Phosphosite-specific co-regulation networks of MELK kinase: insights from integrative global phosphoproteomes

Maternal embryonic leucine zipper kinase (MELK) is a serine/threonine kinase frequently overexpressed in aggressive cancers, yet the precise mechanisms governing its activation and signaling specificity remain poorly understood. Here, we present the first phosphosite-resolved co-regulation atlas of MELK through integrative meta -analysis of 3,825 global human phosphoproteomics datasets. Three phosphosites-S356, S505, and S529-emerge as dominant regulatory nodes, exhibiting high detection frequency and distinct co-regulation patterns. S356 and S505 form a tightly coupled proliferative-mitotic axis controlled by convergent Mitogen-Activated Protein Kinase Kinase (MAPK), Ribosomal S6 Kinase (RSK), Calcium/Calmodulin-dependent protein Kinase (CaMK), Hippo-related, and spindle-checkpoint kinases (NIMA-related kinase 4 (NEK4), Threonine Tyrosine Kinase/Monopolar Spindle 1 Kinase (TTK/MPS1)), whereas S529 functions as a partially antagonistic stress- and polarity-responsive module. Marker of Proliferation Ki-67 (MKI67) phosphosites co-vary with all three MELK sites across virtually all proliferative contexts, establishing a direct mechanistic link between MELK activity and clinical proliferation markers. Extensive networks of co-regulated upstream kinases, phosphatases, binary interactors, and downstream substrates further reveal functional segregation: S356/S505 primarily drive cell-cycle progression and chromatin organization, while S529 integrates calcium, metabolic, and cytoskeletal polarity signals. Kaplan–Meier survival analysis across TCGA cohorts further revealed that high expression of MELK, MKI67, and the mitotic checkpoint kinase TTK consistently predicts poor overall and disease-free survival in lung adenocarcinoma and hepatocellular carcinoma, reinforcing the strong phosphodynamic coupling between MELK activity and clinical proliferation markers. By demonstrating that MELK signaling is orchestrated through modular, site-specific phosphorylation logic rather than total protein abundance, this work establishes a new paradigm for understanding and therapeutically targeting this enigmatic oncogenic kinase.