Simultaneous Visualization of Distinct Posttranslational Modification States of β‐Catenin Using Genetic Code Expansion and Click Chemistry

A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β-catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O-GlcNAcylated, or left unmodified. Using site-specific incorporation of the unnatural phenylselenocysteine, a β-catenin-EGFP fusion protein is engineered allowing selective installation of a S-GlcNAc moiety via oxidative elimination and thiol-Michael ligation. Additional β-catenin variants, phosphomimetic T41E-mCherry and wild-type-blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfully introduced into Hep3B and HeLa cells by lipofection or TAT-mediated transduction. Fluorescence microscopy revealed distinct subcellular localization profiles for each PTM form. Notably, the S-GlcNAcylated β-catenin exhibited enhanced resistance to proteasomal degradation, consistent with known roles of O-GlcNAcylation in protein stability. This approach provides a versatile platform to functionally probe PTMs in a comparative, cell-based context.