Green Judith L, Rees-Channer Roxanne R, Howell Stephen A, Martin Stephen R, Knuepfer Ellen, Taylor Helen M, Grainger Munira, Holder Anthony A
Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
J Biol Chem. 2008 Nov 7;283(45):30980-9. doi: 10.1074/jbc.M803129200. Epub 2008 Sep 3.
Calcium-dependent protein kinases (CDPKs) of Apicomplexan parasites are crucial for the survival of the parasite throughout its life cycle. CDPK1 is expressed in the asexual blood stages of the parasite, particularly late stage schizonts. We have identified two substrates of Plasmodium falciparum CDPK1: myosin A tail domain-interacting protein (MTIP) and glideosome-associated protein 45 (GAP45), both of which are components of the motor complex that generates the force required by the parasite to actively invade host cells. Indirect immunofluorescence shows that CDPK1 localizes to the periphery of P. falciparum merozoites and is therefore suitably located to act on MTIP and GAP45 at the inner membrane complex. A proportion of both GAP45 and MTIP is phosphorylated in schizonts, and we demonstrate that both proteins can be efficiently phosphorylated by CDPK1 in vitro. A primary phosphorylation of MTIP occurs at serine 47, whereas GAP45 is phosphorylated at two sites, one of which could also be detected in phosphopeptides purified from parasite lysates. Both CDPK1 activity and host cell invasion can be inhibited by the kinase inhibitor K252a, suggesting that CDPK1 is a suitable target for antimalarial drug development.
顶复门寄生虫的钙依赖性蛋白激酶（CDPKs）在寄生虫整个生命周期的存活中至关重要。CDPK1在寄生虫的无性血液阶段表达，特别是在晚期裂殖体中。我们已经鉴定出恶性疟原虫CDPK1的两个底物：肌球蛋白A尾域相互作用蛋白（MTIP）和滑行体相关蛋白45（GAP45），这两者都是产生寄生虫主动侵入宿主细胞所需力的运动复合体的组成部分。间接免疫荧光显示CDPK1定位于恶性疟原虫裂殖子的周边，因此其定位适合在内膜复合体上作用于MTIP和GAP45。在裂殖体中，GAP45和MTIP都有一定比例被磷酸化，并且我们证明这两种蛋白在体外都能被CDPK1有效磷酸化。MTIP的主要磷酸化发生在丝氨酸47处，而GAP45在两个位点被磷酸化，其中一个位点也能在从寄生虫裂解物中纯化的磷酸肽中检测到。激酶抑制剂K252a可以抑制CDPK1的活性和宿主细胞入侵，这表明CDPK1是抗疟药物开发的合适靶点。
