of BRAF, MEK1/2, ERK1/2, rictor, raptor, or combined knock-down. (B) Western blot analysis of AKT and ERK phosphorylation in CHL1, A375 and LOX melanoma cell lines treated with RG7167 or vemurafenib for 4 hours.
Figure 5.
BRAFV600E interacts with rictor complex (mTORC2) and impairs the enzymatic activity.
(A) Western blot analysis of BRAF and rictor or raptor complex in respective rictor and raptor immunoprecipitation in A375. (B) Western blot analysis of in vitro phosphorylation of recombinant AKT by rictor complex purified from NIH3T3 isogenic pair or CHL1 and A375 melanoma cell lines. (C) Left panel: Western blot analysis of AKT phosphorylation in A375 melanoma cell line 24 hours after control or BRAF siRNA treatment. Right panel: Western blot analysis of in vitro phosphorylation of recombinant AKT by rictor complex purified from A375 melanoma cell line 24 hours after control or BRAF siRNA treatment.
Figure 6.
Schematic representations of the cross-talk between MAPK and AKT pathways in cells with different genetic backgrounds.
(A) In cells with “wild-type” AKT signaling, the presence of BRAFV600E exerts a negative impact through mTORC2 on AKT phosphorylation and pathway activation. Suppression of AKT activity leads to a de-repression of AKT substrates PRAS40, FOXO and GSK3β due to the reduction of AKT-mediated phosphorylation. (B) In cells where AKT signaling is aberrantly activated (by PTEN loss, PI3K activation, or RTK amplification), the negative impact of BRAFV600E is countered by a dominant input upstream of AKT. Consequently, activated AKT phosphorylates substrates PRAS40, FOXO and GSK3β and suppresses their activity.