Wnt signaling inhibits casein kinase 1α activity by modulating its interaction with protein phosphatase 2A

Abstract

The mechanism by which Wnt signaling, an essential pathway controlling development and disease, stabilizes β-catenin has been a subject of debate over the last four decades. Casein kinase 1α (CK1α) functions as a pivotal negative regulator of this signaling pathway, initiating the events that destabilize β-catenin. However, whether and how CK1α activity is regulated in Wnt-off and Wnt-on states remains poorly understood. We now show that CK1α activity requires its association with the α catalytic subunit of protein phosphatase 2A (PPP2CA) on AXIN, the scaffold protein of the β-catenin destruction complex. Wnt stimulation induces the dissociation of PPP2CA from CK1α, resulting in CK1α autophosphorylation and its consequent inactivation. Moreover, autophosphorylated CK1α is enriched in a subset of colorectal cancers (CRCs) harboring constitutive Wnt activation. Our findings identify a mechanism by which Wnt stimulation inactivates CK1α, filling a critical gap in our understanding of Wnt signaling, with relevance for CRC.

Keywords: CK1α; CP: Cancer; CP: Molecular biology; Wnt signaling; autophosphorylation; colorectal cancer; protein phosphatase 2A.

Figure 1.. Wnt signaling induces the autophosphorylation and consequent inactivation of CK1α

(A and B) HEK293T cells were treated with Wnt3a ligand for the indicated time periods. CK1α or immunoglobulin (Ig)G (control) immunoprecipitated from lysates of these cells were used for protein kinase reactions in the presence of ATP and recombinant β-catenin. After incubation at 30°C for 30 min, the reactions were stopped by the addition of the Laemmli sample buffer and used for immunoblotting. Representative immunoblots are shown in (A), and the quantification of three such experiments (mean ± SEM) is shown in (B). Asterisks indicate statistical significance (****p < 0.0001). (C) A schematic of the LC-MS/MS experiment used to determine CK1α post-translational modifications (PTMs) and interacting proteins from HEK293T cells, treated with or without Wnt3a. (D) A schematic showing LC-MS/MS results that identified two Wnt-responsive phosphorylation sites in CK1α: S3 and T321. Phosphorylated residues are highlighted in red. (E and F) HEK293T cells were transfected with plasmids encoding HA-tagged CK1α wild type (WT) or CK1α phospho-mutants S3A or T321A. Forty-eight hours later, HA-tagged CK1α or an IgG control was immunoprecipitated from the lysates of cells and used for kinase reactions in the presence of ATP and recombinant β-catenin. After incubation at 30°C for 30 min, the reactions were stopped by the addition of the Laemmli sample buffer and used for immunoblotting. Representative immunoblots are shown in (E), and the quantification of the p-β-catenin S45 level normalized to that of HA-CK1α in three such experiments (mean ± SEM) is shown in (F). The asterisk indicates statistical significance (*p < 0.05). (G) HEK293T cells were transfected with plasmids encoding HA-tagged CK1α WT or the CK1α kinase-inactive mutant K46A, followed by Wnt3a treatment for 4 h in the presence of the proteasome inhibitor, MG-132. HA-tagged CK1α or IgG control was immunoprecipitated from cell lysates, followed by immunoblotting. Representative immunoblots are shown (n = 3).

Figure 2.. Wnt-dependent CK1α autophosphorylation occurs via dissociation from PPP2CA

(A) A heatmap showing the interaction between CK1α and its five most abundant serine/threonine protein phosphatase components, in the absence or presence of Wnt3a, using the LC-MS/MS experimental approach described in Figure 1C. PPP2CA and/or PPP2CB (red box) showed decreased binding to CK1α in response to Wnt activation. The average fold change shows the enrichment of protein phosphatase components in CK1α immunoprecipitates versus IgG control immunoprecipitates. (B) HEK293T cells were transfected with small interfering RNAs (siRNAs) for a non-targeting control gene (CTRL) or PPP2CA (#1 or #2) for 48 h, followed by treatment of Wnt3a and MG-132 for 2 h. CK1α or IgG control was immunoprecipitated from these cell lysates, followed by immunoblotting. Representative immunoblots are shown (n = 3). (C and D) HEK293T cells were treated with Wnt3A for the indicated time periods and used in an in situ PLA to quantify the interactions between PPP2CA and CK1α. Interactions are indicated in red, F-actin staining in green, and nuclear staining in blue (DAPI). Representative microscopy images are shown in (C) (scale bar: 25 μm). The quantification of interaction signals in five random fields (mean ± SD) is shown in (D). Asterisks indicate statistical significance (***p < 0.001). (E) HEK293T cells were transfected with siRNAs for a non-targeting control gene (CTRL) or PPP2CA (#1 or #2) for 72 h. CK1α or IgG (control) immunoprecipitates from the lysates were used for protein kinase reactions in the presence of ATP and recombinant β-catenin. After incubation at 30°C for 30 min, the reactions were stopped by the addition of the Laemmli sample buffer and then used for immunoblotting. Representative immunoblots are shown (n = 3).

