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

Colorado State University, Fort Collins CO

Investigators

Abstract

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.