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

钙调素（Calmodulin，CaM）是一种广泛存在于真核生物细胞内的多功能钙受体，通过与多种关键酶和结构蛋白相互作用并调节其活性，介导了一系列钙调节过程。为了鉴定与钙调素相互作用的蛋白质，从拟南芥和其他植物中分离出一种新的驱动蛋白样钙调素结合蛋白（KCBP）。 KCBP的C-末端区域含有类似于驱动蛋白和驱动蛋白样蛋白（KLP）的马达结构域。然而，KCBP是驱动蛋白超家族的新成员，其具有与马达结构域相邻的CaM结合结构域（CBD）。此外，KCBP的N-末端区域与肌球蛋白尾同源性（MyTH 4）和一些肌球蛋白中存在的talin样区域具有显著的相似性，这表明KCBP是部分驱动蛋白和部分肌球蛋白。KCBP已从双子叶植物（马铃薯和烟草）和单子叶植物（玉米）中分离出来，表明它在开花植物中普遍存在;然而，在S. cerevisiae和C.优雅 几条证据表明KCBP在植物中的有丝分裂作用。 此外，遗传研究表明，KCBP（也称为Zwichel，ZWI）对于毛状体的正常发育是必需的（Oppenheimer et al.，1997年）。用zwi突变体进行的抑制筛选强烈表明KCBP与几种其他尚未鉴定的蛋白质相互作用（Krishnakumar和Oppenheimer 1999）。 植物中微管马达蛋白的活性、定位和功能的调控机制还知之甚少。 本项目的长期目标是阐明这种新型钙调蛋白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控。 这种独特的KLP与CBD和肌球蛋白同源区域提供了一个很好的机会，研究的机制，其中Ca 2 +/CaM调节活性和精确的作用，这种蛋白质在细胞分裂和毛状体形态发生。 本项目的具体目标是：1）利用多种遗传学、生物化学和分子生物学方法分离与KCBP相互作用的蛋白质伴侣，特别是氨基末端区域，并对这些伴侣进行表征。 KCBP相互作用蛋白的分离不仅可以帮助我们了解KCBP在细胞分裂和毛状体形态发生中的功能，而且还可以深入了解某些肌球蛋白尾部区域的功能。 2)利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，组成型活性KCBP（KCBP缺乏钙调素结合结构域）的过度表达应该有助于我们了解的作用，钙/钙调素在功能的KCBP在毛形态发生和细胞分裂。动态定位的全长和不同的截断版本的KCBP在分裂细胞和毛状体的KCBP的功能和调节提供了重要的见解。 还将研究操纵胞浆钙水平对KCBP定位的影响。 3)确定在存在和不存在Ca 2 +/CaM的情况下KCBP马达结构域与CaM结合结构域的晶体结构。KCBP中的CaM结合结构域赋予Ca 2 +/CaM调节，并且生化数据表明激活的CaM与KCBP的结合影响马达上的微管结合位点。 了解KCBP马达结构域的晶体结构，在自由和钙调素结合的形式应该提供洞察的机制（S），其中Ca 2 +/钙调素调节KCBP与微管的相互作用。总之，这些研究将阐明这种独特的钙调素结合KLP的功能的各个方面。 了解钙调钙结合马达的钙调节不仅与其他农业重要植物有关（因为KCBP似乎在开花植物中无处不在），而且与最近发现钙调钙结合KLP（尽管具有非常不同的性质）的动物有关（Rogers et al.，1999年）。 鉴定与KCBP的MyTH 4和talin样区域相互作用的蛋白质也有助于了解这些结构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.