Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization.

丝裂原活化蛋白激酶（Mitogen-activated protein kinases, MAPKs）可调控真核细胞内的关键信号事件。在作为疟疾致病病原体的原生动物疟原虫属（Plasmodium）寄生虫的基因组中，已鉴定出两个与其他真核生物MAPKs具有显著同源性的激酶编码基因（mapk1、mapk2）。本研究证实，两个基因均在伯氏疟原虫（Plasmodium berghei）肝期发育过程中发生转录，并对肝期寄生虫中的PbMAPK1蛋白的表达与亚细胞定位（subcellular localization）进行了分析。对表达绿色荧光蛋白标签（green fluorescent protein, GFP）PbMAPK1的转基因寄生虫开展活细胞成像结果显示，PbMAPK1在早期裂殖体期定位于细胞核，该定位过程由C端结构域中的核定位信号介导。与之相反，在寄生虫发育的胞质体阶段以及未成熟血液期裂殖体中，可观察到PbMAPK1定位于靠近寄生虫细胞核与内陷寄生虫细胞膜的逗号/环状结构内。研究发现，PbMAPK1的定位不受以下因素影响：假定参与ATP结合的基序的完整性、假定的激活基序的完整性，以及C端结构域中预测的卷曲螺旋结构域的存在。尽管PbMAPK1基因敲除寄生虫的肝期发育未见异常，但该激酶仍可能通过其动态且具有阶段依赖性的亚细胞定位，在肝期寄生虫的裂殖生殖与芽殖生殖过程中发挥双重功能。