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

钙调蛋白（Calmodulin, 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的相互作用和调节活性，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出一种新的激酶样钙调素结合蛋白（KCBP）。KCBP的c端区域包含一个类似于激酶和激酶样蛋白（KLPs）的运动结构域。然而，KCBP是激酶蛋白超家族的新成员，其cam结合结构域（CBD）与马达结构域相邻。此外，KCBP的n端区域与某些肌球蛋白中存在的肌球蛋白尾部同源区（MyTH4）和talin-like区域具有显著的相似性，表明KCBP是部分肌动蛋白和部分肌球蛋白。KCBP已从其他三种植物系统中分离出来，包括双子叶（马铃薯和烟草）和单子叶（玉米），这表明它在开花植物中普遍存在；然而，在酿酒葡萄球菌和秀丽隐杆线虫完全测序的基因组中尚未发现同源物。一些证据表明KCBP在植物有丝分裂中起作用。此外，遗传学研究表明，KCBP（也称为Zwichel， ZWI）对毛状体的正常发育至关重要（Oppenheimer et al., 1997）。zwi突变体的抑制因子筛选强烈提示KCBP与其他几种尚未确定的蛋白质相互作用（Krishnakumar和Oppenheimer 1999）。植物微管运动蛋白的活性、定位和功能调控机制尚不清楚。本项目的长期目标是阐明这种新型cam结合微管运动蛋白在细胞分裂和毛状体形态发生中的功能和调控。这种独特的具有CBD和肌球蛋白同源区域的KLP为研究Ca2+/CaM调节活性的机制以及该蛋白在细胞分裂和毛状体形态发生中的精确作用提供了极好的机会。该项目的具体目标是：1)利用各种遗传、生化和分子方法分离与KCBP相互作用的蛋白伙伴，特别是氨基末端区域，并对这些伙伴进行表征。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发生中的功能，而且有助于我们进一步了解一些肌球蛋白中尾部区域的功能。2)利用绿色荧光蛋白标签在活细胞中表达KCBP的不同区域，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（缺乏CaM结合结构域的KCBP）的过表达应该有助于我们理解Ca2+/CaM在KCBP在毛状体形态发生和细胞分裂中的作用。KCBP全长和不同截断版本在分裂细胞和毛状体中的动态定位为了解KCBP的功能和调控提供了重要的见解。胞质钙水平对KCBP定位的影响也将被研究。3)测定Ca2+/CaM存在和不存在时KCBP马达域与CaM结合域的晶体结构。KCBP中的CaM结合域赋予Ca2+/CaM调控，生化数据表明，活化的CaM与KCBP的结合影响了马达上的微管结合位点。了解自由和CaM结合形式的KCBP马达结构域的晶体结构，有助于深入了解Ca2+/CaM调节KCBP与微管相互作用的机制。总的来说，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重要的农业植物相关（因为KCBP似乎在开花植物中无处不在），而且与最近发现cam结合KLP（尽管具有非常不同的性质）的动物相关（Rogers等人，1999）。鉴定与KCBP的MyTH4和talin-like区域相互作用的蛋白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.