Coordinated action of CK1 isoforms in canonical Wnt signaling

Abstract

Activation of the Wnt pathway promotes the progressive phosphorylation of coreceptor LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6), creating a phosphorylated motif that inhibits glycogen synthase kinase 3β (GSK-3β), which in turn stabilizes β-catenin, increasing the transcription of β-catenin target genes. Casein kinase 1 (CK1) kinase family members play a complex role in this pathway, either as inhibitors or as activators. In this report, we have dissected the roles of CK1 isoforms in the early steps of Wnt signaling. CK1ε is constitutively bound to LRP5/6 through its interaction with p120-catenin and E-cadherin or N-cadherin and is activated upon Wnt3a stimulation. CK1α also associates with the LRP5/6/p120-catenin complex but, differently from CK1ε, only after Wnt3a addition. Binding of CK1α is dependent on CK1ε and occurs in a complex with axin. The two protein kinases function sequentially: whereas CK1ε is required for early responses to Wnt3a stimulation, such as recruitment of Dishevelled 2 (Dvl-2), CK1α participates in the release of p120-catenin from the complex, which activates p120-catenin for further actions on this pathway. Another CK1, CK1γ, acts at an intermediate level, since it is not necessary for Dvl-2 recruitment but for LRP5/6 phosphorylation at Thr1479 and axin binding. Therefore, our results indicate that CK1 isoforms work coordinately to promote the full response to Wnt stimulus.

Fig. 1.

CK1ε mediates transient CK1α interaction with p120-catenin upon Wnt3a stimulation. (A) SW-480 cells stimulated with Wnt3a-conditioned medium for the indicated times were lysed, and p120-catenin was immunoprecipitated (IP). Associated proteins were analyzed by Western blotting (WB). In the “input” lane, a sample corresponding to 5% of the lysates used was loaded. (B) SW-480 cells were transfected with scrambled shRNA or shRNA specific for CK1ε. Cells were treated with control (Ctl) or Wnt3a-conditioned medium for 30 min. p120-catenin was immunoprecipitated, and immunocomplexes were analyzed by Western blotting. (C) The subcellular distribution of CK1α was determined in SW-480 cells infected with scrambled shRNA or shRNA specific for CK1ε. Cells were treated with Wnt3a-conditioned medium for 0 min (upper panels) or 30 min (lower panels). The analysis was performed by immunofluorescence using an antibody against CK1α. No signal was obtained when the same analysis was performed in the absence of the primary antibody. The scale bar in each panel corresponds to 20 μm. All of the data presented in this figure are representative results from at least three different experiments.

Fig. 2.

CK1α is required for p120-catenin Ser268 phosphorylation and disruption of E-cadherin–p120-catenin interaction. (A) SW-480 cells were transfected with scrambled shRNA or an shRNA specific for CK1α and treated with control (Ctl) or Wnt3a-conditioned medium for 2 h. Cell extracts were analyzed by Western blotting (WB) with the indicated antibodies. (B) SW-480 cells depleted of CK1α as indicated above were stimulated or not with Wnt3a-conditioned medium for 2 h. Cells were lysed, and p120-catenin or LRP5/6 was immunoprecipitated (IP) with specific antibodies. Associated proteins were analyzed by Western blotting. In the “input” lane, a sample corresponding to 5% of each total cell extract was used. The results from a representative experiment out of three performed are shown. (C) SW-480 cells were treated with control or Wnt3a-conditioned medium for 30 min. Total cell extracts were immunoprecipitated with anti-CK1α antibody. Immunocomplexes were analyzed against CK1α or incubated with 2 pmol of recombinant GST-p120-catenin (positions 102 to 911) under CK1 phosphorylation conditions. Phosphorylation of Ser268 was analyzed by Western blotting with a specific phospho Ser268 antibody. The average ± standard deviation (SD) of the densitometric analysis of the results of the three experiments performed is presented in panel D. Phosphorylation of Ser268 was normalized considering the amount of substrate and referred to the value obtained in unstimulated cells. All of the data shown in this figure are representative of at least three independent experiments. Irr. IgG, an irrelevant IgG used as a control in the immunoprecipitation.

Fig. 3.

CK1ε but not CK1α is required for very early events in Wnt3a signaling. (A and B) SW-480 cells were incubated with control (Ctl) or Wnt3a-conditioned medium for the indicated times, and total cell extracts were analyzed by Western blotting (WB) with the indicated antibodies. Representative blots are presented in panel A; shown is the average ± SD of the results of the three experiments performed in panel B. Protein phosphorylation was normalized considering the amount of each susbtrate and referred to the value obtained after 1 h of stimulation with Wnt. (C) SW-480 cells were transfected with scrambled or CK1ε- or CK1α-specific shRNAs. After selection with 2 μg/ml puromycin, cells were treated with control or Wnt3a-conditioned medium for 30 min. LRP5/6 was immunoprecipitated (IP) from total cell extracts, and associated proteins were analyzed by Western blotting. A representative Western blot of four different experiments is shown.

Fig. 4.

Axin binds and recruits CK1α to the LRP5/6 receptor. (A) SW-480 cells stimulated or not with Wnt3a-conditioned medium for the indicated times were lysed, and LRP5/6 was immunoprecipitated (IP). Associated proteins were analyzed by Western blotting (WB) with the indicated antibodies. In the “input” lane, a sample corresponding to 5% of the lysates used was loaded. (B) SW-480 cells were treated with control (Ctl) or Wnt3a-conditioned medium for 30 min. Axin was immunoprecipitated, and immunocomplexes were analyzed against CK1α by Western blotting. (C and D) SW-480 cells were depleted of axin by using a specific shRNA. After selection with 2 μg/ml puromycin, cells were stimulated with control or Wnt3a-conditioned medium for 30 min (C) or 2 h (D). p120-catenin (C and D) or LRP5/6 (C) was immunoprecipitated from whole-cell extracts, and the associated proteins were analyzed by Western blotting. β-Actin was used as a negative control. All of the data presented in this figure are representative of at least three independent experiments.

