Abstract
β-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, β-catenin is a proto-oncogene and activating β-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of β-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of β-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related β-catenin function. The present paper describes a novel suspension bead array assay panel for β-catenin, which requires minimal amounts of sample and is able to relatively quantify total β-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free β-catenin. This is the first study to combine three biochemical methods--sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay--in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different β-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in β-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of β-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.
Highlights
The Wnt signaling pathway is involved in the regulation of physiological processes, embryonic development, and tissue
The use of a sandwich immunoassay format allows the simultaneous measurement of the (i) total amount of -catenin, (ii) four phosphorylated -catenin forms and the detection of (iii) E-cadherin-complexed -catenin in a miniaturized co-immunoprecipitation
The Wnt signaling pathway was analyzed in HEK293 cells treated either with recombinant Wnt3a ligand or the GSK3 inhibitor SB216763
Summary
The Wnt signaling pathway is involved in the regulation of physiological processes, embryonic development, and tissue. Multiplexed Monitoring of Different -Catenin Characteristics—For a relative quantification of -catenin states involving post-translational modifications, binding to other proteins, and transcriptional activity a suspension bead array panel was developed involving the following sandwich immunoassays (Fig. 1).
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