Snapshots of Protein Dynamics and Post-translational Modifications In One Experiment-β-Catenin and Its Functions

beta-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, beta-catenin is a proto-oncogene and activating beta-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of beta-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 beta-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related beta-catenin function. The present paper describes a novel suspension bead array assay panel for beta-catenin, which requires minimal amounts of sample and is able to relatively quantify total beta-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free beta-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 beta-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in beta-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of beta-catenin on Ser-552 and Ser-675 with canonical Wnt signaling. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.007377, 1-10, 2011.