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.