Casein kinase 1 epsilon mutants act as loss of function in the Wnt/β-catenin pathway. Casein kinase 1 epsilon (CK1ε) mutants activate small GTPase Rac1 and the transcriptional activity of AP1 and NFAT. (a) Lysates from HEK293 cells transfected with Rac1-Myc were subjected to pull-down of the active GTP-Rac1 form with agarose-PAK beads. HEK293 cells were either co-transfected with wild-type (WT) or mutant CK1ε or treated with 100 μM D4476 inhibitor for 4 hours prior to lysis. The P3 mutant promotes Rac1 activation, as a higher amount of GTP-Rac1 was pulled from the lysate (as compared with WT CK1ε). Consistently, D4476 inhibits the function of WT CK1ε and elevates the level of GTP-Rac1 in cells. Rac1 protein was detected with an anti-Myc antibody. Western blots were quantified by densitometry, and the GTP-Rac1/Rac1 ratios are indicated by the numbers below the panel. After probing, membranes were stained with amidoblack to confirm equal protein loading. (b) HEK293 cells or (c) MCF7 cells were transfected with an AP1-luciferase reporter, Dvl2-Myc, and CK1ε as indicated. Cells were lysed, and luciferase activity was analyzed the next day. (d) HEK293 cells were transfected with a NFAT-luciferase reporter and CK1ε as indicated. Cells were lysed, and luciferase activity was analyzed the next day. (b) to (d) Data represent the mean ± standard deviation. *P < 0.05, **P < 0.01; one-way analysis of variance, Tukey post-test, n ≥ 3.