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

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.

在真核生物（eukaryotic organisms）中，蛋白激酶（protein kinases）通过对特定蛋白底物的磷酸化修饰，调控多样化的蛋白活性与信号通路。分离并鉴定激酶底物，对于解析下游信号通路至关重要。激酶底物鉴定（Kinase Client, KiC）实验是一种体外合成肽液相色谱-串联质谱（LC-MS/MS）磷酸化检测体系，可用于鉴定多种蛋白激酶的蛋白底物（即Kinase Client）。例如，此前使用包含2100条肽段的（2k）肽库，成功鉴定出胞外ATP受体样激酶P2K1的底物。诸多P2K1的底物已通过额外的体外及植物体内（in planta）研究得到验证，其中整合素连接激酶4（Integrin-Linked Kinase 4, ILK4）的相关验证证据已在本文中呈现。此外，我们基于主要来自拟南芥（Arabidopsis thaliana）数据集的磷酸化位点，构建了包含8000条（8k）肽段的新型KiC肽库。该8k肽库富集了在其他被子植物中保守的磷酸化位点，其设计目标兼具两点：一是覆盖功能保守的磷酸化位点，二是可用于筛选不同植物类群的蛋白激酶。使用活性P2K1激酶结构域对该8k肽库进行筛选，共鉴定到177条磷酸化肽段，其中包括参与细胞钙信号传导的钙调神经磷酸酶B类蛋白9（calcineurin B-like protein, CBL9）。我们通过体外激酶实验验证了P2K1可直接在CBL9的苏氨酸196（Thr196）位点进行磷酸化。这款扩展后的8k KiC实验体系，将成为鉴定不同植物蛋白激酶新型底物的有力工具，有助于探索此前未被发掘的信号通路。