Dose-Dependent Inhibition of Protein Glycosylation Reveals Crosstalk between Glycosylation and Phosphorylation in EGF Signaling Pathways

Protein glycosylation and phosphorylation play critical roles in regulating diverse cellular processes, yet their interplay remains underexplored. This study employed dose-dependent glycosylation inhibition to investigate the function of glycosylation, especially in phosphorylation in epidermal growth factor (EGF) signaling. Using quantitative proteomics, we analyzed global protein expression, glycopeptides, and phosphopeptides in EGF-stimulated and unstimulated conditions. Results revealed that glycosylation inhibition disrupted phosphorylation patterns in EGF-regulated pathways. Notably, 215 EGF-regulated phosphopeptides exhibited dose-dependent changes, with pathways such as ERBB, MAPK, and mTOR signaling being significantly affected. Protein-protein interaction analysis highlighted glycosylation-dependent modulation of phosphorylation in cytoskeletal remodeling and growth factor signaling. Correlation analysis identified site-specific glycopeptides, such as Asn568 of EGFR, that influenced phosphorylation of downstream signaling proteins. Additionally, individual proteins like RCN1 and SLC4A7 demonstrated glycosylation- and phosphorylation-regulated patterns. These findings underscored the role of glycosylation in maintaining phosphorylation-dependent signaling fidelity, providing insights into the function of glycosylation on cell signaling pathways.