Deciphering O‑GlcNAc-Dependent Signaling Via Integrated Proteomics and Phosphoproteomics

Post-translational modifications (PTMs) on proteins play crucial roles in various biological processes. Two highly dynamic modifications, phosphorylation and O-linked N-acetylglucosamine modification (O-GlcNAcylation), are essential for cellular physiology and pathology. Emerging evidence suggests intimate crosstalk between phosphorylation and O-GlcNAcylation on multiple proteins. However, the precise nature of their crosstalk remains largely unknown. In this study, we explored the crosstalk between phosphorylation and O-GlcNAcylation using the pancreatic ductal cell line PANC-1 as a model. Proteome and phosphoproteome changes were measured for cells treated with OSMI-1, a specific inhibitor of O-GlcNAc transferase, and Thiamet G, a specific inhibitor of O-GlcNAcase. Among the 8938 phosphorylation sites quantified, 2289 phosphosites on 1225 proteins and 2201 phosphosites on 1199 proteins were significantly altered by OSMI-1 and TMG treatment, respectively, demonstrating extensive crosstalk between O-GlcNAcylation and phosphorylation. Further analysis revealed widespread phosphorylation changes of the kinome and phosphatome, even after a short-term perturbation with inhibitors to O-GlcNAc cycling enzymes. Moreover, phosphoproteomic profiling, kinase inhibition experiments, and in vitro kinase assays identified that phosphorylation of OGA itself at S364 is specifically mediated by casein kinase 2 α (CK2α). These results uncover glycosylation-dependent cellular signaling through the potentially multilayer crosstalk between phosphorylation and O-GlcNAcylation.