PMC

CKI abrogates β-catenin degradation in Xenopus egg extracts. Reticulocyte lysate-expressed [³⁵S]β-catenin and luciferase were incubated in Xenopus egg extracts with or without 1.15 μM CKIɛΔ319. Protein degradation was assessed by SDS/PAGE and autoradiography. Data from three separate experiments are plotted as the ratio of β-catenin to luciferase ± SEM.

CKI phosphorylates multiple components of the Wnt pathway. The indicated epitope-tagged Wnt pathway components were expressed in 293 cells and then immunoprecipitated and incubated with [γ-³²P]ATP with or without CKIδΔ319. Phosphorylation was assessed by SDS/PAGE and PhosphorImager analysis, and tagged proteins in the pellet were assessed by immunoblotting. The experiments were repeated twice with similar results.

CKIδ/ɛ interacts with Dvl-1. (A) CKI coimmunoprecipitates Dvl-1. In vitro translated proteins were loading directly (Input), or incubated either alone (-), or with CKIɛ or K38R. The samples were immunoprecipitated with anti-HA and coimmunoprecipitating proteins were detected by SDS/PAGE and PhosphorImager analysis. (B) Dvl-1 coimmunoprecipitates CKIδ/ɛ, but not CKIα. HA: Dvl-1 was immunoprecipitated from 293 cells with anti-HA and the lysate and pellet were probed with anti-HA, anti-CKIδ/ɛ, and anti-CKIα antibodies. The experiments were repeated twice with similar results.

CKI phosphorylates β-catenin and axin on major in vivo sites. Tryptic phosphopeptide maps of β-catenin (A–C) and axin (E–G). The major phosphopeptides are indicated with letters; uppercase letters denote shared peptides, whereas lowercase letters denote unique peptides. (D) The immunoblot confirms the effects of Wnt3a on endogenous β-catenin accumulation in 293 cells. α-Tubulin serves as a loading control. (H) The diagram represents the directions of electrophoresis and chromatography. The experiments were repeated twice with similar results.

CKIɛ destabilizes the β-catenin degradation complex in vitro and in vivo. Lysates from 293 cells expressing (A) Myc:axin or (B) Myc:β-catenin were incubated alone (-), with 180 nM CKIɛ (CKIɛ), or with 480 nM K38R and 250 μM CKI inhibitor CKI-7 (K38R) in the presence of anti-Myc-Sepharose. The pellet and 10% of the supernatant were analyzed by SDS/PAGE and immunoblotting. (C) 293 cells expressing Myc:β-catenin were incubated with DMSO or 40 μM IC261 before harvest and anti-Myc-Sepharose immunoprecipitation. The immunopellets were analyzed by SDS/PAGE and immunoblotting. Each experiment was repeated twice with similar results.

B56 and kinase-dead CKI synergistically inhibit Wnt signaling. Xenopus embryos were injected with Xwnt-8 and (A) β-gal, (B) B56α, (C) K38A, or (D) B56α/K38A RNA. Equal amounts of each RNA were injected per embryo, except for β-gal, which was used to normalize total RNA injected per embryo. (E) The graph is a plot of the fitted distribution of responses across the phenotypes from a representative experiment. The injections were performed eight times for an n of 365 (Xwnt-8), 365 (Xwnt-8/B56α), 369 (Xwnt-8/K38A), or 368 (Xwnt-8/K38A/B56α). The statistical analysis shows that there is a synergistic interaction between K38A and B56α, because the phenotypic shift is greater than that predicted for an additive interaction (P value = 0.0066).

B56 acts downstream of CKI. Xenopus embryos were injected with CKIδ and (A) β-gal or (B) B56α; or CKIɛ and (D) β-gal or (E) B56α RNA. (C and F) Diagrams depicting the degree of axis formation in CKIδ and CKIɛ injected embryos, respectively. Vent., ventralized; WT, wild type; Vest., vestigial secondary axis or slightly dorsalized; Incom., incomplete secondary axis; Com., complete secondary axis. The data shown are from a representative experiment which was performed five times for an n of 236 (CKIδ/β-gal) or 231 (CKIδ/B56α), or six times for an n of 290 (CKIɛ/β-gal) or 288 (CKIɛ/B56α). The statistical analysis was carried out on the cumulative experiments, and the difference between the phenotypes resulting from β-gal and B56α RNA injections in both the CKIδ and CKIɛ experiments is highly significant (P value < 10⁻⁴).