SPA1的激酶活性及在蓝光信号传递中识别特异底物的机理研究

Arabidopsis SPA1 protein is a key 'molecular switch' in the signal transduction system by photoreceptors, which is involved in controlling the flowering time, leaf expansion and circadian clock, et al., and many details of its functions remain unknown. As seen in our previous studies, SPA1 can interact with Cry1 and Cry2 individually at the different binding sites of protein sequence, meaning that its functions may be affected differently by Cry1 and Cry2. To indentify this hypothesis, the affection of SPA1 downstream signals by Cry1 and Cry2 is required for research. Based on bioinformatic analysis, SPA1 contains a conservative PKC-like domain at N-terminal sequence, displaying that it can transfer blue light signal as a protein kinase. Thus, in this work, a series of studies are necessary, including: 1) to examin SPA1 kinase activity through finding its substrates using proteomic methods; 2) to understand how Cry1 and Cry2 to regulate SPA1 kinase activity individually, it is required to study Cry2-SPA1 and Cry2-SPA1 interactions using Co-IP technique in cry1- or cry2- lacking mutants; and 3) to research whether these substrates directly mediate light-controlled key factors, studies that their effects on COP1 degradation and their protein-protein interactions with COP1-controlled HY5/CO transcriptors are needed. In a word, this work will contribute to elucidate furtherly the mechanism that SPA1 involves in and regulates Cry signal transduction under blue light.

拟南芥SPA1蛋白是控制光信号传递关键的 "分子开关"，参与调控花期、叶片大小以及生物钟等重要方面，其具体作用机制尚不清楚。我们前期研究显示它直接和两个蓝光受体Cry1、Cry2发生蓝光依赖的相互作用，但其互作位点完全不同。我们推测SPA1的功能可能受到了Cry1、Cry2在蓝光下的差异调控。为证实这一假设，必须调查蓝光信号通路中Cry1、Cry2分别对SPA1下游信号的影响。生物信息学分析显示SPA1 N-端具有一个保守的PKC类激酶域，意味着SPA1可能作为激酶参与对蓝光信号的调控。因此，本项目首先通过蛋白质组学策略筛选SPA1激酶底物，研究Cry1 、Cry2对SPA1激酶活性的影响,并利用反向遗传学手段调查SPA1底物与蓝光信号下游关键蛋白COP1的降解关系以及与COP1调控的重要转录因子HY5、CO等的相互作用，为阐明SPA1调控Cry介导蓝光信号传递的分子机制提供线索和依据。

拟南芥SPA1蛋白是控制光信号传递关键的"分子开关"，参与调控花期、叶片大小以及生物钟等重要方面，其具体作用机制尚不清楚。我们前期研究显示它直接和两个蓝光受体Cry1、Cry2发生蓝光依赖的相互作用，但其互作位点完全不同。据此推测SPA1的功能可能受到了Cry1、Cry2在蓝光下的差异调控。生物信息学分析显示SPA1 N-端具有一个保守的PKC类激酶域，由此推测SPA1可能作为激酶参与对蓝光信号的调控。本项目利用免疫沉淀方法(IP)分别富集Cry1、Cry2以及SPA1在黑暗和蓝光处理下的蛋白分子，然后通过磷酸化检测手段和生物质谱磷酸化鉴定方法分别比较他们在蓝光下的存在蛋白磷酸化差异。同时利用酵母双杂交的方法，筛选获得了与SPA1互作的蛋白AT5G10520。同时分析了蓝光下SPA1与蓝光信号下游关键蛋白COP1 的降解关系以及与COP1 调控的重要转录因子HY5、CO 等的相互作用，为阐明SPA1 调控Cry 介导蓝光信号传递的分子机制提供线索和依据。