Systematic Phosphoproteomic Analysis of Co-Differentially Associated Phosphosites Within the WNK1 C-Terminal Disordered Region

With-No-Lysine Kinase 1 (WNK1) is an atypical serine/threonine kinase involved in ion homeostasis, blood pressure regulation, migration and proliferation. Its intrinsically disordered regions (IDRs) support dynamic phosphorylation patterns. Here, we compiled 895 human phosphoproteomic profiling datasets and 352 differential phosphoproteomic datasets to systematically characterise the phosphorylation landscape associated with WNK1. Among reported WNK1 phosphosites, S2027, S2029, and S2032, located within an IDR, were the most frequently detected. Using a one-sided Fisher’s Exact Test followed by three sequential bias-correction filters, we identified 387 phosphosites in other proteins (PsOPs) showing positive co-differential association and 34 showing negative co-differential association with these three WNK1 sites. Computational approaches and database integration identified two candidate upstream kinases (EIF2AK1 and MAPK3), eight binary interactors, and 26 candidate substrate proteins. GO enrichment revealed site-specific functional categories: S2027-associated proteins were enriched in Ras/small GTPase signaling, chromatin organization, and cell-cycle-related processes; S2029-associated proteins in chromosome organization and transcriptional regulation; and S2032-associated proteins mainly in GTPase-related molecular functions. These findings provide a phosphosite-centric resource and computationally supported candidate associations that generate testable hypotheses for further experimental investigation of WNK1-associated signaling.