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 is phosphorylation analysis ofβ-catenin in SW480 cells using by Zn2+–Phostag SDS-PAGE. β-Catenin is regulated by various protein kinases in vivo, and its various phosphorylation states are closely involved with specific cellular events. In this study, we used a whole lysate of human colon adenocarcinoma cells(SW480).
The 85-kDa β-catenin was detected together with several degraded forms by immunoblotting with anti-β-catenin antibody of normal gel (1-D) and the phosphorylation at the S33, S37, T41, S45, S552, and S675 were identified with four kinds of anti-p-β-catenin antibodies against the
pS33/pS37/pT41, pT41/pS45, pS552, and pS675, respectively (top panels). We next compared the analysis of the same lysate by Zn2+–Phos-tag SDS-PAGE. To specify the correlations between the intact and degraded forms, we performed 2-DE (normal SDS-PAGE/ Phos-tag SDS-PAGE (leftmost panels). The 2-DE allowed us to distinguish between the intact β-catenin (arrowed at 85 kDa in the first dimension) and the degraded forms (left-side area from the position of 85 kDa). On the 2-D Zn2+–Phos-tag SDS-PAGE gel, ten phosphoisotypes derived from the intact were identified in the Rf range of 0.2–0.8 The fastest-migrating spot was assigned to a nonphosphorylated form (open arrowhead). All the spots identified on the 2-D gel correlated with 1-D Phos-tag SDS-PAGE banding images (shown in five right-hand panels). All of the upshifted
intact β-catenin bands showed cross-activity with these site-specific p-β-catenin antibodies. Therefore, the results support that all the up-shifted bands contain the phosphorylated residues.