Characterization of two T. gondii CK1 isoforms
- 作者：Donald ; Robert G.K. ; Zhong ; Tanya ; Meijer ; Laurent ; Liberator ; Paul A.
- 关键词：Casein kinase 1 ; Toxoplasma ; Cyclin-dependent protein kinase ; Purvalanol
- 刊名：Molecular & Biochemical Parasitology
- 出版年：2005
- 期刊代码：108_01666851
- 类别：bio
- 出版时间：May, 2005
- 卷：141
- 期：1
- 页码：15-27
- 文件大小：606 K
摘要
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 (TgCK1α) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1β). Enzymatically active recombinant FLAG-epitope tagged TgCK1α and TgCK1β enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1α expression was found to be cytosolic, TgCK1β was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1β confers this membrane-association. Recombinant TgCK1α and TgCK1β isoforms were also expressed in E. coli and biochemically characterized. A 38 kDa 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 TgCK1α. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1β 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1α, 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.