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.