Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor.
Mitogen-activated protein (MAP) kinases are key regulators of cellular signalling systems that mediate responses to a wide variety of extracellular stimuli and should also play a central role in developmental mechanisms of parasitic helminths. Until now, however, no MAP kinase orthologue has been characterised in a member of this parasite group. Here, we report the identification and characterisation of such a molecule, EmMPK1, from the human parasitic cestode Echinococcus multilocularis. Using a degenerative PCR approach, we isolated and completely sequenced the 1.2kb cDNA for EmMPK1 which displays significant homologies to known MAP kinases of different phylogenetic origin. EmMPK1 contains all amino acid residues which are characteristic for MAP kinases, including a conserved TEY motif which identifies the protein as a member of the ERK subfamily of MAP kinases. The corresponding gene, emmpk1 (6.9 kb), was characterised and contained 10 introns. Southern blot hybridisation studies showed that emmpk1 is present as single copy locus in E. multilocularis. Using RT-PCR analyses we demonstrated that emmpk1 is expressed in form of three different transcripts which derive from alternative splice acceptor site utilisation at intron 9. Using EmMPK1-specific antibodies in Western blot studies and immunohistochemistry, we detected the Echinococcus protein and its phosphorylated form in the larval stages metacestode and protoscolex during in vitro cultivation and during an infection of the intermediate host. EmMPK1, immunoprecipitated from Echinococcus lysate, was able to phosphorylate myelin basic protein in activity assays, indicating that it is a functionally active MAP kinase. Finally, we also show that phosphorylation of EmMPK1 is specifically induced in vitro-cultivated E. multilocularis metacestode vesicles in response to exogenous host serum and upon addition of human epidermal growth factor. These data indicate that the E. multilocularis metacestode is able to sense epidermal growth factor from the host which results in an activation of the parasite's MAP kinase cascade.
Spiliotis
Markus
M
Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany.
Konrad
Christian
C
Gelmedin
Verena
V
Tappe
Dennis
D
Brückner
Stefan
S
Mösch
Hans-Ulrich
HU
Brehm
Klaus
K
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth; D015153:Blotting, Western; D048210:Echinococcus multilocularis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics; D017238:Genes, Helminth; D006801:Humans; D007150:Immunohistochemistry; D007438:Introns; D007814:Larva; D008099:Liver / Q000469:parasitology; D020935:MAP Kinase Signaling System / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D000909:Antibodies, Helminth; D004815:Epidermal Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases