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DOE OSTI · 2573929

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

Kim, Daewon [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000156691451)·Jorge, Gabriel Lemes [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000335415129)·Xu, Chunhui [Univ. of Missouri, Columbia, MO (United States)]·Su, Lingtao [Univ. of Missouri, Columbia, MO (United States); Shandong University of Science and Technology, Qingdao (China)]·Cho, Sung-Hwan [Univ. of Missouri, Columbia, MO (United States); Seoul National Univ. (Korea, Republic of)] (ORCID:0000000322302037)·Ahsan, Nagib [Univ. of Oklahoma, Norman, OK (United States)]·Chen, Dongqin [Univ. of Missouri, Columbia, MO (United States); China Agricultural University, Beijing (China)] (ORCID:0000000247154491)·Zhou, Lijuan [Univ. of Missouri, Columbia, MO (United States); Nanjing Forestry Univ. (China)] (ORCID:0000000301075648)·Gritsenko, Marina A. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000199929829)·Zhou, Mowei [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Zhejiang Univ., Hangzhou (China)] (ORCID:0000000335753224)·Wan, Jinrong [Univ. of Missouri, Columbia, MO (United States)]·Pasa-Tolic, Ljiljana [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000198535457)·Xu, Dong [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000248090514)·Bartley, Laura E. [Washington State Univ., Pullman, WA (United States)] (ORCID:0000000186107551)·Thelen, Jay J. [Univ. of Missouri, Columbia, MO (United States)]·Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000159142247)
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Abstract

In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The kinase-client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including integrin-linked kinase 4, for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B–like protein and G protein alpha subunit 1, which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates calcineurin B–like protein and G protein alpha subunit 1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways.

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59 BASIC BIOLOGICAL SCIENCESArabidopsis thalianaKinase-client assayP2K1phosphopeptidesphosphoproteomicsproteomicsreceptor-like kinase

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Kim, Daewon [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000156691451), Jorge, Gabriel Lemes [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000335415129), Xu, Chunhui [Univ. of Missouri, Columbia, MO (United States)], Su, Lingtao [Univ. of Missouri, Columbia, MO (United States); Shandong University of Science and Technology, Qingdao (China)], Cho, Sung-Hwan [Univ. of Missouri, Columbia, MO (United States); Seoul National Univ. (Korea, Republic of)] (ORCID:0000000322302037), Ahsan, Nagib [Univ. of Oklahoma, Norman, OK (United States)], Chen, Dongqin [Univ. of Missouri, Columbia, MO (United States); China Agricultural University, Beijing (China)] (ORCID:0000000247154491), Zhou, Lijuan [Univ. of Missouri, Columbia, MO (United States); Nanjing Forestry Univ. (China)] (ORCID:0000000301075648), Gritsenko, Marina A. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000199929829), Zhou, Mowei [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Zhejiang Univ., Hangzhou (China)] (ORCID:0000000335753224), Wan, Jinrong [Univ. of Missouri, Columbia, MO (United States)], Pasa-Tolic, Ljiljana [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000198535457), Xu, Dong [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000248090514), Bartley, Laura E. [Washington State Univ., Pullman, WA (United States)] (ORCID:0000000186107551), Thelen, Jay J. [Univ. of Missouri, Columbia, MO (United States)], Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)] (ORCID:0000000159142247). 2025-02-07. Identifying Receptor Kinase Substrates Using an 8000 Peptide Kinase Client Library Enriched for Conserved Phosphorylation Sites. https://doi.org/10.1016/j.mcpro.2025.100926

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