Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3
Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3
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DOI：
10.1016/j.parint.2009.08.005
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发表时间：
2009-12-01
影响因子：
1.9
通讯作者：
Akashi, Hiroomi
中科院分区：
文献类型：
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作者：
Sugi, Tatsuki;Kato, Kentaro;Akashi, Hiroomi
Ca~(2+)信号被认为在弓形虫的运动中起重要作用，包括宿主细胞的入侵和排出。最近，有报道称弓形虫在入侵过程中，其胶质小体部分发生了磷酸化。为了阐明弓形虫钙调素样域蛋白激酶在连接钙离子刺激和运动的信号通路中的作用，我们研究了弓形虫钙调素样域蛋白激酶异构体3(TgCDPKif3)。TgCDPKif3与恶性疟原虫钙依赖蛋白激酶1同源，已有报道可使恶性疟原虫胶质小体成分磷酸化。TgCDPKif3被纯化为[Gamma-P-32]ATP标记的融合蛋白，然后用磷酸酶处理去除标记。当TgCDPKif3的催化赖氨酸残基被丙氨酸取代时，磷酸化被消除。在高钙浓度下，TgCDPKif3以钙离子依赖的方式磷酸化组蛋白IIAS作为代表底物。TgCDPKif3定位于速殖子的顶端。TgCDPKif3显示胞内和胞外速殖子之间的易位。TgCDPKif3在体外可磷酸化弓形虫醛缩酶1(TgALD1)。哺乳动物细胞免疫共沉淀实验证实了TgCDPKif3与TgALD1的相互作用。提示TgCDPKif3可能通过胶体复合体成员的磷酸化参与弓形虫的运动。(C)2009爱思唯尔爱尔兰有限公司。保留所有权利。
Ca2+ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T gondii occurs during invasion. To elucidate the role of T gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca2+ stimulation and motility, we characterized T gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-de pendent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [gamma-P-32]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone IIAS as a representative substrate in a Ca2+-dependent manner at a high Ca2+ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T gondii through the phosphorylation of glideosome complex member. (C) 2009 Elsevier Ireland Ltd. All rights reserved.