Exploring the AAK1 Phospho-signaling Landscape Through Phospho-centric Co-regulation Network.

Phosphorylation is a crucial regulatory mechanism in cellular signaling, yet phosphoproteomic data are often analyzed at the protein or gene level, neglecting site-specific regulation that defines kinase activity and signaling specificity. AAK1 (AP2-associated protein kinase 1) regulates endocytosis and contributes to tumorigenesis, but its phosphosite-level signaling network remains unclear. To address this, over 3,800 PubMed-indexed phosphoproteomic studies were curated, identifying 1,042 qualitative and 338 quantitative datasets containing Class I AAK1 phosphosites. Analysis revealed four predominant phosphosites S624, S637, S678, and T606 located outside the kinase domain. Co-phosphoregulation analysis of these potential hub phosphosites identified hundreds of phosphosites in other proteins (PsOPs) consistently co-regulated with AAK1. Their protein-level enrichment significantly associated AAK1 to endocytosis-associated functions, while limited to the current phosphosite-centric annotations, their phosphosites enriched pathways related to apoptosis, carcinogenesis, cell cycle regulation, cytoskeletal dynamics, membrane trafficking, and transcription. Notably, phosphosites in 5 predicted upstream kinases and 81 predicted AAK1 substrates showed coordinated regulation. The experimentally-validated substrate AP2M1 T156 positively co-regulated with AAK1 S624, reinforcing the robustness of this analytical strategy. This work presents the first phosphosite-resolved signaling framework of AAK1, expanding understanding of its regulatory network and providing a foundation for future studies on its upstream kinases, downstream substrates, and roles in both normal and disease contexts.