title: Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog.
abstract: Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.
  
  
  
  An in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.
  
  
  
  We identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.
  
  
  
  Our data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. kkato@obihiro.ac.jp.|Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. kkato@obihiro.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.|Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Sugi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.|Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takemae
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Takano
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Gong
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.|Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishiwa
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Horimoto
authors.affiliation: Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Akashi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006801:Humans; D008018:Life Cycle Stages; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014122:Toxoplasma / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014123:Toxoplasmosis / Q000469:parasitology
chemical_list: D002147:Calmodulin; D015800:Protozoan Proteins; D011494:Protein Kinases; D002118:Calcium
keywords: Calcium calmodulin-dependent protein kinase homolog; GAP45; Phosphorylation; T. gondii CaMK-related kinase; Toxoplasma gondii