Characterization of two T. gondii CK1 isoforms.
Previous affinity chromatography experiments have described the unexpected binding of an isoform of casein kinase I (CK1) from Leishmania mexicana, Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii to an immobilized cyclin-dependent kinase (CDK) inhibitor (purvalanol B). In order to further evaluate CK1 as a potential anti-parasitic target, two T. gondii CK1 genes were cloned by PCR using primers derived from a putative CK1 gene fragment identified from a T. gondii EST database. The genes are predicted to encode a smaller polypeptide of 38 kDa (TgCK1alpha) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1beta). Enzymatically active recombinant FLAG-epitope tagged TgCK1alpha and TgCK1beta enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1alpha expression was found to be cytosolic, TgCK1beta was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1beta confers this membrane-association. Recombinant TgCK1alpha and TgCK1beta isoforms were also expressed in E. coli and biochemically characterized. A 38kDa native CK1 activity was partially purified from T. gondii tachyzoites by ion-exchange and hydrophobic interaction chromatography with biochemical and serological properties closely resembling those of recombinant TgCK1alpha. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1beta 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1alpha, confirming the existence of potentially exploitable structural differences between host and parasite CK1 enzymes. Since the more cell-permeable aminopurvalanol also inhibits parasite growth, these results provide further impetus to investigate inhibitors of CK1 as anti-parasitic agents.
Donald
Robert G K
RG
Department of Human/Animal Infectious Disease Research, Merck Research Laboratories, Merck & Co., P.O. Box 2000, R80Y-260, Rahway, NJ 07065-0900, USA. robert_donald@merck.com
Zhong
Tanya
T
Meijer
Laurent
L
Liberator
Paul A
PA
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D015153:Blotting, Western; D047389:Casein Kinase I / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D048128:Casein Kinase Ialpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C502582:aminopurvalanol A; C113584:purvalanol B; D047389:Casein Kinase I; D048128:Casein Kinase Ialpha; D000225:Adenine