泛素修饰对渗透胁迫调节子CPK4活性影响的分子机制研究

钙依赖的蛋白激酶（CPK）作为细胞Ca2+信号的感受器，在调节植物生长发育和响应环境胁迫方面具有重要作用。目前对CPK的研究集中在鉴定CPK的磷酸化底物，研究CPK基因家族成员特定的生理功能，然而对CPK自身的活性调节机制了解甚少。前期工作中，我们发现拟南芥CPK4通过下调GEF-ROP-PP2C信号来调节植物在渗透胁迫环境下的生长。为了鉴定CPK4激酶活性的调节因子，利用免疫沉淀和质谱分析，我们发现泛素连接酶CAE3能够与CPK4免疫共沉淀。CPK4并不磷酸化CAE3；相反，CAE3能够泛素化修饰CPK4，暗示CAE3可能调节CPK4蛋白的稳定性。本项目将深入研究CAE3介导的泛素化修饰对CPK4功能的调节机制。并以此为基础，利用免疫沉淀和质谱鉴定CAE3的互作蛋白，进一步发掘调节植物渗透胁迫响应的重要基因，为阐明植物应答渗透胁迫的早期信号转导机制提供有价值的线索。

Calcium-dependent protein kinases (CPKs) comprise a gene family of serine/threonine protein kinases specific to plants and some protists. CPKs act as sensors of calcium signals to mediate plant development and responses to environmental stimuli. In the past, studies on elucidating CPK's function were mainly focused on identifying their substrates and deciphering their roles in specific physiological processes through investigating the phenotype of cpk mutants and CPK overexpressing transgenic lines. However, mechanisms implicated in the in vivo regulation of CPK activity remain elusive. Previously, our work demonstrated that CPK4 down-regulates plant growth under high osmotic conditions through compromising the GEF-ROP-PP2C signal control loop. To understand how osmotic stress modulates the activity of CPK4, we performed immunoprecipitation and Mass Spectrometry analysis to identify interactors of CPK4. An E3 ligase named CAE3 (CPK4-associated E3 ligase), was identified. In-vitro purified CPK4 shows no kinase activity to CAE3, instead, CAE3 exhibits an E3 ligase activity towards CPK4 in in-vitro ubiquitination assay, suggesting that CAE3 may regulate CPK4 protein stability. The aim of this research is to elucidate the molecular mechanism of how CAE3 modulates CPK4 activity. Moreover, yeast two-hybrid and IP-MS will be performed to identify CAE3 interacting proteins that may exhibit regulatory function in plant responses to osmotic stresses. This study will contribute to the understanding of early signal transduction events in plant sensing osmotic stimuli.