Characterizing the Effects of Protein Glycosylation Perturbation on Phosphorylation Signaling.

Protein glycosylation and phosphorylation constitute two pervasive regulatory layers in mammalian cells, yet the effects of protein glycosylation on phosphorylation signaling remain poorly understood. Here, we show that controlled perturbation of N-linked glycan biosynthesis through multiplex glycoengineering fundamentally rewires phosphorylation signaling networks in human cells. Using comprehensive proteomics approaches, we simultaneously profiled the global proteome, glycoproteome, and phosphoproteome in engineered HEK293 cells designed to force the glycan processing network into a defined "boundary-state" glycome that eliminates fucosylation while enhancing sialylation and reducing GlcNAc branching complexity. Glycoengineering emerged as the dominant source of molecular variation across all data sets, with over 9600 intact glycopeptides identified, of which over 3400 are significantly altered, establishing a remodeled cellular state. Upon serum stimulation, engineered cells not only exhibited markedly differentiated phosphorylation responses compared to wild-type cells but also comprehensively rewired away from canonical RTK/MAPK/mTOR-Rho growth pathways toward calcium/PLC-linked signaling and cell cycle programs. These findings establish a systematic and scalable framework for targeting glycosylation-phosphorylation regulation and nominate glycan-dependent signaling nodes as potential therapeutic vulnerabilities in glycosylation-remodeled disease states.