Identifying Receptor Kinase Substrates Using an 8000 Peptide Kinase Client Library Enriched for Conserved Phosphorylation Sites

• DOI: 10.1016/j.mcpro.2025.100926
• Publication Date: 2025-02-08T00:07:58Z
• Authors: Daewon Kim; Gabriel Lemes Jorge; Chunhui Xu; Lingtao Su; Sung-Hwan Cho; Nagib Ahsan; Dongqin Chen; Lijuan Zhou; Marina A. Gritsenko; Mowei Zhou; Jinrong Wan; Ljiljana Pasa-Tolic; Dong Xu; Laura E. Bartley; Jay J. Thelen; Gary Stacey

In eukaryotic organisms, protein kinases regulate diverse activities and signaling pathways through phosphorylation of specific substrates. Isolating characterizing kinase substrates is vital for defining downstream pathways. The Kinase Client (KiC) assay an in vitro synthetic peptide LC-MS/MS that has enabled identification (i.e., clients) various kinases. For example, previous use a 2,100-member (2k) library identified the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional planta studies, including Integrin-Linked 4 (ILK4), which we provide evidence herein. addition, developed new KiC containing 8,000 (8k) peptides based on sites primarily from Arabidopsis thaliana datasets. 8k are enriched with conservation other angiosperm plants, paired goals representing functionally conserved usefulness screening plants. Screening active domain 177 phosphopeptides, calcineurin B-like (CBL9) G alpha subunit 1 (GPA1), functions cellular calcium signaling. We directly phosphorylates CBL9 GPA1 assays. This expanded will be useful tool identifying novel across plant kinases, ultimately facilitating exploration previously undiscovered