Molecular Cloning, Expression, and Characterization of Novel Calcium/calmodulin-dependent Protein Kinase from a Lower Eukaryote, Physarum polycephalum

We cloned novel calcium/calmodulin-dependent protein kinase (CaMK) from plasmodia of Physarum polycephalum. The full-length cDNA of CaMK was 1,710 nucleotides long. The amino acid sequence predicated a 42,650-dalton protein. The deduced amino acid sequence is highly homologous to that of MLCK from higher eukaryotes and lower eukaryotes including Dictyostelium MLCK. We also expressed regulatory light chain of Physarum myosin and found that the Physarum CaMK phosphorylated it in a calcium/calmodulin-dependent manner. However, in the Physarum CaMK sequence, we could not identify the consensus sequences for the calmodulin-binding domains, which are present in many MLCKs. The calmodulin-binding sequence differs from those reported previously with several CaMK and MLCK. To find such a domain in the Physarum CaMK, we designed the full-length enzyme and truncated form lacking COOH-terminus, expressed them in bacterial cells, and purified them. We try to determine the important amino acid residues in the calmodulin-binding domain. Discussion will be made about the role of regulatory domain, which was also identified in various MLCKs, in regulating the Physarum CaMK.