Figure 4
Casein kinase 1 epsilon mutants downregulate Wnt/β-catenin signaling at the level of the Dishevelled protein. HEK293 or MCF7 cells were transfected with the indicated plasmids, and after 24 hours TCF/LEF-driven transcription was measured using the SuperTopFlash dual luciferase reporter assay. (a) Dvl2 enhances TCF/LEF-dependent transcription when co-transfected with wild-type (WT) casein kinase 1 epsilon (CK1ε) in HEK293 cells. In contrast, all CK1ε mutants are unable to promote Dvl-mediated TopFlash. (b) Similar results to those obtained for HEK293 cells have been obtained for the breast cancer cell line MCF7. (c) β-Catenin induces the TCF/LEF transcriptional response in the cell by recruiting transcriptional cofactors directly to the TCF/LEF complex. Neither WT CK1ε nor the CK1ε mutants could influence the high TopFlash signal caused by constitutively active β-catenin, confirming that CK1ε and its mutants operate upstream of β-catenin (HEK293 cells). (d) The Wnt signaling pathway was induced by seeding B1A-wnt3a cells (fibroblasts that constitutively produce the Wnt3a protein) over transfected HEK293 cells. Wnt3a induces the Wnt/β-catenin/TCF/LEF pathway in control cells transfected with empty plasmid. The Wnt3-induced signal is enhanced by overexpression of WT CK1ε but is strongly depleted by overexpression of the CK1ε mutants. (e) Constitutively activation of the co-receptor LRP6 in transfected HEK293 cells confirmed the regulatory function of CK1ε downstream of the Wnt-receptor complex. WT CK1ε transduces the signal from the activated receptor towards TCF/LEF-driven transcription, while mutant CK1ε proteins abolish downstream signaling. Neither WT nor mutant CK1ε are able to markedly activate the TCF/LEF signal when Wnt stimulation is blocked by the mutant LRP6. (f) Nonphosphorylatable (T44A) and phospho-mimicking (T44D) mutants of CK1ε were co-expressed with Dvl2-Myc in HEK293 cells. The effects on TCF/LEF-dependent transcription show that T44 D behaves as overactive CK1ε. (a) to (f) Data represent the mean ± standard deviation from normalized values. *P < 0.05, ***P < 0.001; n.s., not significant; one-way analysis of variance, Tukey post-test, n ≥ 3.