The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1

Abstract

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

Fig. 1.

Schematic representation of full-length β-catenin, and deletion and point mutants that were used in this work.

Fig. 2.

CD spectrum of the β-catenin fragments used for competition assays. The percentage of α-helix was calculated as described in Methods.

Fig. 3.

Inhibition of β-catenin phosphorylation by nonphosphorylatable β-catenin fragments depends on the first armadillo repeat. Phosphorylation of β-catenin^WT and deletion mutants (100 nM) by CK1α was determined in the presence of increasing concentrations of GST-β-Cat^131–422 (A) or fragment GST-β-Cat^181–422 (B), as described in Methods.

Fig. 4.

Phosphorylation efficiency of β-catenin Ser-45 by CK1 is increased by the Y142E mutation. (A) β-Catenin^WT and the Y142E mutant were subjected to phosphorylation with CK1α as described in Methods. (B) Twenty micromolar GST-β-Cat^WT and the Y142E mutant were phosphorylated by CK1α. Phosphorylated products were detected by Western blotting with an anti-phosphoserine-45 (anti-pS45) antibody, and β-catenin was detected by using an antibody that recognizes full-length β-catenin. *, V_max is expressed as picomoles of phosphate transferred per minute per microgram of enzyme.

Fig. 5.

α-Catenin prevents β-catenin phosphorylation by CK1 in a dose-dependent manner. (A) Phosphorylation of β-catenin^WT by CK1α was tested in the presence of increasing concentrations of α-catenin. (B) The phosphorylation by CK1 of either β-catenin^WT and deletion mutants (all 100 nM) was assayed in the absence or presence of 1 μM α-catenin. In both A and B, radioactive products were subjected to SDS/PAGE and autoradiography.