Isolation and characterization of casein kinase I from Dictyostelium discoideum

In the present study, the molecular cloning and characterization of a 49-kD a form of casein kinase (CK)I from Dictyostelium discoideum is reported. Th e predicted amino acid sequence shares 70% identity with the catalytic doma in of the mammalian delta and epsilon isoforms, Drosophila CKI epsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as de tected by Northern-blot analysis. The level of DdCKI expression did not cha nge during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Prote in expression paralleled the pattern found for the RNA. The expression of D dCKI in Escherichia coli resulted in an active enzyme that autophosphorylat ed and phosphorylated casein. Immunofluorescence assays showed thar DdCKI w as localized in the cytoplasm and nuclei of Dictyostelium cells. The lack o f disruptants of the CKI gene suggests that this protein is essential for t he vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair.