LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5.
The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.
John von Freyend
Simona
S
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G40NR, UK.
Rosenqvist
Heidi
H
Fink
Annette
A
Melzer
Inga Maria
IM
Clos
Joachim
J
Jensen
Ole Nørregaard
ON
Wiese
Martin
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015151:Immunoblotting; D007894:Leishmania mexicana / Q000201:enzymology*; D013058:Mass Spectrometry; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D010802:Phylogeny; D025941:Protein Interaction Mapping; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D019311:Staurosporine / Q000494:pharmacology
D016054:DNA, Protozoan; D047428:Protein Kinase Inhibitors; D015800:Protozoan Proteins; D020928:Mitogen-Activated Protein Kinases; D019311:Staurosporine