Figure 1

CK1δ positively regulates Wnt/β-catenin signaling. Panel A, siRNA targeting CK1δ attenuates Lef/Tcf-sensitive transcription. F9 cells were transfected with siRNA targeting CK1δ one day before co-transfection with Rfz1 and Super8xp TOPFlash (M50) reporter. On the following day, cells were either unstimulated or stimulated with Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Results are displayed relative to the unstimulated cells (set to “1”). Results shown are mean values ± s.e.m. from 5 independent experiments. Statistical significance is indicated (*, p < 0.005). Panel B, treatment of CK1δ ιnhibitor attenuates Lef/Tcf-sensitive transcription. F9 cells were co-transfected with Rfz1 and M50. One day post transfection, cells were either untreated or pretreated with IC261 (20 μM, selective inhibitor of CK1δ/ϵ) for 1 hr prior to stimulation either with or without Wnt3a (20 ng/ml) for 7 hr in the continued presence (or absence) of IC261. Cell lysates were prepared and assay of Lef/Tcf-sensitive transcription was performed. Results are displayed relative to the unstimulated cells (set to “1”). Results shown are mean values ± s.e.m. from 3 independent experiments. Statistical significance is indicated (*, p < 0.005). Panel C, expression of CK1δ enhances Lef/Tcf-sensitive transcription. F9 cells were transfected with Rfz1, M50 and either wild-type CK1δ or K38M-CK1δ (kinase inactive mutant). On the following day, cells were either unstimulated or stimulated with Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Results are displayed relative to the unstimulated cells (set to “1”). Statistical significance is provided, * denotes p < 0.005 (versus control cells). Results shown are mean values ± s.e.m. from 4 independent experiments. Panel D, wild-type CK1δ rescues Lef/Tcf-sensitive transcription in the CK1δ-deficient cells. F9 cells stably expressing Rfz1 and M50 were treated with siRNA targeting CK1δ one day before transfection with either wild-type CK1δ or K38M-CK1δ. On the following day, cells were either unstimulated or stimulated with Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Results are displayed relative to the unstimulated cells (set to “1”). Statistical significance is provided, * denotes p < 0.005 (versus control cells). Results shown are mean values ± s.e.m. from 3 independent experiments. Panel E, role of serine/threonine sites of Dvl2 in Lef/Tcf-sensitive transcription. Phosphorylation of rDvl2 (purified from Sf9 cells) catalyzed by purified CK1δ was carried out in vitro and phosphorylation sites were identified by mass spectrometry. The phosphorylation sites (S/T) identified then were mutated to alanine using Quickchange Mutagenesis System (Stratagene, La Jolla, CA). F9 cell were co-transfected with Rfz1, M50 and either wild-type Dvl2 or a specific Dvl2 mutants. On the following day, assay for Lef/Tcf-sensitive transcription was performed. Statistical significance is provided, * denotes p < 0.005 (versus wild-type Dvl2 expressed cells). Results shown are mean values ± s.e.m. from 6 independent experiments.