Calcium/calmodulin regulation of a novel microtubule motor protein from Arabidopsis

Abstract

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from other known kinesins and kinesin-like proteins (KLPs) in having a calmodulin-binding domain (CBD). This CBD, 23 amino-acid long, is located at the C-terminus adjacent to motor domain. The C-terminal motor of the KCBP, with or without CBD, binds to tubulin subunits and MTs and bundles MTs in an ATP-dependent manner. In the presence of Ca2+-CaM, the motor with CBD does not bind to tubulin and MTs and cannot bundle MTs. The KCBP lacking the CBD show no effect of Ca2+-CaM. These results indicate that Ca2+-CaM modulates the interaction of KCBP with MTs and this modulation is due to the CBD in the KCBP. When motor domain and calmodulin-binding domain are in two separate peptides, there is no Ca2+/CaM regulation. NCD (with a C-terminal motor) and DK (with an N-terminal motor) are KLPs from Drosophila and do not bind CaM. The addition of CBD to these two KLPs confers CaM-binding and similar Ca2+-CaM regulation on binding MTs. In Arabidopsis, there are six CaM-isoforms (AtCaMs). Three AtCaMs and one bovine CaM were used to study Ca2+/calmodulin modulation of KCBP interaction with MTs. All AtCaMs and bovine CaM have inhibited the binding of KCBP to MTs in a calcium-dependent manner. However, AtCaM-2 appears to be more effective in inhibiting KCBP interaction with MTs. A part of the tail domain in KCBP shows sequence similarity to tail domain of some myosins (MyTH4) and a region in talin (talin-like domain). The tail domain contains a microtubule-binding site and binds unpolymerized and polymerized actin, suggesting that KCBP may connect microtubule and actin cytoskeletal systems. The KCBP is unique in having features of actin-based motors (myosins) and microtubule based motors (kinesins). These results of calcium/CaM modulation via CBD suggest regulation of KCBP function in vivo by calcium, the first such regulation among all the known kinesins and KLPs. KCBP localizes to MT arrays including the preprophase band, the spindle apparatus and the phragmoplast. The bundling results suggest a role for KCBP in establishing these mitotic MT arrays during different stages of cell division and that Ca2+/CaM is likely regulate the formation of these MT arrays.