Signal Transduction by Calcium Dependent Protein Kinases in Higher Plants

该研究的长期目标是了解Ca2+依赖性蛋白激酶（CDPKs）在植物生长发育中的作用。我们的方法是首先了解CDPKs解码和转导Ca2+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。本文使用拟南芥的CPK-1异构体作为CDPK模型。CDPKs的独特之处在于它们的结构安排：在单个多肽中，一个激酶融合到c端钙调蛋白样调节结构域（CaM-LD）。CaM-LD含有4个Ca2+结合ef -手，这使得CDPKs成为Ca2+信号的直接靶标（解码器）。先前NSF支持的研究表明，激酶激活涉及CaM-LD和相邻连接域（自抑制）之间的分子内结合。目前的研究建立在这一观察的基础上，分为以下3个目标：1。测试CaM-LD总是与连接处结合的假设（即使在没有Ca2+的情况下），并且Ca2+触发该复合物的变化，其功能是脱离自身抑制剂（即激活激酶）。该方法是使用多维核磁共振来解决在Ca2+存在和不存在的情况下分离的结/CaM-LD蛋白的结构。2. 测试连接CaM-LD与其上游结合序列的序列（tether）提供重要的结构约束的假设（即tether不仅仅是两个结构域之间的简单柔性连接器）。该方法是通过位点特异性诱变增加系链的长度和灵活性，并评估这些变化对Ca2+激活机制的影响。3. 筛选温度敏感的激酶突变体，在高温下（例如25℃）变得不依赖于Ca2+。方法是引入突变，削弱假底物自身抑制剂与激酶的结合，从而使自身抑制剂对热不稳定更敏感。目的是获得一种温度敏感的激酶，该激酶可以引入转基因植物并用于有条件地激活CDPK途径。提出的研究的主要意义应该来自于从载脂蛋白和Ca2+负载的结/CaM-LD复合物的三维结构中获得的见解。短期目标是利用这些结构提出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。在细胞表面被细胞感知的许多外在信号，是诱导生理和发育事件的信号。活细胞中最广泛使用的信号系统之一是钙依赖性蛋白激酶CDPK，其中钙信号导致CDPK将磷酸盐附着在靶蛋白上，将钙信号传递到蛋白质的修饰中，通常会改变其活性。CDPK如何发挥作用对于阐明细胞对信号的感知至关重要。该奖项将资助蛋白激酶领域和核磁共振领域（NMR）专家的合作研究。核磁共振对确定蛋白质的确切结构很有用。该研究将阐明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.