Proximity-Based Phospho-Interactome (Prob-PhI) Characterization Reveals Distinct Signaling Activities of MEK1 and MEK2

Protein kinases play a key role in regulating cellular processes through protein phosphorylation. Comprehensive identification of kinase-specific substrates is essential for elucidating mechanisms of health and disease, yet remains a significant challenge. Here, we present the proximity-based phospho-interactome (Prob-PhI) platforma novel and streamlined method for dissecting kinase interactomes and substrate profiles. Prob-PhI utilizes the rapid biotin ligase BASU to label proteins in proximity to a kinase of interest. Phosphorylation events among these biotinylated interactors are then enriched and analyzed under conditions with and without kinase inhibition, enabling the identification of differential phosphorylation and corresponding substrates. We applied Prob-PhI to MEK1 and MEK2, central components of the mitogen-activated protein kinase (MAPK) pathway, and delineated their distinct interactomes and phosphoproteomes. Notably, functional validation revealed that MEK2, but not MEK1, specifically interacts with and phosphorylates lysosome-associated membrane glycoprotein 3 (LAMP3) at threonine 201, thereby modulating lysosomal function. This study highlights the unique substrate profiles of MEK1 and MEK2 and demonstrates the applicability of Prob-PhI in mapping kinase signaling networks.