Figure 3. Loss of RAF dependency predicts enhanced ERK signaling output by MEK1 mutants.

A, NIH 3T3 cells stably expressing doxycycline-inducible WT or indicated mutant MEK1 were treated with doxycycline (300 ng/ml) for 24 hr. Expression and phosphorylation of the indicated proteins was assayed by western blot. B, ERK signaling ouptput was analyzed by real-time quantitative RT-PCR of nine validated ERK downstream target genes in NIH 3T3 cells inducibly expressing WT or mutant MEK1. The boxplot shows the log fold changes of gene expression (y axis) between 3 classes of MEK1 mutants (grouped in × axis and filled with three colors) and WT, which is overlaid with detailed changes of each gene plotted as colored points. Significant increasing changes were evaluated by T-tests among three groups. C, Co-mutation of MEK1 with RTK/RAS/RAF/NF1 in MEK1 mutant cancer patients. The data were collected from https://cbioportal.mskcc.org. Upper histogram showing RAF independent activity ratio (RAF independent in vitro kinase activity divided by kinase activity when RAF is added to the assay) RAF independent in vitro kinase activity was estimated by pERK in the absence of RAF, pERK in the presence of RAF was quantified as total MEK activity from Figure 2A. D, Weighted Pearson correlation between frequency of co-mutation and RAF independent activity ratio from c. The weights are based on the number of samples for each MEK1 mutation.