Molecular characterisation of MEK1/2-and MKK3/6-like mitogen-activated protein kinase kinases (MAPKK) from the fox tapeworm Echinococcus multilocularis
Molecular characterisation of MEK1/2-and MKK3/6-like mitogen-activated protein kinase kinases (MAPKK) from the fox tapeworm Echinococcus multilocularis
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DOI：
10.1016/j.ijpara.2009.10.009
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发表时间：
2010-04-01
影响因子：
4
通讯作者：
Brehm, Klaus
中科院分区：
文献类型：
--
作者：
Gelmedin, Verena;Spiliotis, Markus;Brehm, Klaus
丝裂原活化蛋白激酶激酶（MAPKKs）是进化保守的信号转导模块的重要组成部分，调节各种基本的细胞过程，以响应环境刺激。到目前为止，没有MAPKK直系同源物已在自由生活或寄生扁形虫物种的特点。在这里，我们报告了两个这样的分子在人类寄生绦虫泡状棘球蚴病的病原体多房棘球绦虫的鉴定和分子特征。利用退化PCR和3 '-和5'-快速扩增cDNA末端（RACE）技术，获得了编码两个不同的E.已鉴定并完全克隆了多房菌MAPKKs EmMKK 1和EmMKK 2。在结构上，EmMKK 1和EmMKK 2分别非常类似于来自各种脊椎动物和无脊椎动物生物的MKK 3/6-和MEK 1/2-MAPKK亚家族的成员，并且在相应位置含有MAPKK的所有催化重要残基。通过逆转录-PCR分析，EmMKK 2编码基因，emmkk 2，观察到在幼虫阶段，后囊蚴和原头节，而emmkk 1显示一个protoplastiex-specific表达模式。在酵母双杂交分析中，EmMKK 1与先前鉴定的棘球蚴MAPKK激酶EmRaf强烈相互作用，但不与ERK样MAP激酶EmMPK 1或p38样MAP激酶EmMPK 2相互作用。另一方面，EmMKK 2不仅与EmRaf和寄生虫14-3-3蛋白家族的成员相互作用，而且与EmMPK 1相互作用，这一点已通过免疫共沉淀试验得到证实。孵育在体外培养的后绦虫囊泡与Raf-和MEK-激酶的小分子抑制剂导致在一个显着的去磷酸化EmMPK 1和负面影响寄生虫的生长，但在囊泡杀死无效。综上所述，我们的结果定义EmRaf，EmMKK 2和EmMPK 1为Erk样E.本研究为进一步研究Erk样MAPK信号在寄生虫发育和干细胞功能中的作用提供了坚实的基础。(C)2009年澳大利亚寄生虫学会由爱思唯尔有限公司出版。保留所有权利。
Mitogen-activated protein kinase kinases (MAPKKs) are essential components of evolutionary conserved signalling modules that regulate a variety of fundamental cellular processes in response to environmental stimuli. To date, no MAPKK ortholog has been characterised in free-living or parasitic flatworm species. Here, we report the identification and molecular characterisation of two such molecules in the human parasitic cestode Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Using degenerative PCR approaches as well as 3'- and 5'-rapid amplification of cDNA ends (RACE), the cDNAs encoding two different E. multilocularis MAPKKs, EmMKK1 and EmMKK2, have been identified and fully cloned. Structurally, EmMKK1 and EmMKK2 closely resemble members of the MKK3/6- and the MEK1/2-MAPKK sub-families, respectively, from a variety of vertebrate and invertebrate organisms, and contain all catalytically important residues of MAPKKs at the corresponding positions. By reverse transcriptase-PCR analyses, expression of the EmMKK2-encoding gene, emmkk2, was observed in the larval stages, metacestode and protoscolex while emmkk1 displayed a protoscolex-specific expression pattern. In yeast two-hybrid analyses, EmMKK1 strongly interacted with the previously identified Echinococcus MAPKK kinase EmRaf but not with the Erk-like MAP kinase EmMPK1 or the p38-like MAP kinase EmMPK2. EmMKK2, on the other hand, not only interacted with EmRaf and a member of the parasite's 14-3-3 protein family, but also with EmMPK1, which was confirmed by co-immunoprecipitation assays. Incubation of in vitro cultivated metacestode vesicles with small-molecule inhibitors of Raf- and MEK-kinases resulted in a marked dephosphorylation of EmMPK1 and negatively affected parasite growth, but was ineffective in vesicle killing. Taken together, our results define EmRaf, EmMKK2 and EmMPK1 as the three components of the Erk-like E. multilocularis MAPK cascade module and provide a solid basis for further investigations into the role of Erk-like MAPK signalling in parasite development and stem cell function. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.