Chimeric Calcium/Calmodulin-Dependent Protein Kinase Mediated Signaling in Plants

这个更新项目的重点是这个实验室长期以来对了解植物中钙/钙调蛋白介导的信号的兴趣。细胞内钙离子浓度的变化可以通过钙调素(CaM)的作用影响许多生理过程，CaM是一种普遍存在的钙结合蛋白。钙/钙调素调节的蛋白磷酸化被认为在钙介导的信号作用的放大和多样化中起着关键作用。尽管钙/钙调素依赖的蛋白磷酸化参与调控植物的许多细胞过程，但对钙/钙调素依赖的蛋白激酶及其在钙信号转导中的作用知之甚少。本实验室从发育中的花药(proc.娜塔莉。阿卡德。SCI。92：4897-4901,1995，由诺贝尔奖获得者Edwin Krebs教授传达)。CCaMK的特征是在单个多肽中存在一个激活域、一个自抑制域、一个CaM结合域和一个神经粘蛋白样钙结合域。CCaMK的独特特征之一是存在一个类似Visinin的钙结合结构域，这是一种以前未知的在激酶中存在的额外的钙感应机制(J.Biol。化学，271：8126-8132,1996；J.Biochem 121：984-990,1997)。以35S标记的CaM和丹参CaM为探针，研究了CCaMK的自磷酸化作用及其与CaM的相互作用。CCaMK对CaM(CaM Trapping)的亲和力在自动磷酸化后增加了8倍以上。这些结果表明，当CCaMK与CaM具有较高的亲和力时，它存在一种新的状态。CCaMK在花药发育过程中以阶段特异性的方式在花粉母细胞和绒毡层细胞中表达(Planta209：161-171,1999)。我们推测，CCaMK以钙/钙调素依赖的方式磷酸化这些靶细胞中的蛋白(S)，并在小孢子发生和小孢子成熟过程中触发特定阶段的变化。为了研究CCaMK的功能意义，我们获得了携带CCaMK反义结构的转基因植株。结果表明，转基因植株是雄性不育的，表明CCaMK在花药发育中起着重要的作用。在这项工作的基础上，华盛顿州立大学研究基金会已经获得了一项名为《生产雄性不育植物的组合物和方法》的美国专利，一项全球专利申请正在申请中。为了研究CCaMK的生物学功能，利用酵母双杂交系统分离了与CCaMK相互作用的蛋白编码基因。其中一个克隆(Lef-1α1)与真核细胞延伸因子-1α(EF-1α)有很高的相似性。CCaMK以钙/钙调素依赖的方式磷酸化LlEF-1α1。本实验室克隆和鉴定了一种CDPK亚型(CRPK2)，它以钙依赖但不依赖CaM的方式磷酸化Lef-1α1的多个位点。与CDPK不同，CCaMK只磷酸化一个位点(Thr-257)，该位点与CDPK的磷酸化位点不同。有趣的是，Thr-257位于Lef-1α1(J.Biol.化学，274：12001-12008,1999年)。我们建议继续这方面的研究：通过鉴定反义植物(T1、T2、T3和T4代)和获得T-DNA插入突变体，CCaMK在花药发育中的功能意义；CCaMK与其目标蛋白在花粉母细胞和绒毛细胞中的蛋白质-蛋白质相互作用；CCaMK的结构/功能关系，特别是粘蛋白样钙结合结构域在自磷酸化和底物磷酸化中的作用。更好地了解CCaMK的调节和功能将增加我们对植物中钙/钙调素介导的信号转导的了解，同时促进各个水平的教学、训练和学习。

This renewal project is focussed on this laboratory's long standing interest in understanding the calcium/calmodulin-mediated signaling in plants. Changes in intracellular calcium concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous calcium-binding protein. Calcium/CaM-regulated protein phosphorylation is believed to play a pivotal role in amplifying and diversifying the action of calcium-mediated signals. Although calcium/CaM-dependent protein phosphorylation is implicated in regulating a number of cellular processes in plants, not much is known about calcium/CaM-dependent protein kinases and their role in calcium signaling. This laboratory has cloned a chimeric calcium/CaM-dependent protein kinase (CCaMK) gene from developing anthers (Proc. Natl. Acad. Sci. 92: 4897-4901, 1995, communicated by Professor Edwin Krebs, nobel laureate). CCaMK is characterized by the presence of a kinase domain, an autoinhibitory domain, a CaM-binding domain and a neural visinin-like calcium-binding domain in a single polypeptide. One of the unique features of CCaMK is the presence of a visinin-like calcium-binding domain, an additional calcium sensing mechanism not previously known to exist in kinases (J. Biol. Chem., 271: 8126-8132, 1996; J. Biochem 121: 984-990, 1997). The effect of autophosphorylation of CCaMK and its interaction with CaM was studied by using 35S-labeled CaM and dansylated CaM as probes. The affinity of CCaMK for CaM (CaM trapping) increases over eight-fold upon autophosphorylation. These results suggest the existence of a new state of CCaMK when it has a higher affinity for CaM. CCaMK is expressed in a stage-specific manner in pollen mother cells and tapetal cells during anther development (Planta 209: 161-171, 1999). We hypothesize that CCaMK phosphorylates protein(s) in these target cells in a calcium/CaM-dependent manner and the phosphorylation triggers stage-specific changes during microsporogenesis and microspore maturation. To study the functional significance, transgenic plants carrying the antisense construct of CCaMK were produced. Results indicate that the transgenic plants are male sterile, suggesting an important role for CCaMK in anther development. Based on this work, Washington State University Research Foundation has obtained a U.S. patent entitled 'Compositions and Methods for Production of Male Sterile Plants' and a global patent application is pending. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LIEF-1 alpha 1) has high similarity with the eukaryotic elongation factor-1 alpha (EF-1 alpha). CCaMK phosphorylates LlEF-1 alpha 1 in a calcium/CaM-dependent manner. A CDPK isoform cloned and characterized in this laboratory (CRPK2) phosphorylates multiple sites of LIEF-1 alpha 1 in a calcium-dependent but CaM-independent manner. Unlike CDPK, CCaMK phosphorylates only one site (Thr-257), and this site is different from CDPK phosphorylation sites. Interestingly, Thr-257 is located in the putative tRNA-binding region of LIEF-1 alpha 1 (J. Biol. Chem., 274: 12001-12008, 1999). We propose to continue this investigation by emphasizing the following: the functional significance of CCaMK in anther development by characterizing antisense plants (T1, T2, T3 and T4 generations) and by obtaining T-DNA insertion mutants; protein-protein interaction between CCaMK and its target proteins in pollen mother cells and tapetal cells; and the structure/function relationships of CCaMK, especially the role of the visinin-like calcium-binding domain in autophosphorylation and substrate phosphorylation. A better understanding of the regulation and function of CCaMK will increase our knowledge of calcium/CaM-mediated signaling in plants while at the same time promoting teaching, training and learning at all levels.