Figure 3.. Wnt-dependent, PPP2CA-mediated CK1α autophosphorylation requires AXIN, the scaffold protein of the β-catenin destruction complex

(A–C) HEK293T cells were transfected with siRNA for a non-targeting control gene (CTRL) or pooled siRNAs for AXIN1 for 48 h and then used in an in situ PLA to quantify the interactions between PPP2CA and CK1α (A and B) or directly lysed for immunoblotting (C). Representative microscopy images (A) (scale bar: 25 μm) and immunoblots (C) are shown. The quantification of interaction signals in five random fields (mean ± SD) is shown in (B). Asterisks indicate statistical significance (**p < 0.01). (D and E) HEK293T cells were treated with PBS or Wnt3a in the presence or absence of the Axin stabilizer, XAV-939 (10 μM), for 1 h. Cells were then used in an in situ PLA to determine the interaction between PPP2CA and CK1α in the absence or presence of Axin stabilization. Interactions are indicated in red and nuclear staining with DAPI in blue. Representative microscopy images are shown in (D) (scale bar: 25 μm). The quantification of interaction signals in five random fields (mean ± SD) is shown in (E). Asterisks indicate statistical significance (****p < 0.0001). (F and G) HEK293T cells were treated with PBS or Wnt3a for 1 h. An in situ PLA was then performed to determine the interaction between PPP2CA, CK1α, and AXIN1. Interactions are indicated in red and nuclear staining with DAPI in blue. Representative microscopy images are shown in (F) (scale bar: 25 μm). The quantification of interaction signals in five random fields (mean ± SD) is shown in (G). Asterisk indicates statistical significance (*p < 0.05).

Figure 4.. Phosphorylated CK1α-T321 enriches in a subset of CRC

(A and B) Wild-type or CK1α-T321A knockin HEK293FT cells were treated with PBS or Wnt3a (100 ng/mL) for 2 h. Lysates from cells were used for immuno-blotting. Representative blots are shown in (A) and the quantification of levels of Wnt activation biomarkers (mean ± SEM, n = 3) in (B). The asterisk indicates statistical significance (*p < 0.05). (C and D) Lysates from HEK293T and five Wnt activity-driven CRC cell lines were used for immunoblotting. Representative blots are shown in (C) and the quantification of autophosphorylated CK1α levels (mean ± SEM, n = 3) in (D). Asterisks indicate statistical significance (*p < 0.05 and **p < 0.01). (E and F) The CRC cell line Caco2 was transfected with plasmids encoding HA-tagged CK1α WT or the CK1α mutant lacking the autophosphorylation site (T321A) for 72 h. Transfected cells were then seeded at 1,000 cells/well and grown for 10 days, followed by crystal violet staining of cell colonies. Representative bright-field images are shown in (E) and the quantification of colony number (mean ± SEM, n = 3) in (F). The asterisk indicates statistical significance (*p < 0.05). (G) Peptide abundance of p-CK1α T321 in normal colon (n = 26) and primary CRC tumor (n = 40) was mined from the proteomic and phosphoproteomic data generated by CPTAC. To quantitate autophosphorylated CK1α, the abundance of p-CK1α T321 peptides was normalized to that of total CK1α protein. Asterisks indicate statistical significance (**p < 0.01). (H and I) A tissue microarray containing samples from normal colon, primary colorectal cancer, or metastatic colorectal cancer (N = 60 from 49 patients) was used to determine the levels of autophosphorylated CK1α by immunohistochemistry. Representative images of H&E and autophosphorylated CK1α staining are shown in (I) (scale bar: 50 μm). The quantification of CK1α autophosphorylation levels across the tissue microarray are shown in (H).