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基于数据独立采集的磷酸化蛋白质组学全面揭示I型酪蛋白激酶在植物发育中的作用 被引量:1

Data-independent acquisition-based global phosphoproteomics reveal the diverse roles of casein kinase 1 in plant development
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摘要 I型酪蛋白激酶(CKI)是一类真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶,通过介导底物的磷酸化调节生长发育和信号转导:拟南芥蛋白AELs,是植物特异的CK1,具有多种功能,但其内源底物的准确识别和验证极大地限制了对CK1生理功能的系统阐明.本研究通过数据独立采集模式(DIA)开展的磷酸化蛋白质组学研究系统揭示了CKI的内源底物:从AELs过表达及多种突变体的幼苗中提取蛋白并进行质谱检测,与野生型比较分析获取了3985个在AELs过表达材料中丰度高于野生型(Col-0)以及在缺失突变体中丰度低于Col-0的磷酸化肽段(AEL依赖的磷酸化肽段).对应获得1032个磷酸化蛋白。进一步从磷酸化肽段中富集了8个底物识别基序并对其中4个新基序进行了实验验证,用于预测CK1的潜在候选底物,基于富集到的一个参与生殖发育的锌指转录因子,C3H17,我们通过生化和遗传分析进行了底物验证和功能鉴定,证明AEL通过介导C3H17磷酸化以增强蛋白稳定性和转录活性进而调控拟南芥的胚胎发育,基于CK1在真核生物中的高度保守性,进一步利用CK1的底物识别基序搜索了水稻、小鼠和人类的蛋白数据库,获得了远超已知数目的众多候选底物,极大地扩展了对CK1在动植物中发挥的共同和独特功能的理解,为跨物种全面揭示CK1的保守功能提供了重要线索. 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 wildtype 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.
作者 渠莉 刘默洋 郑玲丽 王旭 薛红卫 Li Qu;Moyang Liu;Lingli Zheng;Xu Wang;Hongwei Xue(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)
出处 《Science Bulletin》 SCIE EI CAS CSCD 2023年第18期2077-2093,M0004,共18页 科学通报(英文版)
基金 supported by the National Natural Science Foundation of China(91954206,31721001,and 32100588).
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