Signal Transduction by Calcium Dependent Protein Kinases in Higher Plants

小行星9723539哈珀 该研究的长期目标是了解Ca2+依赖性蛋白激酶（CDPKs）在植物生长和发育中的作用。我们的方法是首先了解CDPKs如何解码和抑制Ca2+信号的结构基础，并利用这些信息开发研究其体内功能的工具。 来自拟南芥的同种型CPK-1在此用作模型CDPK。 CDPKs是独特的，因为它们的结构安排：在一个单一的多肽，激酶融合到C-末端钙调素样调节结构域（CaM-LD）。 CaM-LD含有四个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（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的基础上，分为以下3个目标：1。 检验CaM-LD始终与连接处结合（即使在不存在Ca2+的情况下）的假设，以及Ca2+触发该复合物的变化，该复合物的功能是使自身抑制剂脱离（即激活激酶）。 该方法是使用多维NMR来解决在存在和不存在Ca 2+的情况下分离的连接/CaM-LD蛋白的结构。2. 检验以下假设：连接CaM-LD与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接头）。 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. 筛选在高温（例如25 ℃）下变得不依赖Ca2+的温度敏感性激酶突变体。 该方法是引入突变，其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去稳定化更敏感。 本发明的目的是获得一种温度敏感性激酶，其可被引入转基因植物中并用于条件性激活CDPK途径。所提出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连接/CaM-LD复合物的三维结构获得的见解。 短期目标是使用这些结构来提出关于Ca2+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来测试替代模型。 细胞在细胞表面感知的许多外在信号引起生理和发育事件。活细胞中最广泛使用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将磷酸盐连接到靶蛋白，将钙信号传递到通常改变其活性的蛋白质的修饰中。CDPK如何发挥作用在阐明细胞对信号的感知方面具有根本的重要性。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。NMR可用于确定蛋白质的确切结构。将要进行的研究将阐明CDPK在其功能期间发生的结构变化。

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.