Ca 2+ ‐dependent protein kinases of Paramecium

Two genes for Ca 2+ ‐dependent protein kinases, PCaPK‐α and PCaPK‐β, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK‐α encoded 481 amino acids and that of PCaPK‐β encoded 493 amino acids, predicting molecular masses of 55 603 Da and 57 131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK‐α and PCaPK‐β were closely related to those of the Ca 2+ ‐dependent protein kinases (CDPKs) from Plasmodium , Eimeria , and several plants, and the catalytic region of the Ca 2+ /calmodulin‐dependent protein kinase family (35−48 % identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C‐terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30−34 % identity). Two Ca 2+ ‐dependent protein kinases previously characterized from Paramecium (52 kDa CaPK1, and 50 kDa CaPK‐2) are activated by Ca 2+ in the micromolar concentration range and they directly bind Ca 2+ in a 45 Ca 2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca 2+ ‐binding motifs encoded in PCaPK‐α and PCaPK‐β, and the immunological cross‐reaction of expressed cloned fragments of these genes with CaPK‐2, suggest that they encode proteins of the same family.