Abstact

AAK1 and BMP2K are serine/threonine kinases traditionally known for phosphorylating AP2 during clathrin-mediated endocytosis (CME), but their broader roles remained incompletely defined. Here, using motif-guided in silico, biochemical, and phosphoproteomic screens, we identify PDLIM5 and Talin1 as direct AAK1/BMP2K substrates. Despite high kinase-domain similarity, only AAK1 promotes cell migration and potentiates focal adhesion (FA) turnover. Live-cell imaging shows that AAK1 recruitment to FAs peaks as disassembly begins. The conserved AAK1 C-terminal PDZ-binding motif mediates direct, low-affinity binding to PDLIM5, providing a plausible mechanism for localized substrate access. Dynamic analyses of phospho-mimetic and phospho-null mutants support a model in which AAK1-dependent phosphorylation promotes timely release of PDLIM5 and Talin1 during FA disassembly. These findings reveal a kinase-driven contribution to FA turnover distinct from protease- and phosphatase-based mechanisms and suggest that functional divergence between AAK1 and BMP2K may provide a strategy to modulate cell migration with reduced impact on CME.