Microtubule interactions and regulation of the mitotic kinesin-like protein-1 and kinesin-like calmodulin-binding protein
Microtubules are essential for many dynamic processes occurring within eukaryotic cells including organelle and vesicular trafficking, motility of cilia and flagella, and mitosis. Microtubules operate in conjunction with the kinesin superfamily of microtubule-dependent motor proteins, which use the energy from ATP hydrolysis to "walk" along microtubule tracks, and in doing so generate force for the transport of cellular cargo and mitosis. The goal of this project was to define the microtubule interactions and regulation of two kinesin-like proteins (KLPs), the Homo sapiens mitotic kinesin-like protein-1 (HsMKLP-1) and the Arabidopsis thaliana kinesin-like calmodulin-binding protein (KCBP). Functional domains of HsMKLP-1 and KCBP were heterogeneously expressed in insect cells (HsMKLP-1) and/or E. coli (HsMKLP-1, KCBP) and used to examine the microtubule binding and ATPase activity of HsMKLP-1 and KCBP catalytic domains.
Overall, the HsMKLP-1 catalytic domain was found to operate in a similar fashion to other KLPs with respect to microtubule binding and ATP hydrolysis, but HsMKLP-1 exhibited enhanced microtubule binding of the dimer and weaker affinity for ATP that functionally distinguishes it from other KLPs. HsMKLP-1 proteins were also used to generate HsMKLP-1 specific antibodies to be used as a tool for characterizing native HsMKLP-1. To define the role of nuclear localization in regulating the activity of HsMKLP-1 during interphase, sequences directing nuclear localization of HsMKLP-1 were identified. Mutation of the nuclear localization sequence 799PNGSRKRR806 to 799PNGSRTSR806 or removal of AA's 830-856 of HsMKLP-1, which contains the nuclear localization sequence 851PKRKKP856, were sufficient to abolish nuclear localization. In the absence of a functional nuclear localization sequence HsMKLP-1 localized to microtubule plus ends, suggesting that nuclear localization serves to limit the interaction of HsMKLP-1 with the interphase microtubule array.
The KCBP catalytic domain, which contains a calmodulin-binding site, was used to determine the effect of Ca2+/calmodulin on the microtubule binding and ATPase activity of KCBP. Ca2+/calmodulin was found to inhibit the binding of KCBP to microtubules and reduced the motor's microtubule-stimulated ATPase activity, which suggests that Ca2+/calmodulin may modulate the activity of KCBP in vivo by regulating the motor's association with microtubules.