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.