Targeted O-GlcNAcylation of CK2α Triggers Its Ubiquitin-Proteasome Degradation and Alters Downstream Phosphorylation.

O-Linked β--acetylglucosamine-modification (O-GlcNAcylation) is an important post-translational modification (PTM), yet dissecting its protein-specific functions has remained challenging. Here, we applied our previously reported chemical biology tool, the O-GlcNAcylation Targeting Chimera (OGTAC), to specifically induce O-GlcNAcylation of the casein kinase II subunit α (CK2α) at Ser347 in living cells. We found that this targeted O-GlcNAcylation destabilized CK2α through ubiquitin-proteasome degradation and enhanced its interaction with cereblon (CRBN). Overexpression and knockdown experiments also indicated CK2α as a substrate of the Cullin-RING E3 ubiquitin ligase 4-CRBN (CRL4) E3 ligase complex. Furthermore, the OGTAC-induced O-GlcNAcylation of CK2α reprogrammed phosphorylation of Akt and PFKP. These findings reveal that a single O-GlcNAc modification can serve as a molecular switch, controlling the protein stability and downstream phosphorylation of CK2α. More broadly, our results highlight the profound utility of the OGTAC-mediated O-GlcNAcylation to interrogate its cellular functions with specificity, overcoming limitations inherent to prior global perturbation methods.