Site-Specific Phosphoproteomics Uncovers Potential Regulatory Networks of NEK4 in DNA Damage Response and Cancer Progression.

NIMA-related kinase 4 (NEK4) is a serine/threonine kinase implicated in microtubule stabilization, cilia function, and DNA damage response (DDR), with emerging roles in cancer progression through context-dependent effects on proliferation, epithelial-to-mesenchymal transition (EMT), and metastasis. Despite its significance, site-specific phosphorylation dynamics of NEK4 remain underexplored. Here, we conducted a comprehensive computational phosphoproteomic analysis by curating Class-1 phosphosites from over 3800 public datasets, identifying NEK4 phosphosites, including four predominant sites (S563, S661, S461, S639) outside the kinase domain that exhibit high detection frequencies and differential regulation. Coregulation analysis revealed phosphosites in other proteins (PsOPs) that coordinate with these NEK4 sites, linking them to DDR pathways (e.g., via interactions with DNA-PK complex components), EMT signaling, microtubule organization, and mitochondrial function. Network mapping integrated predicted upstream kinases (e.g., CDK13, RPS6KA1/3), downstream substrates (e.g., MKI67, INCENP), and binary interactors (e.g., TMPO, RRP1B), highlighting NEK4's integration into cancer-associated networks involving cell cycle regulation, apoptosis, and autophagy. Functional enrichment underscored NEK4's potential in modulating genotoxic stress responses and tumorigenic reprogramming. These findings provide a phospho-centric framework for NEK4 signaling, positioning it as a therapeutic target in DDR-defective and EMT-driven cancers, and lay the groundwork for experimental validation of its site-specific roles.