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

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 2,100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinases, P2K1. Many P2K1 clients have been confirmed by additional in vitro and in planta studies, including Integrin-Linked Kinase 4 (ILK4), for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8,000 (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 (CBL9), which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates CBL9 at the Thr196 residue through in vitro kinase assays. The expanded 8k KiC assay will be a useful tool for identification of novel substrates across diverse plant protein kinases, facilitating the exploration of previously undiscovered signaling pathways.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Gabriel Lemes Jorge  

LAB HEAD: Jay J. Thelen

PROVIDER: PXD056072 | Pride | 2025-02-26

REPOSITORIES: pride