Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii
Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii
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DOI：
10.1046/j.1365-2958.2002.03026.x
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发表时间：
2002-07-01
影响因子：
3.6
通讯作者：
Kim, K
中科院分区：
文献类型：
--
作者：
Khan, F;Tang, JZ;Kim, K
顶复合体寄生虫弓形虫通过内源性复制，这是一种不同寻常的二元分裂形式。我们测试了TPK2在细胞周期调控中的作用，TPK2是CDC2细胞周期依赖蛋白激酶的同系物。用HA标签和PSTAIRE抗体进行Western印迹分析，证实了HA表位标记的TPK2(TPK2-HA-wt)在哺乳动物细胞中的表达。TPK2-HA-wt磷酸化了组蛋白H1的一个多肽，证明了TPK2是一种功能激酶。TPK2-HA-wt与哺乳动物细胞周期蛋白A、B1、D3和E共沉淀，但不能挽救裂殖酵母cdc2和酿酒酵母cdc28突变株。TPK2显性负性突变体(TPK2-HA-dN)在弓形虫速殖子中的过表达抑制了复制。表达TPK2-HA-dN的速殖子的流式细胞仪分析显示，与转TPK2-HA-wt的速殖子相比，S期细胞比例增加。TPK2-HA-wt的表达不能抑制速殖子的复制。未发现明显的G2细胞周期阻滞，表明弓形虫的细胞周期检查点不同于大多数其他真核细胞。这些数据表明，TPK2在弓形虫细胞周期中起着重要的作用，很可能是弓形虫CDC2的同源基因。
The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites. Expression of TPK2-HA-wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.