Functional Analysis of a Novel Calmodulin-Binding Protein from Arabidopsis

Reddy Calmodulin是一种关键的细胞内钙受体，被认为通过与一些关键酶和结构蛋白的相互作用和调节活性来介导许多钙调节过程。然而，对植物中与钙/钙调素复合物相互作用的蛋白质知之甚少。由于缺乏钙调素结合蛋白及其特性的相关信息，限制了在生物化学和分子水平上阐明植物钙调素的作用。本实验室最近通过筛选生物素化钙调素表达文库，从拟南芥中分离出一种新的钙调素结合蛋白（KCBP，激酶样钙调素结合蛋白）全长cDNA （4kb）。推导出的蛋白长1261个氨基酸，羧基末端区域约有340个氨基酸，与酪蛋白重链的马达结构域具有显著的序列相似性，并且含有酪蛋白典型的ATP和微管结合位点。通过分析大肠杆菌中表达的截断蛋白的钙调素结合活性，钙调素结合区域被定位到蛋白质c端附近约50个氨基酸残基的延伸。这个激酶蛋白超家族的新成员是第一个同时是钙调素结合蛋白和激酶蛋白重链的蛋白质。结合钙调素的KCBP同源物已从马铃薯中分离出来，表明该蛋白在植物中普遍存在。这两种蛋白中的钙调素结合域是高度保守的，在迄今为止从动物中发现的40多种动蛋白或动蛋白样蛋白中都不存在。我们对植物中的钙调素结合蛋白和微管运动蛋白都知之甚少。提出的研究旨在阐明这种新蛋白的功能。KCBP在不同条件下与微管的结合将被分析，使用全长或截断的蛋白表达和p从大肠杆菌中纯化。体外运动测定将用于分析KCBP的运动活性和选定的结构域，包括钙和钙调素的作用（与杜克大学的Sharyn Endow博士合作）。KCBP会在细胞周期的不同阶段免疫定位于各种植物组织和培养细胞。钙调素结合区域将通过精细制图和确定结合亲和力来分析。KCBP的表达将被操纵，组成或以组织特异性的方式，使用正义和反义结构。长期目标是了解KCBP的功能以及钙和钙调蛋白在KCBP驱动的运动活动中的调节作用。所提出的研究将增强我们对这种独特蛋白在细胞内运输中的理解，并有助于对钙调蛋白的功能有新的认识。* * *

9630782 Reddy Calmodulin, a key intracellular calcium receptor, is believed to mediate a number of calcium regulated processes by interacting with and regulating the activity of a number of key enzymes and structural proteins. However, little is known about the proteins that interact with calcium/calmodulin complexes in plants. The lack of information on the calmodulin-binding proteins and their identity has been a major limitation in plants in elucidating calmodulin action at the biochemical and molecular level. This laboratory recently isolated a full-length (4 kb) cDNA encoding a novel calmodulin-binding protein (KCBP, kinesin-like calmodulin-binding protein) from Arabidopsis by screening an expression library with biotinylated calmodulin. The deduced protein is 1261 amino acids long, with a carboxyl-terminal region domain of about 340 amino acids that has significant sequence similarity with the motor domain of kinesin heavy chains and contained putative ATP- and microtubule-binding sites typical of kinesins. By analyzing calmodulin-binding activity of truncated proteins expressed in Escherischia coli, the calmodulin-binding region mapped to a stretch of about fifty amino acid residues near the C-terminus of the protein. This new member of the kinesin superfamily is the first protein shown to be both a calmodulin-binding protein and a kinesin heavy chain. A homologue of KCBP that binds calmodulin has been isolated from potato, suggesting that this protein is ubiquitous in plants. The calmodulin-binding domain in these two proteins is highly conserved and not present in any of more than forty kinesins or kinesin-like proteins thus far characterized from animals. Very little is known about either calmodulin-binding proteins or microtubule motor proteins in plants. The proposed studies are aimed at elucidating the function of this novel protein. KCBP binding to microtubules under various conditions will be analyzed, using either full-length or truncated proteins expressed in and p urified from E. coli. In vitro motility assays will be used to analyze the motor activity of KCBP and selected domains, including the effect of calcium and calmodulin (in collaboration with Dr. Sharyn Endow of Duke University). KCBP will be immunolocalized in various plant tissues and cultured cells at different stages of the cell cycle. The calmodulin-binding domain will be analyzed by fine mapping and determination of the binding affinity. The expression of KCBP will be manipulated, constitutively or in a tissue-specific manner, using sense and antisense constructs. The long term goal is to understand the function of KCBP and the regulatory role of calcium and calmodulin in KCBP-driven motor activity. The proposed studies will enhance our understanding of this unique protein in intracellular transport and help gain new insights into calmodulin function. ***