#200-79 Identifying receptor kinase substrates using an 8,000 peptide kinase client library enriched for conserved phosphorylation sites

Plant protein kinases (numbering ~1000 in Arabidopsis) regulate signaling pathways via phosphorylation of specific motifs in protein substrates. As there are nearly 100,000 phosphorylation events in plants, high-throughput discovery of kinase substrates is useful for characterizing kinase specificity and defining cognate signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled the identification of protein substrates (i.e. clients) for a purified protein kinase. We developed a novel KiC peptide library containing ca. 8,000 peptides based on known Arabidopsis phosphorylation sites, enriched for phosphopeptides also found in other species or conserved based on sequence. Given the high functional conservation of these sites among angiosperms, this library will be a valuable resource for screening kinases from the plant community. Screening the 8k library with the active P2K1 kinase domain, a key receptor for extracellular ATP involved in plant stress resistance and immunity, resulted in the identification of 177 phosphopeptides, with varying efficiency. We followed up on one of these candidates, the calcineurin B-like protein (CBL9), which P2K1 phosphoryates at Thr196. The interaction and phosphorylation of CBL9, which functions in cellular calcium signaling, were further verified via bimolecular fluorescence complementation experiments and split-luciferase complementation imaging. Beyond offering insights into establishing efficient selection criteria for further confirmatory experiments, we demonstrate that the expanded 8k KiC assay is a valuable tool for identifying novel substrates for plant protein kinases, facilitating the discovery of previously unknown signaling pathways and substrate interactions.