Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases

Calcium is a critical component in a number of plant signal transduction pathways. A new family of calcium sensors called calcineurin B-like proteins (AtCBLs) have been recently identified from Arabidopsis. These calcium sensors have been shown to interact with a family of protein kinases (CIPKs). Here we report that each individual member of AtCBL family specifically interacts with a subset of CIPKs and present structural basis for the interaction and for the specificity underlying these interactions. Although the C-terminal region of CIPKs is responsible for interaction with AtCBLs, the N-terminal region of CIPKs is also involved in determining the specificity of such interaction. We have also shown that all three EF-hand motifs in AtCBL members are required for the interaction with CIPKs. Several AtCBL members failed to interact with any of the CIPKs presented in this study, suggesting that these AtCBL members either have other CIPKs as targets or they target distinct proteins other than CIPKs. These results may provide structural basis for the functional specificity of CBL family of calcium sensors and their targets.     

Calcineurin B-like proteins in rice can bind with calcium ion and associate with the Arabidopsis CIPK family members

Arabidopsis calcineurine B-like proteins (AtCBLs) are known to sense Ca²⁺ signals induced by a variety of extracellular stimuli such as cold, drought, low K⁺ concentration, and high salinity. The Ca²⁺-bound AtCBLs then associate with and activate a group of serine/threonine protein kinases (CIPKs) to transmit the signals further downstream. In this study, we isolated three rice cDNA clones (OsCBL1, 3, and 5), which respectively encode a polypeptide very similar to the AtCBL family. Deduced amino acid sequence analysis revealed that each OsCBL contains canonical EF-hand Ca²⁺-binding motifs. In fact, our electrophoresis mobility shift assays showed that both OsCBL1 and OsCBL3 proteins purified from Escherichia coli via the GST-fusion expression system can bind with Ca²⁺ in vitro. Using the yeast two-hybrid system and pull-down assays, we also demonstrated that OsCBLs, just like AtCBLs, can interact with the Arabidopsis CIPKs. In addition, deletion analyses indicated that the C-terminal nonkinase domain of the CIPK family is responsible for the interaction with OsCBLs. Taken together, these findings strongly suggest that OsCBLs are very likely to be the rice orthologs of AtCBLs, which may be involved in the various stress responses.     

The crystal structure of plant-specific calcium-binding protein AtCBL2 in complex with the regulatory domain of AtCIPK14

Calcium signals mediate a multitude of plant responses to external stimuli. Calcineurin B-like (CBL) proteins and their target kinases, CBL-interacting protein kinases (CIPKs), represent important relays in plant calcium signaling. CBL interacts with CIPK through a conserved motif (NAF/FISL motif) within the C-terminal regulatory domain. To better understand the functional role of the CBL-CIPK system, we determined the crystal structure of AtCBL2 in complex with the regulatory domain of AtCIPK14 at 1.2 Å resolution. The NAF/FISL motif is inserted into a hydrophobic crevice within AtCBL2, accompanied by a large displacement of the helices and loop on the opposite side of the NAF/FISL motif from the C-terminal region, which shields the hydrophobic crevice in free form. Ca²⁺ are coordinated within four EF hands in AtCBL2 in bound form. This calcium coordination pattern differs from that in the structure of the SOS3-SOS2 complex previously reported. Structural comparison of the two structures shows that the recognition of CBL by CIPK is performed in a similar manner, but inherent interactions confer binding affinity and specificity.     

The Arabidopsis calcium sensor calcineurin B-like 3 inhibits the 5'-methylthioadenosine nucleosidase in a calcium-dependent manner

Calcineurin B-like (CBL) proteins represent a unique family of calcium sensors in plant cells. Sensing the calcium signals elicited by a variety of abiotic stresses, CBLs transmit the information to a group of serine/threonine protein kinases (CBL-interacting protein kinases [CIPKs]), which are currently known as the sole targets of the CBL family. Here, we report that the CBL3 member of this family has a novel interaction partner in addition to the CIPK proteins. Extensive yeast two-hybrid screenings with CBL3 as bait identified an interesting Arabidopsis (Arabidopsis thaliana) cDNA clone (named AtMTAN, for 5'-methylthioadenosine nucleosidase), which encodes a polypeptide similar to EcMTAN from Escherichia coli. Deletion analyses showed that CBL3 utilizes the different structural modules to interact with its distinct target proteins, CIPKs and AtMTAN. In vitro and in vivo analyses verified that CBL3 and AtMTAN physically associate only in the presence of Ca(2+). In addition, we empirically demonstrated that the AtMTAN protein indeed possesses the MTAN activity, which can be inhibited specifically by Ca(2+)-bound CBL3. Overall, these findings suggest that the CBL family members can relay the calcium signals in more diverse ways than previously thought. We also discuss a possible mechanism by which the CBL3-mediated calcium signaling regulates the biosynthesis of ethylene and polyamines, which are involved in plant growth and development as well as various stress responses.     

菜用大豆CIPK基因对逆境胁迫及激素的响应特征

CIPK(calcineurin B-like-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶,在植物响应逆境胁迫和激素信号转导中发挥重要作用。本研究利用大豆基因组数据库,在全基因组水平鉴定获得52个CIPK蛋白激酶。蛋白比对分析发现所有Gm CIPK含有高度保守特征性的N端激酶区、连接区和C端调控区。系统进化树分析发现大豆Gm CIPK与拟南芥、水稻CIPK分类一致,分为4个亚家族,且每个亚家族含有3个不同物种的成员,表明Gm CIPK基因的分化早于物种的分化。启动子分析表明,多数Gm CIPK基因的启动子区含有逆境和激素应答元件。组织表达分析发现,Gm CIPK基因呈现多样化的组织表达特性。进一步选取组织表达量相对较高的14个Gm CIPK进行荧光定量PCR分析,结果表明这些菜用大豆CIPK基因在不同程度上均受高温、干旱、高盐胁迫以及ABA、ACC、SA、Me JA激素的诱导表达。采用蛋白同源比对和蛋白互作在线数据库对拟南芥及大豆同源CIPK蛋白激酶与其他蛋白的互作关系进行了预测分析,发现17对同源CIPK与其他蛋白(激酶、磷酸酶、转录因子等)存在互作。本研究为菜用大豆CIPK基因的功能研究与利用奠定了基础。     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.     

CBL‐mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores

During adaptation and developmental processes cells respond through nonlinear calcium‐decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B‐like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL‐interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N‐terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII‐independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra‐ and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.     

植物钙感受器及其介导的逆境信号途径

钙信号是植物生长发育和逆境响应的重要调控因子, 是植物生理与逆境生物学研究领域中的热点之一。当植物细胞受到外界逆境刺激时, 其胞内会产生具有时空特异性的Ca2+信号变化, 这种变化首先被胞内钙感受器所感知并解码, 再由钙感受器互作蛋白将信号传递到下游, 从而激活下游早期响应基因的表达或相关离子通道的活性, 最终产生特异性逆境响应。植物细胞通过感知胞内钙信号的变化如何识别来自外界不同性质或不同强度的刺激, 是近几年植物生物学家所关注的科学问题。文章主要总结了近几年在植物钙感受器研究领域中的最新进展, 包括钙依赖蛋白激酶(CDPKs)、钙调素(CaMs)、类钙调素蛋白(CMLs)、类钙调磷酸酶B蛋白(CBLs)及其互作蛋白激酶(CIPKs)等的结构、功能及其介导的逆境信号途径, 并提供新的见解和展望。