Figure 6.

Effect of Pairwise Overexpression of a Kinase and a Phosphatase on Signaling

(A) Workflow of the pairwise overexpression. Two plasmids encoding an FLAG-tagged kinase and a GFP-tagged phosphatase were transfected into HEK293T cells successively. Cells were binned into 25 groups according to their FLAG and GFP abundances. The median level of each measured phosphorylation site was computed for each bin.

(B), Kinases MAP2K2, MAPK1, and RPS6KA1 and phosphatases DUSP4, DUSP7, and PTPN2 were selected for the pairwise overexpression, generating nine overexpression combinations in total.

(C) In cells with overexpression of MAP2K2 and DUSP4, median phosphorylation levels of p-MEK1/2 and p-ERK1/2 are plotted for all of the bins over the 1-h EGF stimulation time course.

(D) Signaling trajectories of p-MEK1/2 and p-ERK1/2 plotted as the medians of each individual bin over the 1-h EGF stimulation time course.

(E) Schematic illustrating the modulation of RAF-MEK-ERK cascade signaling states and dynamics upon pairwise overexpression.

(F and G) Analysis of p-ERK1/2 and p-MEK1/2 phosphorylation levels (F) and signaling trajectories (G) on MAPK1-FLAG and DUSP7-GFP abundances.