Characterization of the key region and putative phosphorylation sites of EcaICE1 in its molecular interaction with the EcaHOS1 protein in Eucalyptus camaldulensis,Plant Biology

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Characterization of the key region and putative phosphorylation sites of EcaICE1 in its molecular interaction with the EcaHOS1 protein in Eucalyptus camaldulensis
Plant Biology ( IF 4.2 ) Pub Date : 2020-10-27 , DOI: 10.1111/plb.13205
L. Cheng _{1,

2,

3} , W. Zhang ₄ , J. Hu _{1,

2} , Z. Zhang _{1,

2} , Y. Liu _{1,

2} , Y. Lin _{1,

2}

Affiliation

Inducer of CBF expression 1 (ICE1), a MYC‐like bHLH transcriptional activator, plays an important role in plants under cold stress. The ubiquitination–proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of CBFs and their downstream genes under cold stress response in Arabidopsis, but knowledge of ubiquitination regulation of ICE1 by HOS1 is still limited in woody plants.
In this study, a E3 ubiquitin ligase gene EcaHOS1 were amplified from Eucalyptus camaldulensis and the protein interactions between EcaICE1 and EcaHOS1 were analysed.
Yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay results showed that EcaICE1 can interact with the EcaHOS1 protein in the nucleus and, further, the Y2H assay demonstrated that the 126–185 amino acid region at the N‐terminus of the EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 145, 158 and 184 within the key interaction region were the putative phosphorylation sites of EcaICE1, based on bioinformatics analysis, and only the substitution of serine (Ser) 158 by alanine (Ala) blocked the protein–protein interactions between EcaICE1 and EcaHOS1 based on Y2H and β‐galactosidase activity assays using site‐directed mutagenesis.
We identified Ser 158 of EcaICE1 as the key putative phosphorylation site for its interaction with the EcaHOS1 protein.

中文翻译：

EcaICE1关键区域和推测的磷酸化位点在与桉树EcaHOS1蛋白的分子相互作用中的表征

CBF表达1（ICE1）的诱导物是一种MYC样bHLH转录激活因子，在寒冷胁迫下的植物中起重要作用。拟南芥在寒冷胁迫下，由渗透反应基因1（HOS1）的高表达介导的泛素化-蛋白酶体途径可以有效诱导ICE1的降解并降低CBF及其下游基因的表达，但有关HOS1对ICE1泛素化调控的了解仍然限于木本植物。
本研究从Eucalyptus camaldulensis扩增了一个E3泛素连接酶基因EcaHOS1，并分析了EcaICE1和EcaHOS1之间的蛋白质相互作用。
酵母双杂交（Y2H）和双分子荧光互补（BiFC）分析结果表明，EcaICE1可以与细胞核中的EcaHOS1蛋白相互作用，此外，Y2H分析表明，EcaICE1可以在N-末端的126-185个氨基酸区域EcaICE1蛋白与EcaHOS1蛋白相互作用是必不可少的。此外，根据生物信息学分析，我们发现关键相互作用区内第145、158和184位的氨基酸是EcaICE1的假定磷酸化位点，只有丙氨酸（Ala）取代丝氨酸（Ser）158才能阻断EcaICE1的磷酸化。基于定点诱变的Y2H和β-半乳糖苷酶活性测定，EcaICE1和EcaHOS1之间的蛋白质相互作用。
我们确定EcaICE1的Ser 158是关键推测的磷酸化位点，因为它与EcaHOS1蛋白相互作用。

更新日期：2020-10-27

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