Casein kinase iepsilon in the wnt pathway: regulation of beta-catenin function

Abstract

Wnt and its intracellular effector beta-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, we isolated casein kinase Iepsilon (CKIepsilon). CKIepsilon mimicked Wnt in inducing a secondary axis in Xenopus, stabilizing beta-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIepsilon by kinase-defective CKIepsilon or CKIepsilon antisense-oligonucleotides attenuated Wnt signaling. CKIepsilon was in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIepsilon appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate beta-catenin.

Figure 1

CKIɛ/δ induces a secondary axis in Xenopus embryos. (a) Examples of the embryos injected with CKIɛ or β-galactosidase (β-gal) RNA. (b) Percentage of embryos with duplicated axis injected with β-gal, β-catenin, CKIɛ, CKIδ, CKIα, KN-CKIɛ, or ΔC-CKIɛ RNA as indicated. The number of the embryos with duplicated axis relative to the total number of injected embryos is indicated above each bar. (c) Axin inhibits secondary axis formation induced by CKIɛ. CKIɛ RNA was coinjected with β-gal or axin RNA (1–2 ng). (d) Induction of Siamois by CKIɛ. Siamois expression was analyzed by RT-PCR from dorsal halves of the embryos injected with XWnt-8, CKIɛ, or β-gal. EF-1 expression was a loading control.

Figure 2

β-catenin stabilization induced by CKIɛ. (a) Drosophila S2 Schneider cell lysates blotted with armadillo antibody and hemagglutinin (HA) antibody recognized transfected CKIɛ. Tubulin was a loading control. (b) Cytosolic fraction from 293 cells transfected with vector, Wnt-1, CKIɛ, and KN-CKIɛ blotted with β-catenin antibody. RNA polymerase II was a loading control.

Figure 3

Lef-1 reporter gene activity induced by CKIɛ. Lef-1 reporter gene assay was performed as described (10). Representative data from several independent experiments are shown. (a) The effects of CKI isoforms on Lef-1 activity. (b) Axin inhibits Lef-1 reporter gene activity induced by CKIɛ. (c) KN-CKIɛ blocks Lef-1 activity induced by Wnt-1. (d) CKIɛ antisense-oligonucleotides inhibit Lef-1 reporter gene activity induced by Wnt-1. (Left) Wnt-1 induced Lef-1 reporter activity. CKI-ASa and CKI-ASb are two different antisense-oligonucleotides from the human CKIɛ coding sequence. The control-oligonucleotide is the reverse sequence of ASa. (Right) Endogenous level of CKIɛ normalized to levels of 14-3-3 protein (loading control) after transfection of oligonucleotides.

Figure 4

CKIɛ forms a complex with the other molecules in the Wnt pathway. (a) Endogenous CKIɛ coimmunoprecipitated with transfected myc-tagged axin. (b) The C-terminus domain of CKIɛ is required for binding to axin. Myc-axin and hemagglutinin (HA)-CKIɛ constructs (indicated by arrows) were cotransfected in 293 cells. Myc-axin immune complexes were analyzed by immunoblotting with myc (detect axin) and HA (detect CKIɛ) antibodies. Dye front was marked as ★. (c) GSK-3β is in a complex with CKIɛ and axin. Myc-axin and HA-CKIɛ constructs (indicated) were cotransfected in 293 cells. HA-CKIɛ immune complexes were analyzed by immunoblotting with myc (detect axin) and GSK-3β antibodies. (d) Endogenous CKIɛ is coimmunoprecipitated with transfected myc-tagged Dvl3.