Regulation and Functional Analysis of a Novel Calmodulin-Binding Microtubule Motor Protein From Arabidopsis

钙调素(CaM)是真核生物中普遍存在的一种多功能细胞内钙受体，通过与一些关键酶和结构蛋白相互作用并调节其活性，介导一系列钙调节过程。为了鉴定与CaM相互作用的蛋白，从拟南芥和其他植物中分离到一种新的激动素样钙调素结合蛋白(KCBP)。KCBP的C-末端含有一个运动域，类似于运动蛋白和运动蛋白样蛋白(KLP)。然而，KCBP是Kinesin超家族中的一个新成员，因为它具有与运动域相邻的CaM结合结构域(CBD)。此外，KCBP的N-末端区域与肌球蛋白尾部同源序列(MyTH4)和某些肌球蛋白中存在的Talin样区有显著的相似性，表明KCBP部分是Kinesin，部分是肌球蛋白。从包括双子叶植物(马铃薯和烟草)和单子叶植物(玉米)在内的其他三个植物系统中分离到KCBP，这表明KCBP在开花植物中普遍存在；然而，在完全测序的酿酒酵母和线虫基因组中尚未发现同源基因。有几条证据表明KCBP在植物中具有有丝分裂作用。此外，遗传学研究表明，KCBP(也称为Zwichel，Zwi)对于毛状体的正常发育是必不可少的(Oppenheimer等人，1997)。Zwi突变体的抑制子筛选强烈表明KCBP与其他几种尚未确定的蛋白质相互作用(Krishnakumar和Oppenheimer，1999)。微管马达蛋白在植物中的活性、定位和功能的调控机制知之甚少。本项目的长期目标是阐明这种新的CaM结合微管马达蛋白在细胞分裂和毛状体形态发生中的功能和调节。这种具有CBD和肌球蛋白同源区的独特的KLP为研究钙/钙调素调节该蛋白在细胞分裂和毛状体形态形成中的活性和确切作用提供了一个很好的机会。该项目的具体目标是：1)使用各种遗传、生化和分子方法分离与KCBP相互作用的蛋白质伙伴，特别是氨基末端区域，并对这些伙伴进行表征(S)。KCBP相互作用蛋白的分离不仅有助于我们了解KCBP在细胞分裂和毛状体形态形成中的功能，而且有助于我们深入了解某些肌球蛋白尾部的功能。2)用绿色荧光蛋白标签在活细胞中表达KCBP的不同区域，以阐明KCBP在细胞分裂和毛状体发育中的确切作用。例如，结构性活性KCBP(缺少CaM结合域的KCBP)的过表达应该有助于我们理解钙/CaM在KCBP在毛状体形态发生和细胞分裂中的作用。KCBP全长和不同截短版本在细胞和毛状体分裂中的动态定位将为KCBP的功能和调控提供重要的见解。细胞内钙水平的调节对KCBP定位的影响也将被研究。3)在钙/钙调素存在和不存在的情况下，用钙调素结合区测定KCBP运动区的晶体结构。KCBP中的CaM结合域参与了钙/CaM的调节，生化数据表明，激活的CaM与KCBP的结合影响了发动机上的微管结合部位。了解游离形式和CaM结合形式的KCBP运动域的晶体结构有助于深入了解钙/CaM调节KCBP与微管相互作用的机制(S)。总体而言，这些研究将阐明这种独特的CaM结合KLP功能的各个方面。了解CaM结合马达的钙调节不仅与其他农业上重要的植物有关(因为KCBP似乎普遍存在于开花植物中)，而且还与最近发现CaM结合KLP(尽管具有非常不同的性质)的动物相关(Rogers等人，1999年)。鉴定与MyTH4和KCBP的Talin样区相互作用的蛋白质也有助于理解这些区域在动物肌球蛋白中的作用。

Calmodulin (CaM), a ubiquitous multifunctional intracellular calcium receptor in all eukaryotes, mediates a number of calcium regulated processes by interacting with and regulating the activity of a number of key enzymes and structural proteins. In an effort to identify the proteins that interact with CaM, a novel kinesin-like calmodulin-binding protein (KCBP) was isolated from Arabidopsis and other plants. The C-terminal region of KCBP contains a motor domain that is similar to kinesins and kinesin-like proteins (KLPs). However, KCBP is a novel member of the kinesin superfamily in having a CaM-binding domain (CBD) adjacent to the motor domain. In addition, the N-terminal region of KCBP has significant similarities to the myosin tail homology (MyTH4) and talin-like regions present in some myosins, suggesting that the KCBP is part kinesin and part myosin. KCBP has been isolated from three other plant systems including dicots (potato and tobacco) and monocots (maize), suggesting that it is ubiquitous in flowering plants; however, homologues have not been found in the completely sequenced genomes of S. cerevisiae and C. elegans. Several lines of evidence indicate a mitotic role for KCBP in plants. In addition, genetic studies have shown that KCBP (also called Zwichel, ZWI) is essential for normal development of trichomes (Oppenheimer et al., 1997). Suppressor screens with a zwi mutant strongly suggest the interaction of KCBP with several other yet unidentified proteins (Krishnakumar and Oppenheimer 1999). Very little is known about the mechanisms that regulate the activity, localization and function of microtubule motor proteins in plants. The long-term goal of this project is to elucidate the function and regulation of this novel CaM-binding microtubule motor protein in cell division and trichome morphogenesis. This unique KLP with a CBD and myosin homology regions offers an excellent opportunity to study the mechanisms by which Ca2+/CaM regulates the activity and the precise role of this protein in cell division and trichome morphogenesis. The specific objectives of this project are: 1) To isolate protein partners that interact with the KCBP, especially the amino-terminal region, using a variety of genetic, biochemical and molecular approaches and characterize these partner(s). Isolation of KCBP interacting proteins should not only help us understand the function of KCBP in cell division and trichome morphogenesis but also provide insights into the function of the tail region in some myosins. 2) To express different regions of KCBP in live cells with green fluorescent protein tag to elucidate precise roles of KCBP in cell division and trichome development. For example, overexpression of constitutively active KCBP (KCBP lacking the CaM-binding domain) should help us understand the role of Ca2+/CaM in functioning of the KCBP in trichome morophogenesis and cell division. Dynamic localization of full-length and different truncated versions of KCBP in dividing cells and trichomes should provide important insights into the function and regulation of KCBP. The effects of manipulation of cytosolic calcium levels on the localization of KCBP will also be investigated. 3) To determine crystal structure of KCBP motor domain with the CaM-binding domain in the presence and absence of Ca2+/CaM. CaM-binding domain in KCBP confers Ca2+/CaM regulation, and biochemical data suggest that the binding of activated CaM to KCBP influences microtubule binding sites on the motor. Knowing the crystal structure of KCBP motor domain in free and CaM-bound form should provide insight into the mechanism(s) by which Ca2+/CaM regulates the interaction of KCBP with microtubules. Overall, these studies will elucidate various aspects of the function of this unique CaM-binding KLP. Understanding the calcium regulation of a CaM-binding motor will be relevant not only to other plants of agricultural importance (as KCBP appears to be ubiquitous in flowering plants) but also to animals where a CaM-binding KLP (albeit with very different properties) has been discovered recently (Rogers et al., 1999). Identification of proteins that interact with MyTH4 and talin-like regions of KCBP should also help understand the role of these domains in animal myosins.