β-catenin is a key com- ponent of the Wnt signaling pathway as a transcriptional activator that affects cell proliferation and differentiation in many types of cell. In the absence of a Wnt signal, β-catenin interacts with Axin, casein kinase Iα (CKIα), glycogen synthase kinase-3β(GSK-3β, and adenomatous polyposis coli gene product. In the complex, CKIα serves as a priming kinase that phosphorylates the S45 residue of β-catenin and enhances subsequent phosphorylation at the S33, S37, and T41 residues by GSK-3β. The multi- phosphorylated β-catenin is ubiquitinated and degraded by the proteasome pathway. In this phosphorylation- dependent manner, β-catenin in the cytoplasm is maintained at low levels in quiescent cells. On the other hand, the phosphorylation-dependent ubiquitin-proteasome pathway is frequently disordered in cancer cells, where the level of β-catenin protein increases. In addition to phospho- rylation by CKIα and GSK-3β, β-catenin can also be phosphorylated at the S552 and S675 residues by protein kinases A and B (AKT), respectively, and each phosphoryation reaction induces transcriptional activity of β-catenin without affecting the ubiquitin-proteasome pathway. Thus, β-catenin is regulated by various protein kinases in vivo, and its various states of phosphorylation are closely related to specific cellular events.This data describes two-dimensional Phos-tag affinity electrophoresis for profiling of double post-translational modifications of phosphorylation and ubiquitination of β-catenin.
To detect phosphorylation of β-catenin as shifts in the mobility, we analyzed the lactacystin (proteasome inhibitor) treated HEK293 and SW480 cells by Zn2+– Phos-tag SDS-PAGE. Both samples showed similar banding patterns (left-hand panel). When referred to our previous data(link；β-catenin), we could assign nine bands of phosphorylated species as indicated by cross lines of #1–#9 . The fastest-migrating band was assigned to the nonphosphorylated form of the protein (shown by the open arrowhead).
(Each band were assigned previously by using the anti-phospho-β-catenin antibodies) link；β-catenin
To determine the phosphorylated species of β-catenin that are responsible for the polyubiquitination, the lactacystin-treated HEK293 lysate sample was analyzed by two-dimensional electrophoresis (2-DE) with normal SDS-PAGE coupled with Zn2+–Phos-tag SDS-PAGE (center panel). The 2-DE image of the lactacystin-treated HEK293 lysate gave more-detailed information, not only on the phosphorylation of β-catenin, but also on the phosphorylation-dependent polyubiquitination. All up-shifted spots on the 2-DE gel were correlated with nine bands in the one-dimensional electrophoresis (1-DE) gel. Multiple up-shifted right-diagonal spots in the area to the right-hand side of the 85-kDa position (arrowed) were ubiquitinated forms of β-catenin and they were derived from two phosphorylated species assigned as #5 and #8, indicating that the two species are responsible for polyubiquitination. Both #5 and #8 showed cross-activity with the anti-phospho-β-catenin anti- bodies against phosphorylated S33/S37/T41 and against phosphorylated T41/S45. Moreover, #5 showed highly cross-activity with the anti-phospho-β-catenin antibody against phosphorylated S675, but #8 did not do so at all. In the SW480 cells (ubiquitin-proteasome pathway is disordered), on the other hand, ubiquitinated forms were not observed in the 2-DE gel (right-hand panel), irrespective of any treatment with lactacystin. These results are consistent with the widely accepted phosphorylation-dependent ubiq- uitination model of β-catenin. Thus, this 2-DE strategy using Zn2+–Phos-tag SDS-PAGE permitted the profiling of double post-translational modifications of phosphorylation and ubiquitination.