2. Academic Validation
  3. Proximity-Based Phospho-Interactome (Prob-PhI) Characterization Reveals Distinct Signaling Activities of MEK1 and MEK2

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

  • Anal Chem. 2026 Feb 10;98(5):3688-3698. doi: 10.1021/acs.analchem.5c05557.
Ying Wang 1 2 3,  Ping Xiao 3 4,  Ligang Fan 5,  Yilin Pan 3,  Haiying Ma 3,  Rui Wang 6,  Cuixiang Xu 1 2,  Liang Zhang 3 7
Affiliations

Affiliations

  • 1 Shaanxi Provincial Key Laboratory of Infection and Immune Diseases, Shaanxi Provincial People's Hospital, Xi'an 710068, China.
  • 2 Shaanxi Engineering Research Center of Cell Immunology, Shaanxi Provincial People's Hospital, Xi'an 710068, China.
  • 3 Department of Biomedical Sciences, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong China.
  • 4 College of Biological Science and Food Engineering, Southwest Forestry University, Kunming 650224, China.
  • 5 Ministry of Education Key Laboratory of Resource Biology and Biotechnology in Western China; Shaanxi Provincial Key Laboratory of Biotechnology; School of Medicine, Northwest University, Xi'an 710069, China.
  • 6 Shenzhen Bay Laboratory, PKU-Princeton Research Center for Drug Development, 5F No.9 Duxue Rd. Nanshan District, Shenzhen 518000, China.
  • 7 Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China.
PMID: 41611650 DOI: 10.1021/acs.analchem.5c05557
Abstract

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.

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