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多个富含亮氨酸重复序列的受体样蛋白(LRR RLK)，其功能结构域的组织方式与动物受体蛋白类似。一些LRR RLK已被认为是调节植物发育和对环境反应的信号通路中的关键元件，但这个推测的受体大家族中的大多数成员的生物学功能仍然未知。该项目将通过使用Gateway克隆和高通量液体机器人来体外分析自动磷酸化活性、底物偏好和配对相互作用，获得所有拟南芥LRR RLK激动域的基本生化知识。从不同生理条件下生长的拟南芥植物中分离出的膜蛋白的蛋白质组学分析将被用来鉴定30个LRR RLK的子集，以便用质谱仪详细分析体内的自磷酸化位点。我们将使用遗传学和生物化学方法来研究选定的磷酸化位点的功能意义，也将研究植物中选定的LRR RLK之间的相互作用。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--识别特定的磷酸化位点及其功能意义将促进我们对RLK信号机制的理解，并提供膜上定位的激酶的磷酸化位点的比较数据库。对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，对体内相互作用的研究可能会发现新的异源二聚体，提示信号途径之间可能存在串扰。拟议活性的广泛影响--在本研究中确定的磷酸化位点将定期发布在PlantsP数据库(http://plantsp.sdsc.edu)，)上，从而创建一个独特的社区资源用于比较研究。用于拟南芥LRR RLKs体外和体内研究的Gateway载体中的各种标记构建体将与大量转基因植物一起保存在拟南芥生物资源中心。鉴于已知的几个LRR RLK对植物发育的重要性，增加对该基因家族的了解可能会对农业产生实际影响。该项目将在鼓励代表性不足的少数民族参与的环境中，在从高中生实习生到博士后科学家的所有级别提供蛋白质生物化学、质谱学和拟南芥分子遗传学方面的出色培训。

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.