A Novel Calcium Sensor and Its Target Kinase in Arabidopsis

一种新的拟南芥Ca ~（2+）传感器及其靶激酶干旱、低温、盐胁迫等胁迫因子严重阻碍植物的生长发育。为了在这些极端条件下生存，植物已经进化出复杂的机制来“监测”胁迫条件，并通过改变其生理和发育程序做出反应。该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，致病或共生诱导子，可以引发Ca 2+脉冲或瞬变，作为“第二信使”在进一步的信号传导过程。由于不同的信号往往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的刺激产生的Ca 2+信使，并相应地作出反应。研究表明，“感知”和“解释”Ca 2+参数的信号成分是关键。 该实验室已经确定了一个新的Ca 2+传感器蛋白家族，它们在植物对胁迫信号的反应中发挥作用。该家族的一个成员AtCBL 1对包括干旱、寒冷和创伤在内的应激因素反应强烈。另一个成员AtCBL 4或SOS 3是耐盐性所必需的。为了了解AtCBL 1功能的分子机制，该实验室已经取得了一项关键发现，AtCBL 1特异性地与一个仅在高等植物中发现的新型蛋白激酶家族相关。这些AtCBL 1相互作用蛋白激酶（CIPKs）彼此高度相关，并且它们的激酶结构域与蛋白激酶的SNF 1亚家族最相似。然而，所有CIPK都含有一个独特的C-末端非激酶结构域，负责与AtCBL 1相互作用。对CIPK之一CIPK 1的进一步研究表明，AtCBL 1与CIPK 1的相互作用需要微摩尔水平的Ca 2+，这表明Ca 2+结合改变了AtCBL 1的构象并触发了与CIPK 1的缔合。使用重组CIPK 1的激酶测定确定CIPK 1是丝氨酸/苏氨酸激酶，但具有独特的辅因子偏好。例如，CIPK 1由于其活性而高度偏好Mn 2+，并且作为Mn 2+结合蛋白发挥功能。 本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AtCBL 1-CIPK 1复合物的功能意义。在第一个目标中，生物化学和细胞生物学方法将用于解剖天然形式的CIPK 1全酶，并确定CIPK 1的功能底物。在第二个目标中，将使用转基因植物和“敲除”突变体模型的分子遗传学方法来阐明AtCBL 1-CIPK 1在植物生长和发育过程中的功能，特别是在胁迫条件下。本研究将为钙离子信号转导建立一个新的范式，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.