Functional analysis of a calcium sensor protein/serine-threonine kinase signalling network

钙信号调节真核生物的多种生物反应和细胞过程。尽管在阐明植物钙信号的生理过程方面已经取得了相当大的进展，但大多数相关成分的身份以及在这种信号级联反应中产生特异性的机制才刚刚开始被理解。本项目旨在全面探索拟南芥钙信号系统的机制。基于最近鉴定的一组特定的丝氨酸-苏氨酸蛋白激酶（AtCIPKs）作为拟南芥钙调神经磷酸酶b样钙传感器蛋白（AtCBLs）的相互作用靶点，本项目重点研究了CBL-CIPK蛋白-蛋白质相互作用网络的功能特征及其对整合和特异性解码植物细胞中各种钙信号的贡献。提出的研究解决以下主要目标：(1)CBL和CIPK蛋白家族的表达，亚细胞定位，生化特性和相互作用特异性的彻底分子特征。(2)通过差异钙传感器/激酶复合物的形成来识别特定的生理过程。这种方法将依赖于由保留遗传方法产生的突变系的分离和详细的表型生理和遗传特征，以及酵母双杂交试验的相互作用分析。(3)利用大规模双杂交和三杂交筛选技术对CBL-CIPK复合物调控的蛋白进行鉴定和分析。

Calcium signals regulate a multitude of biological responses and cellular processes in eukaryotic organisms. Although considerable progress has been made in elucidating physiological processes involving calcium signals in plants, the identity of most of the components involved and the mechanisms generating specificity in this signalling cascades are only beginning to be understood. This project aims to comprehensively explore the mechanisms of a novel calcium signalling system from Arabidopsis. Based on the recent identification of a specific group of serine-threonine protein kinases (AtCIPKs) as interacting targets of the Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) this project is focused on the functional characterisation of the CBL-CIPK protein-protein interaction network and its contribution to integrate and specifically decode the various calcium signals in plant cells. The proposed research addresses the folowing main objectives: (1) A thorough molecular characterisation of the CBL and CIPK protein families by their expression, subcelluar localisation, biochemical properties and interaction specificy. (2) The identification of the specific physiological processes by the diffrential calcium sensor/kinase complex formation. This approach will rely on the isolation and detailed phenotypical physiological and genetic charakterisation of mutant lines generated by reserve genetic approaches as well as on interaction analyses by yeast two-hybrid assays. (3) The identification and analyses of proteins regulated by the CBL-CIPK complexes by means of large scale two- and three-hybrid screens.