Signal Transduction by Calcium Dependent Protein Kinases in Higher Plants

高等植物中钙依赖性蛋白激酶的信号转导

• 批准号: 9723539
• 负责人: Jeffrey Harper
• 金额: $ 37.19万
• 依托单位: The Scripps Research Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723539&HistoricalAwards=false
• 关键词: Signal; Transduction; Calcium; Dependent; Protein

9723539 Harper The long range goal of the proposed research is to understand the role of Ca2+-Dependent Protein Kinases (CDPKs) in plant growth and development. Our approach is to first understand the structural basis for how CDPKs decode and transduce Ca2+ signals, and to use that information to develop tools for investigating their in vivo functions. Isoform CPK-1 from Arabidopsis is used here as a model CDPK. CDPKs are unique because of their structural arrangement: Within a single polypeptide, a kinase is fused to a C-terminal calmodulin-like regulatory domain (CaM-LD). The CaM-LD contains four Ca2+ -binding EF-hands, which makes CDPKs a direct target (decoder) of Ca2+ signals. Prior NSF supported research indicated that kinase activation involves intramolecular binding between the CaM-LD and the adjacent junction (autoinhibitory) domain. The current research builds on that observation and is divided into the following 3 objectives: 1. Test the hypothesis that the CaM-LD is always bound to the junction (even in the absence of Ca2+), and that Ca2+ triggers a change in this complex which functions to disengage the autoinhibitor (i.e. activate the kinase). The approach is to use multi-dimensional NMR to solve the structure of an isolated junction/CaM-LD protein in the presence and absence of Ca2+. 2. Test the hypothesis that the sequence (tether) which connects the CaM-LD to its upstream binding sequence provides an important structural constraint, (i.e. the tether is not just a simple flexible linker between two domains). The approach is to increase the length and flexibility of the tether by site specific mutagenesis and evaluate the impact of these changes on the Ca2+ activation mechanism. 3. Screen for a temperature sensitive kinase mutant which becomes Ca2+ independent at high temperatures (e.g. 25 oC). The approach is to introduce mutations which weaken the binding of the pseudosubstrate autoinhibitor to the kinase, thereby making the autoinhibitor more sensitive to therma l destabilization. The purpose is to obtain a temperature sensitive kinase which can be introduced into transgenic plants and used to conditionally activate a CDPK pathway. The primary significance of the proposed research should come from insights obtained from the 3 dimensional structures of an apo and Ca2+ loaded Junction/CaM-LD complex. The short term goal is to use these structures to propose detailed models on how Ca2+ activates a CDPK, and to test alternative models using a molecular genetic and biochemical approach. Many extrinsic signals that are perceived by cells at the cell surface signal that induce physiological and developmental events. One of most widely used signaling systems in living cells is the calcium-dependent-protein kinase CDPK in which a calcium signal results in the CDPK attaching a phosphate to a target protein transmitting the calcium signal into a modification of a protein that usually alters its activity. How the CDPK functions is of fundamental importance in elucidating the perception of signals by cells. This award will fund the collaborative research by experts in the protein kinase field and the nuclear magnetic resonance field (NMR). NMR is useful to determine the exact structure of proteins. The research to be conducted will elucidate the changes in structure of CDPK that occur during its function.

9723539 HARPER拟议的研究的远距离目标是了解Ca2+依赖性蛋白激酶（CDPK）在植物生长和发育中的作用。我们的方法是首先了解CDPK如何解码和传递Ca2+信号的结构基础，并使用该信息开发工具来研究其体内功能。 来自拟南芥的同工型CPK-1在这里用作Model CDPK。 CDPK由于其结构排列而独一无二：在单个多肽中，激酶融合到C端钙调蛋白样调节域（CAM-LLD）。 CAM-LD包含四个Ca2+结合的EF手，这使CDPK成为Ca2+信号的直接目标（解码器）。 先前的NSF支持的研究表明，激酶激活涉及CAM-LD和相邻连接（自抑制）结构域之间的分子内结合。 当前的研究以该观察结果为基础，并分为以下3个目标：1。测试CAM-LLD始终与连接点结合的假设（即使在没有Ca2+的情况下），并且CA2++ CA2+会触发该复合物的变化，这起作用可启动自动抑制剂（即激活激酶）。 该方法是使用多维NMR在存在和不存在Ca2+的情况下求解分离的连接/CAM-LD蛋白的结构。 2。检验以下假设：将CAM-LLD连接到其上游结合序列的序列（系绳）提供了重要的结构约束（即，系绳不仅是两个域之间的简单灵活链接器）。 该方法是通过特定于位置的诱变来提高系绳的长度和柔韧性，并评估这些变化对Ca2+激活机制的影响。 3.温度敏感激酶突变体的屏幕在高温下独立于Ca2+（例如25 OC）。 该方法是引入突变，从而削弱了假基底自身抑制剂与激酶的结合，从而使自身抑制剂对热稳定性更敏感。 目的是获得温度敏感的激酶，该激酶可引入转基因植物并用于有条件地激活CDPK途径。拟议的研究的主要意义应来自从Apo和Ca2+负载连接/CAM-LLD复合物的3维结构中获得的见解。 短期目标是使用这些结构提出有关CA2+如何激活CDPK的详细模型，并使用分子遗传和生化方法测试替代模型。 细胞表面信号中细胞感知的许多外部信号诱导生理和发育事件。活细胞中使用最广泛的信号传导系统之一是钙依赖性蛋白激酶CDPK，其中钙信号导致CDPK连接到磷酸盐将磷酸盐连接到靶蛋白，从而将钙信号传递到通常改变其活性的蛋白质的修饰中。 CDPK的功能在阐明细胞对信号的感知方面至关重要。该奖项将资助蛋白激酶场和核磁共振场（NMR）专家的合作研究。 NMR可用于确定蛋白质的确切结构。进行的研究将阐明其功能过程中发生的CDPK结构的变化。