Characterization of Proteins that Interact with CDPK Gamma

9604647 Harmon Technology钙调素样结构域蛋白激酶(又称钙依赖蛋白激酶或CDPK)是一类新的蛋白激酶家族，直接受钙调节。它们不仅含有一个催化结构域，而且还有一个类似于钙调蛋白的钙结合结构域。这些酶最大限度地受到微摩尔浓度的钙离子的刺激，并被认为在细胞对不同刺激的反应中发挥作用，如光、生长调节剂、病原体攻击和环境应激。在植物中存在着一个CDPKs大家族。由于这些酶的活性受游离钙的调节，它们能够感知和传递由多种刺激引起的细胞内钙信号。然而，关于这些激酶在信号通路中的具体作用的信息仍然缺乏。CDPKs的蛋白质底物很少，目前还不清楚这些酶是否与调节蛋白或锚定/靶向蛋白相互作用。为了确定作为CDPK底物或调节子的蛋白质或用于将它们靶向于特定的细胞位置，进行了相互作用克隆。用3个~(32)P标记的重组CDPKs筛选大豆表达文库，鉴定了12个编码CDPKg相互作用蛋白的克隆。这些克隆中有八个编码已知的蛋白质，其中几个在体外被CDPKg磷酸化。相互作用的两个蛋白质是丝氨酸乙酰转移酶和谷胱甘肽S转移酶，它们分别参与半胱氨酸的合成和谷胱甘肽的代谢。另一种是多泛素，它是参与蛋白质降解途径的单体泛素的前体。目前的证据表明，谷胱甘肽(一种含有半胱氨酸的三肽)代谢和蛋白质降解在细胞对应激或病原体攻击的反应中发挥了作用。本研究的目的是在体外研究我们筛选的蛋白质与CDPKg之间的相互作用，并利用大豆细胞培养模型系统来研究它们在体内的相互作用和作用。我们将验证CDPKg活性及其与泛素、SAT、GST和其他蛋白质的相互作用在大豆细胞对逆境或病原体攻击的响应中发挥作用的假设。这些研究是朝着确定CDPKg在体内的特定作用迈出的重要一步，并可能为深入了解参与植物防御反应的细胞成分提供帮助。这个项目的目标是首先表征CDPKg与通过相互作用克隆确定的蛋白质的相互作用。编码已知蛋白质的克隆将被表达为与麦芽糖结合蛋白或另一个亲和标签的融合蛋白。将制造针对相互作用蛋白质的抗体，并确定蛋白质的亚细胞位置。相互作用的蛋白质与重组CDPKg在蛋白质印迹和溶液中的结合将被鉴定。将对底物蛋白质的磷酸化进行表征，并确定磷酸化对这些蛋白质的影响。最初的工作将集中在丝氨酸乙酰转移酶上，它的活性是在培养细胞或原生质体上调节的，以表征CDPKg在体内的作用。细胞或原生质体中CDPKg的自动磷酸化和底物蛋白的磷酸化，以磷酸化的方式响应变化。2.使用基于人工诱导或对刺激反应的细胞内游离钙浓度的模型系统将通过免疫沉淀代谢标记的~(32)P蛋白来测量。通过相互作用克隆确定的CDPKg的激活和底物蛋白的磷酸化参与细胞对应激或病原体攻击的反应的假设将得到检验。最初的工作将集中在丝氨酸乙酰转移酶上。如何检测环境变化以便在植物中引起特定的反应，这是植物生物学的一个大问题，目前对此知之甚少。钙离子似乎在将信号从传感器传递到蛋白质合成和变化的下游效应方面发挥着重要作用。这个项目将分析钙依赖的蛋白激酶如何将磷酸盐分子连接到特定的蛋白质上，以响应生物和非生物的压力。这种特定蛋白质的修饰被认为是逆境反应的关键步骤。这项研究很重要，因为它将解决植物如何感知逆境并传递信号以启动反应的一个基本方面。

9604647 Harmon Technical Calmodulin-like domain protein kinases (also called calcium-dependent protein kinases or CDPK) are a new family of protein kinases that are directly regulated by calcium. They contain not only a catalytic domain, but also a calcium-binding domain that is similar to calmodulin. These enzymes are maximally stimulated by micromolar concentrations of Ca2+ and are thought to play a role in cellular responses to diverse stimuli such as light, growth regulators, pathogen attack and environmental stress. A large family of CDPKs is present in plants. Since the activity of these enzymes is regulated by free calcium, they are capable of perceiving and transducing intracellular calcium signals evoked by numerous stimuli. However, information about the specific roles of these kinases in signaling pathways is still lacking. Few protein substrates of CDPKs have been identified, and it is unknown if the enzymes interact with regulatory or anchoring/targeting proteins. To identify proteins that are substrates or regulators of CDPK or serve to target them to specific cellular locations, interaction cloning was undertaken. Screening of a soybean cDNA expression library with three 32P-labeled recombinant CDPKs, identified twelve cDNA clones that encode proteins that interact with CDPKg. Eight of these clones encode known proteins, and several are phosphorylated by CDPKg in vitro. Two of the interacting proteins are serine acetyl transferase (SAT) and glutathione S-transferase (GST), which are involved in cysteine synthesis and glutathione metabolism, respectively. Another is polyubiquitin, which is the precursor of monomeric ubiquitin that is involved in protein degradation pathways. Current evidence points to roles for glutathione (a cysteine-containing tripeptide) metabolism and protein degradation in cellular responses to stress or pathogen attack. The objective of this proposal is to characterize the interaction between the proteins identified in our screen and CDPKg in vitro and t o use a soybean cell culture model system to study their interactions and roles in vivo. We will test the hypothesis that CDPKg activity and its interaction with ubiquitin, SAT, GST and other proteins play roles in the response of soybean cells to stress or pathogen attack. These studies are an important step towards identifying specific roles of CDPKg in vivo and may provide insight into cellular components involved in plant defense responses. The objectives of this project are to first characterize the interaction of CDPKg with the proteins identified by interaction cloning. Clones encoding known proteins will be expressed as a fusion proteins with maltose binding protein or another affinity tag. Antibodies to interacting proteins will be made and the subcellular location of the proteins determined. Binding of the interacting proteins to recombinant CDPKg on protein blots and in solution will be characterized. Phosphorylation of substrate proteins will be characterized, and the effect of phosphorylation on these proteins will be determined. Initial work will focus on serine acetyltransferase whose activity is regulated on cultured cells or protoplasts for characterization of the role of CDPKg in vivo. Autophosphorylation of CDPKg and phosphorylation of substrate proteins in cells or protoplasts in response to changes in by phosphorylation. 2. Use a model system based intracellular free calcium concentration induced artificially or in response to stimuli will be measured by immunoprecipitation of proteins metabolically labeled with 32P. The hypothesis that activation of CDPKg and phosphorylation of substrate proteins identified by interaction cloning are involved in cellular responses to stress or pathogen attack will be tested. Initial work will focus on serine acetyltransferase. Nontechnical How environmental changes are detected in order to evoke specific responses in plants is one of the large questions of plant biology for which there is presently little information. Calcium ions appear to have an important role in transmitting the signal from sensors to downstream effects of protein synthesis and alterations. This project will analyze how a calcium-dependent protein kinase attaches phosphate molecules to specific proteins in response to biotic and abiotic stress. This modification of specific proteins is proposed to be a critical step in the stress reponse. This research is important because it will address a fundamental aspect of how plant perceive stress situation and transmit the signal to initiate a response.