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

拟南芥 2010：拟南芥中富含亮氨酸重复受体样激酶的功能分析和磷酸化位点定位

• 批准号: 0419819
• 负责人: Steven Clouse
• 金额: $ 200万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0419819&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Functional; Analysis; Phosphorylation

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.

拟南芥基因组编码超过200种富含亮氨酸重复序列的受体样激酶（LRR RLK），其功能结构域的组织类似于动物受体激酶。已知几种LRR RLK是调节植物发育和对环境的响应的信号传导途径中的关键元件，但是这个推定受体大家族的大多数成员的生物学功能仍然未知。本项目将通过使用Gateway克隆和高通量液体机器人技术对所有拟南芥LRR RLKs的激酶结构域进行自磷酸化活性、底物偏好和成对相互作用的体外分析，获得基本的生物化学知识。将使用从在各种生理条件下生长的拟南芥植物中分离的膜蛋白的蛋白质组学分析来鉴定30个LRR RLK的子集，用于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物化学方法进行检查，并在植物中选择的LRR RLK之间的相互作用也将进行检查。 有关该项目的信息，包括研究的基因列表，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--特异性磷酸化位点及其功能意义的鉴定将促进我们对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。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揭示新的异源二聚体，提示信号转导通路之间可能的串扰。plantsp.sdsc.edu用于拟南芥LRR RLKs的体外和体内研究的Gateway载体中的多种标记构建体将与许多转基因植物系一起沿着保藏在拟南芥生物资源中心。鉴于已知几种LRR RLK对植物发育的重要性，很可能增加对该基因家族的了解会产生实际的农业影响。该项目将在鼓励代表性不足的少数群体参与的环境中，为从高中实习生到博士后科学家的各级人员提供蛋白质生物化学、质谱法和拟南芥分子遗传学方面的出色培训。