The role of CDPKs in calcium sensing and ABA signaling

描述(由申请人提供)： 钙依赖蛋白激酶(CDPKs)是一个特殊的蛋白激酶家族，包含一个激活域和一个钙结合域。在拟南芥基因组中编码了34个CDPKs，但还没有发表任何功能丧失的cdpk突变体的表型。发起人实验室的初步结果表明，在ABA诱导的气孔关闭过程中，两个保卫细胞表达的CDPKs(CDPK-GCs)中的双突变体存在缺陷。这种cdpk-gc双突变体在ABA信号中的表型为分析CDPKs在钙敏感和ABA信号中的作用提供了一个遗传学工具。[Ca~(2+)]Cyt振荡是气孔长期关闭所必需的。CDPK-GCs在ABA信号通路中与[Ca~(2+)]细胞振荡的相对位置将通过钙成像和气孔运动分析来确定。CDPK-GCs的亚细胞定位将通过共聚焦荧光显微镜和免疫金电子显微镜来确定。通过酵母双杂交和串联亲和纯化/质谱法分离CDPK-GCs的底物和相互作用蛋白。长期目标是使用标记的CDPK底物来测试CDPKs作为[Ca2+]细胞振荡的解码器的假设。了解[Ca~(2+)]-Cyt振荡的解码机制将有助于人类包括记忆研究在内的Ca~(2+)信号转导的广泛领域。

DESCRIPTION (provided by applicant): Calcium-dependent protein kinases (CDPKs) constitute a specific protein kinase family containing both a kinase domain and a Ca2+ binding domain. There are 34 CDPKs encoded in Arabidopsis genome, but no phenotypes have been published for any loss-of-function cdpk mutants. The preliminary results of the sponsor's lab suggest that double mutants in two guard cell expressed CDPKs (CDPK-GCs) exhibit defects in ABA-induced stomatal closure. The phenotype of this cdpk-gc double mutant in ABA signaling provides a genetic tool to analyze the role of CDPKs in Ca2+ sensing and ABA signaling. [Ca2+]cyt oscillations are essential for long-term stomatal closure. The relative location of CDPK-GCs to [Ca2+]cyt oscillations in ABA signaling pathway will be determined by calcium imaging and stomatal movement analyses. The subcellular localization of CDPK-GCs will be determined by confocal fluorescent microscopy and immunogold electron microscopy. Substrates and interacting proteins of CDPK-GCs will be isolated by yeast two-hybrid assay and tandem affinity purification/mass spectrometry. The long-term goal is to use tagged CDPK substrates to test the hypothesis that CDPKs function as decoders of [Ca2+]cyt oscillations. Understanding the decoding mechanism of [Ca2+]cyt oscillation will contribute to the broad field of Ca2+ signaling including memory study in human.