Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin binding protein.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca2+-dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca2+-calmodulin. This Ca2+-calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca2+-calmodulin, suggesting an essential role for the calmodulin binding region in Ca2+-calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.类驱动蛋白钙调蛋白结合蛋白（kinesin-like calmodulin binding protein，KCBP）是驱动蛋白超家族的新成员，目前仅在植物中被发现存在。KCBP的独特之处在于其能够以钙依赖性方式与钙调蛋白相互作用。为研究KCBP与微管的相互作用，我们对拟南芥KCBP的不同区段进行了重组表达，并将纯化后的蛋白用于与微管的共沉淀实验。无论是否包含钙调蛋白结合结构域，其马达结构域均可与微管结合。携带钙调蛋白结合区域的马达结构域与微管的结合可被钙-钙调蛋白复合物抑制。若加入针对钙调蛋白结合区域的特异性抗体，则钙-钙调蛋白复合物对马达结构域与微管相互作用的调控作用会被消除。此外，缺失钙调蛋白结合区域的马达结构域与微管的结合不受钙-钙调蛋白复合物的抑制，这表明钙调蛋白结合区域在钙-钙调蛋白复合物的调控过程中发挥关键作用。针对N端尾部区域的共沉淀实验结果表明，KCBP上存在第二个微管结合位点。但KCBP的N端尾部区域与微管的相互作用不受三磷酸腺苷（ATP）的调控。上述关于KCBP与微管相互作用的研究数据，为阐明KCBP在植物体内的功能提供了新的视角。