Arabidopsis 2010: Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases in Arabidopsis

拟南芥基因组编码200多种富含亮氨酸的重复受体样激酶（LRR RLKs），其功能域组织与动物受体激酶相似。已知一些LRR RLKs是调节植物发育和对环境反应的信号通路中的关键元件，但这一假定的受体大家族中大多数成员的生物学功能尚不清楚。该项目将通过Gateway克隆和高通量液体机器人技术，获得所有拟南芥LRR RLKs激酶结构域的基本生化知识，用于自磷酸化活性、底物偏好和成对相互作用的体外分析。对在各种生理条件下生长的拟南芥植物分离的膜蛋白进行蛋白质组学分析，以鉴定30个LRR RLKs子集，并通过质谱法详细分析体内自磷酸化位点。选择的磷酸化位点的功能意义将使用遗传和生化方法进行检查，并在植物中选择的LRR RLKs之间的相互作用也将进行检查。有关该项目的信息，包括所研究的基因列表，可以在http://www.cals.ncsu.edu/hort_sci/faculty/clouse.html上获得。对2010年项目目标的意义——鉴定特定磷酸化位点及其功能意义将促进我们对RLK信号传导机制的理解，并提供膜定位激酶磷酸化位点的比较数据库。LRR RLK激酶结构域的全家族分析可能揭示多基因家族功能进化的新方面，体内相互作用的研究可能揭示新的异源二聚体，提示信号通路之间可能存在串扰。拟议活动的更广泛影响——本研究确定的磷酸化位点将定期发布在PlantsP数据库（http://plantsp.sdsc.edu）上，为比较研究创建一个独特的社区资源。各种用于拟南芥LRR RLKs体外和体内研究的Gateway载体标记构建物将与众多转基因植物系一起存放在拟南芥生物资源中心。鉴于已知几种LRR RLKs对植物发育的重要性，增加对该基因家族的了解可能会产生实际的农业影响。该项目将在鼓励未被充分代表的少数民族参与的环境中，为从高中生实习生到博士后科学家的各个层次提供蛋白质生物化学、质谱和拟南芥分子遗传学方面的优秀培训。

The Arabidopsis genome encodes more than 200 Leucine-Rich Repeat Receptor-like kinases (LRR RLKs) with an organization of functional domains similar to that of animal receptor kinases. Several LRR RLKs are known to be critical elements in signaling pathways regulating plant development and response to the environment, but the biological functions of most members of this large family of putative receptors remain unknown. This project will acquire fundamental biochemical knowledge of the kinase domains of all Arabidopsis LRR RLKs by using Gateway cloning and high-throughput liquid robotics for in vitro analysis of autophosphorylation activity, substrate preference and pair-wise interactions. A proteomic analysis of membrane proteins isolated from Arabidopsis plants grown under a variety of physiological conditions will be used to identify a subset of 30 LRR RLKs for detailed analysis of in vivo autophosphorylation sites by mass spectrometry. The functional significance of selected phosphorylation sites will be examined using genetic and biochemical approaches, and interaction between selected LRR RLKs in planta will also be examined. Information about the project, including a list of genes studied can be obtained at http://www.cals.ncsu.edu/hort_sci/faculty/clouse.html. Significance to 2010 Project Objectives--Identification of specific phosphorylation sites and their functional significance will advance our understanding of RLK signaling mechanisms and provide a comparative database of phosphorylation sites for membrane localized kinases. The family-wide analysis of LRR RLK kinase domains may reveal new aspects of the evolution of function in multigene families and studies of in vivo interactions may uncover novel heterodimers, suggesting possible cross-talk between signaling pathways.Broader Impact of the Proposed Activity--Phosphorylation sites determined in this study will be posted regularly on the PlantsP database (http://plantsp.sdsc.edu), creating a unique community resource for comparative study. A variety of tagged constructs in Gateway vectors for in vitro and in vivo studies of Arabidopsis LRR RLKs will be deposited in the Arabidopsis Biological Resource Center, along with numerous lines of transgenic plants. Given the known importance of several LRR RLKs to plant development, it is likely that increased knowledge of this gene family could have practical agricultural impact. The project will provide excellent training in protein biochemistry, mass spectrometry, and Arabidopsis molecular genetics at all levels from high school student interns through postdoctoral scientists in an environment that encourages the participation of underrepresented minorities.