A Novel Calcium Sensor and Its Target Kinase in Arabidopsis

拟南芥中一种新型的钙离子感受器及其靶标蛋白激酶一系列胁迫因素，如干旱、寒冷、盐度等，都会严重阻碍植物的生长发育。为了在这些极端条件下生存下来，植物进化出了复杂的机制来“监控”胁迫条件，并通过改变它们的生理和发育程序来做出反应。这个项目的长期目标是了解高等植物中将环境胁迫因素与细胞反应联系起来的信号通路。几乎所有的细胞外信号，包括植物激素、光、胁迫因子和致病或共生激发子，都可以诱导钙脉冲或瞬时信号，在进一步的信号过程中充当“第二信使”。由于不同的信号通常会引起不同的和特定的细胞反应，一个有趣的问题是细胞如何区分不同刺激产生的钙信使并做出相应的反应。研究表明，“感觉”和“解释”钙离子参数的信号成分是关键。该实验室已经确定了一个新的钙离子感受器蛋白家族，它们在植物对胁迫信号的反应中发挥作用。该家族的一名成员AtCBL1对干旱、寒冷和伤害等应激因素反应强烈。另一个成员AtCBL4或SOS3是耐盐性所必需的。为了了解AtCBL1功能的分子机制，实验室取得了一个关键发现，即AtCBL1特定地与一个仅在高等植物中发现的新的蛋白激酶家族有关。这些AtCBL1相互作用的蛋白激酶(CIPK)相互之间高度相关，它们的激活域与SNF1亚家族的蛋白激酶最为相似。然而，所有的CIPK都含有一个独特的C末端非激酶域，负责与AtCBL1的相互作用。对其中一种CIPKs CIPK1的进一步研究表明，AtCBL1与CIPK1的相互作用需要微摩尔水平的钙离子，这表明钙结合改变了AtCBL1的构象并触发了与CIPK1的结合。用重组CIPK1进行的激酶分析表明，CIPK1是一种丝氨酸/苏氨酸激酶，但具有独特的辅因子偏好。例如，CIPK1高度偏好Mn2+的活性，并作为一种Mn2+结合蛋白发挥作用。该项目将结合生物化学、细胞生物学和分子遗传学方法，进一步探索AtCBL1-CIPK1复合体的功能意义。在第一个目标中，将使用生化和细胞生物学的方法来剖析CIPK1全酶的天然形式，并鉴定CIPK1的功能底物。在第二个目标中，我们将利用转基因植物和“基因敲除”突变模型的分子遗传学方法来揭示AtCBL1-CIPK1在植物生长发育过程中的功能，特别是在胁迫条件下。这项研究将建立一个新的钙信号转导范式，这将对细胞生物学特别是信号转导研究产生重大影响。

A Novel Ca2+ Sensor and Its Target Kinase in Arabidopsis A number of stressful factors such as drought, cold, salinity, can severely hamper plant growth and development. To survive these extreme conditions, plants have evolved complex mechanisms to "monitor" the stress conditions and respond by changing their physiological and developmental programs. The long term goal of this project is to understand the signaling pathways that link the environmental stress factors to cellular responses in higher plants. Almost all extracellular signals, including plant hormones, light, stress factors, and pathogenic or symbiotic elicitors, can elicit Ca2+ pulses or transients which serve as "second messengers" in further signaling processes. Because different signals often induce distinct and specific cellular responses, an interesting question is how cells distinguish the Ca2+ messengers produced by different stimuli and respond accordingly. Studies suggest that signaling components that "sense" and "interpret" the Ca2+ parameters hold the key. The laboratory has identified a new family of Ca2+ sensor proteins that play a role in plant response to stress signals. One member of the family, AtCBL1, is strongly responsive to stress factors including drought, cold, and wounding. Another member, AtCBL4 or SOS3, is required for salt tolerance. To understand the molecular mechanism for AtCBL1 function, the laboratory has made a key finding that AtCBL1 specifically associates with a family of novel protein kinases that are found only in higher plants. These AtCBL1-interacting protein kinases (CIPKs) are highly related to each other and their kinase domains are most similar to SNF1 subfamily of protein kinases. However, all CIPKs contain a unique C-terminal non-kinase domain that is responsible for interaction with AtCBL1. Further studies with one of the CIPKs, CIPK1, show that AtCBL1 interaction with CIPK1 requires micromolar levels of Ca2+, suggesting that Ca2+-binding changes the conformation of AtCBL1 and triggers association with CIPK1. Kinase assays using recombinant CIPK1 determined that CIPK1 is a serine/threonine kinase but has unique cofactor preference. For instance, CIPK1 highly prefers Mn2+ for its activity and functions as a Mn2+-binding protein. This project will further explore the functional significance of AtCBL1-CIPK1 complex using a combination of biochemical, cell biology, and molecular genetic approaches. In the first objective, biochemical and cell biology approaches will be used to dissect the native form of CIPK1 holoenzyme and identify the functional substrates for CIPK1. In the second objective, molecular genetic approaches using transgenic plant and "knockout" mutant models will be used to unravel the function of AtCBL1-CIPK1 in plant growth and developmental processes especially under stress conditions. This study will establish a new paradigm for Ca2+ signal transduction, which will have major impact on cell biology in general and on signal transduction research in particular.