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Fig. 2. CK1 inhibition blocks Wnt-5a-induced phosphorylation of Dvl2 and Dvl3. SN4741 cells were treated with vehicle (control) or Wnt-5a (100 ng/ml) for 2 hours in the presence or absence of D4476 (100 µM) (A) or IC261 (10 µM) (C). Western blot analysis was performed as in Fig. 1. The position of dephosphorylated (
) and phosphorylated (
) Dvl is indicated. Antibodies against phospho-Ser45-
-catenin (CK1
target site), against active (Ser37-Thr41-dephosphorylated) -catenin (ABC) and total -catenin were used. (B) Scheme of phosphorylation sites of -catenin. Ser45 is a CK1 target site, which serves as a priming for sequential phosphorylation of Thr41, Ser37 and Ser33 by GSK3. (D) SN4741 cells were transfected with plasmids encoding Dvl2-Myc and either CK1
or kinase-dead CK1 (K>R) mutant. Phosphorylation of Dvl2-Myc was detected as a phosphorylation-dependent mobility shift by Myc- and Dvl2-specific antibodies. CK1 levels were monitored with a CK1 specific antibody. Data in A, C and D are representative of at least three independent replicates.
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