Molecular characterisation of MEK1/2- and MKK3/6-like mitogen-activated protein kinase kinases (MAPKK) from the fox tapeworm Echinococcus multilocularis.
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 de-phosphorylation 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.
Gelmedin
Verena
V
University of Würzburg, Institute of Hygiene and Microbiology, Würzburg, Germany.
Spiliotis
Markus
M
Brehm
Klaus
K
D000818:Animals; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D048210:Echinococcus multilocularis / Q000201:enzymology*; D020869:Gene Expression Profiling; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism*; D047468:Immunoprecipitation; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D020929:Mitogen-Activated Protein Kinase Kinases