Signal Transduction by Calcium Dependent Protein Kinases in Higher Plants

9723539哈珀拟议研究的长期目标是了解钙依赖蛋白激酶(CDPKs)在植物生长发育中的作用。我们的方法是首先了解CDPKs如何解码和转导钙信号的结构基础，并利用这些信息开发工具来研究它们在体内的功能。本研究以拟南芥中的CPK-1亚型作为模型CDPK。CDPKs是独一无二的，因为它们的结构排列：在一个单一的多肽中，一个激酶与一个C端钙调素样调制域(CaM-LD)融合在一起。CaM-LD包含四个与钙离子结合的EF-HAND，这使得CDPKs成为钙信号的直接靶点(译码)。先前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。目前的研究建立在这一观察结果的基础上，分为以下3个目标：1.检验CaM-LD总是与连接点结合的假设(即使在没有钙的情况下)，以及钙触发这个复合体的变化，该复合体具有脱离自身抑制物(即激活激酶)的功能。该方法是使用多维核磁共振来解决分离的连接/CaM-LD蛋白在钙存在和不存在的情况下的结构。2.检验如下假设，即连接CaM-LD及其上游结合序列的序列(系链)提供了重要的结构约束(即系链不仅仅是两个结构域之间的简单柔性连接物)。方法是通过定点突变增加系绳的长度和灵活性，并评估这些变化对钙激活机制的影响。3.筛选在高温下(如25oC)不依赖于钙离子的温度敏感激酶突变体。方法是引入突变，削弱假底物自身抑制物与激酶的结合，从而使自身抑制物对L热失稳更加敏感。目的是获得一种可以导入转基因植物并用于有条件地激活CDPK途径的温度敏感蛋白激酶。拟议的研究的主要意义应该来自于从载脂蛋白和钙离子负载的Junction/CaM-LD复合体的三维结构中获得的见解。短期目标是利用这些结构提出关于钙如何激活CDPK的详细模型，并使用分子遗传学和生物化学方法测试替代模型。在细胞表面被细胞感知的许多外在信号，这些信号诱导生理和发育事件。活细胞中最广泛使用的信号系统之一是钙依赖蛋白激酶CDPK，在CDPK中，钙信号导致CDPK将磷酸连接到靶蛋白上，将钙信号传递到通常改变其活性的蛋白质的修饰上。CDPK的功能对于阐明细胞对信号的感知是至关重要的。该奖项将资助蛋白激酶领域和核磁共振领域专家的合作研究。核磁共振有助于确定蛋白质的确切结构。将进行的研究将阐明CDPK在其功能过程中发生的结构变化。

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.