Characterization of Proteins that Interact with CDPK Gamma

与 CDPK Gamma 相互作用的蛋白质的表征

• 批准号: 9604647
• 负责人: Alice Harmon
• 金额: --
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-15 至 2001-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604647&HistoricalAwards=false
• 关键词: Characterization; Proteins; Interact; CDPK; Gamma

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.

钙调素样结构域蛋白激酶（Calmodulin-like domain protein kinase，又称钙依赖性蛋白激酶或CDPK）是一类新的由钙直接调控的蛋白激酶家族。它们不仅含有催化结构域，还含有类似于钙调蛋白的钙结合结构域。这些酶受到微摩尔浓度Ca2+的最大刺激，并被认为在细胞对各种刺激（如光、生长调节剂、病原体攻击和环境胁迫）的反应中发挥作用。CDPKs的一个大家族存在于植物中。由于这些酶的活性受游离钙的调节，它们能够感知和转导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。很少有CDPKs的蛋白质底物被确定，并且不知道这些酶是否与调节或锚定/靶向蛋白相互作用。为了鉴定CDPK的底物或调节蛋白，或将它们定位到特定的细胞位置，进行了相互作用克隆。用3个32p标记的重组CDPKs筛选大豆cDNA表达文库，鉴定出12个编码与CDPKg相互作用蛋白的cDNA克隆。这些克隆中有8个编码已知的蛋白质，其中一些在体外被CDPKg磷酸化。其中两个相互作用的蛋白是丝氨酸乙酰转移酶（SAT）和谷胱甘肽s转移酶（GST），它们分别参与半胱氨酸合成和谷胱甘肽代谢。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。目前的证据指向谷胱甘肽（一种含半胱氨酸的三肽）代谢和蛋白质降解在细胞对应激或病原体攻击的反应中的作用。本研究的目的是表征筛选到的蛋白与CDPKg在体外的相互作用，并利用大豆细胞培养模型系统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等蛋白的相互作用在大豆细胞对胁迫或病原体攻击的反应中发挥作用的假设。这些研究是确定CDPKg在体内具体作用的重要一步，并可能为了解参与植物防御反应的细胞成分提供见解。本项目的目的是首先表征CDPKg与相互作用克隆鉴定的蛋白质的相互作用。编码已知蛋白的克隆将表达为与麦芽糖结合蛋白或其他亲和标签的融合蛋白。针对相互作用的蛋白质的抗体将被制造出来，并确定蛋白质的亚细胞位置。相互作用蛋白与重组CDPKg在蛋白印迹和溶液中的结合将被表征。底物蛋白的磷酸化将被表征，磷酸化对这些蛋白的影响将被确定。最初的工作将集中在丝氨酸乙酰转移酶，其活性在培养细胞或原生质体上受到调节，以表征CDPKg在体内的作用。细胞或原生质体中CDPKg的自磷酸化和底物蛋白的磷酸化对磷酸化变化的响应。2. 使用基于模型系统的细胞内游离钙浓度，人工诱导或响应刺激，将通过代谢标记为32P的蛋白质的免疫沉淀来测量。通过相互作用克隆鉴定的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被验证。初期工作将集中于丝氨酸乙酰转移酶。如何检测环境变化以引起植物的特定反应是植物生物学的一个大问题，但目前所知甚少。钙离子似乎在将信号从传感器传递到蛋白质合成和改变的下游效应中起着重要作用。该项目将分析钙依赖性蛋白激酶如何在生物和非生物胁迫下将磷酸盐分子附着到特定蛋白质上。这种特定蛋白质的修饰被认为是应激反应的关键步骤。这项研究的重要意义在于，它将解决植物如何感知逆境并传递信号以启动反应的基本问题。