Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Sci Bull (Beijing). 2023 Sep 30;68(18):2077-2093. doi: 10.1016/j.scib.2023.08.017. Epub 2023 Aug 9.
Casein kinase 1 (CK1) is serine/threonine protein kinase highly conserved among eukaryotes, and regulates multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species.
酪蛋白激酶 1（CK1）是一种在真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶，通过磷酸化靶蛋白来调节多种发育和信号事件。拟南芥早期开花 1（EL1）样（AELs）是植物特异性 CK1，具有多种功能，但鉴定和验证其底物是阐明其生理作用的主要瓶颈。在这里，我们以数据非依赖性采集模式进行了定量磷酸蛋白质组学分析，以系统地鉴定 CK1 底物。我们从过表达单个 AEL 基因（AEL1/2/3/4-OE）或缺乏 AEL 功能的幼苗（所有 ael 单突变体和两个三突变体）中提取蛋白质，以鉴定与野生型 Col-0 相比丰度有明显变化的高可信度磷酸肽。在这些中，我们选择了 3985 个在 AEL-OE 系中丰度较高或在 ael 突变体中丰度较低的磷酸肽作为 AEL 上调的磷酸肽，并定义了 1032 个磷酸蛋白。在 AEL 上调的磷酸肽中富集了 8 个 CK1 底物基序，并进行了验证，这使我们能够预测 CK1 的其他候选底物和功能。我们通过生化和遗传分析对新鉴定的底物 C3H17 进行了功能表征，C3H17 是一种 CCCH 型锌指转录因子，揭示了 AEL 促进 C3H17 蛋白稳定性和反式激活活性在调节胚胎发生中的作用。由于 CK1 在真核生物中高度保守，我们使用新鉴定的 CK1 底物基序在水稻、小鼠和人类蛋白质数据库中进行了搜索，得到了比目前已知的更多的候选底物，大大扩展了我们对 CK1 在拟南芥和人类中发挥的共同和独特功能的理解，为未来在不同物种中进行 CK1 介导的磷酸化的机制研究提供了便利。