Fig. 5.

CK1γ is required for Wnt3a-induced LRP5/6 T1479 phosphorylation but not for Dvl-2 binding to LRP5/6. SW-480 cells were depleted of CK1γ or p120-catenin by using specific shRNAs or a scrambled shRNA as a control (Ctl). After the selection, cells were treated with control or Wnt3a-conditioned medium for 30 min (B and D) or 2 h (A and C), respectively. (A) Total cell extracts were analyzed by Western blotting (WB) using antibodies against the indicated proteins. (B, C, and D) Cell extracts were immunoprecipitated (IP) with anti-LRP5/6 (B and C) or CK1γ (D), and associated proteins were analyzed by Western blotting with the indicated antibodies. The results from a representative experiment out of three performed are shown.

Fig. 6.

CK1ε is required for CK1α interaction with LRP5/6 in HEK293 cells. HEK293 cells were transfected with scrambled or CK1α- or CK1ε-specific shRNA for 72 h (A) or scrambled, CK1α- CK1ε-, or CK1γ-specific shRNA for 72 h (B). Cells were treated with control (Ctl) or Wnt3a-conditioned medium for 30 min (A) or 2 h (B). LRP5/6 (A) or p120-catenin (B) was immunoprecipitated (IP) from total cell extracts, and immunocomplexes were analyzed by Western blotting (WB). In the “input” lane, a sample corresponding to 5% of the lysates used was loaded. All of the data presented in this figure are representative of at least three independent experiments. Irr. IgG, an irrelevant IgG used as a control in the immunoprecipitation.

Fig. 7.

N-cadherin–LRP5/6 interaction is sensitive to Wnt3a and CK1 phosphorylation. HEK293 cells (A) or MEF cells (B) were treated with control or Wnt3a-conditioned medium for 2 h. N-cadherin was immunoprecipitated (IP) from total cell extracts, and the presence in the immunocomplex of p120-catenin and LRP5/6 was determined by Western blotting (WB). In the “input” lane, 5% of each total cell extract used was loaded. (C) Recombinant p120-catenin corresponding to isoform 1 (aa 1 to 911) or 3 (aa 102 to 911) was in vitro phosphorylated with the CK1 catalytic domain when indicated. In vitro binding assays were performed by incubating recombinant GST-cytoN-cadh (3 pmol) or GST as a control with p120-catenin (aa 1 to 911) (2 pmol) or p120-catenin (aa 102 to 911). Protein complexes were affinity purified with glutathione-Sepharose and analyzed by Western blotting with anti-p120-catenin. Blots were reanalyzed with anti-GST to ensure that a similar amount of fusion protein was present. (D) Recombinant GST fusion proteins containing cytoN-cadh or GST as a control were in vitro phosphorylated with the CK1 kinase domain when indicated. Pulldown assays were performed by incubating fusion proteins (7 pmol) with extracts from 293T cells. Protein complexes were affinity purified and analyzed by Western blotting.

Fig. 8.

CK1ε, -γ, and -α isoforms are required for Wnt3a stimulation of β-catenin/Tcf-4 transcriptional activity. (A) HEK293 cells were depleted of CK1α, CK1ε, or CK1γ by using specific shRNAs and treated with control (Cont) or Wnt3a-conditioned medium for 16 h. Cells were lysed in RIPA buffer to obtain total extracts. Levels of total β-catenin were determined by Western blotting (WB). The results from a representative experiment out of three performed are shown. (B) β-Catenin transcriptional activity was determined using the TOP reporter plasmid in HEK293 cells transfected with scrambled, CK1α, CK1ε, or CK1γ shRNA. pTK-Renilla plasmid was transfected to normalize the efficiency of transfection. Relative luciferase activity was determined 48 after transfection, when the indicated cells were treated for the last 16 h with Wnt3a-conditioned medium. The results show the average ± range of two experiments performed in quadruplicate.

Fig. 9.

Proposed model for the involvement of the different CK1 isoforms in the Wnt pathway. In unstimulated cells (A), inactive CK1ε is bound to p120-catenin and E-cadherin (E-cadh); E-cadherin is also associated with Wnt coreceptor LRP5/6 (LRP). CK1α is interacting with axin. Upon binding of Wnt ligands and the formation of the LRP5/6-Fz complex (B), CK1ε is activated by removal of inhibitory phosphates in its C-terminal tail by the action of an unknown phosphatase. Activated CK1ε phosphorylates Dvl-2 (Dvl), stabilizing the interaction of Dvl-2 with LRP5/6 (C). Binding of Dvl-2 enables LRP5/6 phosphorylation at Thr1479 by the action of CK1γ, which in turn allows the recruitment of axin to the complex (D). Axin-associated CK1α also binds to the Wnt-receptor complex, contributing to LRP5/6 phosphorylation (E). Moreover, CK1α also phosphorylates p120-catenin at Ser268 and E-cadherin, releasing E-cadherin and p120-catenin from the signalosome. CK1ε is released from the complex with p120-catenin, interrupting the input signal. Finally, axin/CK1α complex is released from LRP5/6-Dvl2 complex by the action of a phosphatase that dephosphorylates axin (F).