We recently reported a neutral-pH gel system buffered with 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol hydrochloride (Bis-Tris-HCl) for use in Zn²⁺-Phos-tag sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for advanced profiling of protein phosphorylation. In the current study, we extended the utility of Zn²⁺-Phos-tag SDS-PAGE with the Bis-Tris-HCl buffer system to a detailed analysis of phosphorylated β-catenin, which is closely involved in the ubiquitin-proteasome pathway. The Phos-tag-based approach, followed by Western blotting with an anti-β-catenin antibody, allowed us to assign nine phosphorylated species of β-catenin produced in complicated signaling pathways of cultured HEK293 and SW480 cells. Two-dimensional image coupling with normal Laemmli’s SDS-PAGE as the first dimension gave more detailed information, not only on the phosphorylation of β-catenin, but also on the phosphorylation-dependent polyubiquitination by visualizing multiple ubiquitinated forms derived from two phosphorylated species of β-catenin in lactacystin-treated HEK293 cells. We identified two distinct phosphorylated species of β-catenin that are responsible for polyubiquitination. The first contains phosphorylated residues at S33, S37, T41, and S45, and the second contains these sites and an additional phosphorylated residue at S675. The profiling of double post-translational modifications of β-catenin is consistent with the widely accepted phosphorylation-dependent ubiquitination model in the absence of a Wnt signal.

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