KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM-Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca2 +/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals.
Keywords: Cyanophora paradoxa; calcium; calmodulin; kinesin; microtubules; motor proteins; phylogeny.