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

Abstract

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

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

“…5B), or E-cadherin-associated ␤-catenin (Fig. 5C) (37). Measuring the total fraction of ␤-catenin, we detected a ϳtwofold increase upon induction with CHIR.…”

“…The development of new research tools has always proven to advance the knowledge of ␤-catenin. This includes the generation and application of specific antibodies recognizing different post-translationally modified versions of ␤-catenin (47, 66 -68), reliable immunoassays to study protein dynamics upon compound treatment (37) or the development of fluorescently labeled constructs to trace the dynamics of ␤-catenin in different cellular models (26,27).…”

“…After removal of the bound fraction, remaining supernatants were analyzed using bead arraybased sandwich immunoassays for total and hypo-phosphorylated ␤-catenin (nonphospho Ser33/Ser37/Thr41), as described earlier (37). E-cadherin/␤-catenin complex was measured by miniaturized coimmunoprecipitation as described previously (41).…”

Inhibition of GSK3 by Wnt signalling – two contrasting models

“…3), which provided a conceptually satisfactory answer to a long-standing question, indicating that similar molecular mechanisms underlie GSK3 inhibition by both Wnt and insulin signalling. Notably, this model can readily explain the rapid Wnt-induced block of GSK3-mediated phosphorylation of b-catenin, and of Axin -both of which are detectable within 5-15 minutes after Wnt stimulation (Luckert et al, 2011;Willert et al, 1999). Altogether, this biochemical model is highly persuasive, although some questions remain unanswered.…”

Tools used to study how protein complexes are assembled in signaling cascades

“…[33][34][35] It is also very difficult to replicate post-translational modifications in the system which results in many transient protein-protein interactions being missed. Several adaptations of the protein array system, such as suspension bead arrays, have the potential to circumnavigate this problem 36 and studies investigating post-translational modifications of particular residues such as lysine have been successful and are being used to study ubiqutin (Ub) ligases, SUMOylation E3 ligases and acetyltransferases. 37 Advances in peptide synthesis on nitrocellulose support have also helped overcome some of these challenges and is discussed later in the review.…”