Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G40NR, UK.
Int J Parasitol. 2010 Jul;40(8):969-78. doi: 10.1016/j.ijpara.2010.02.004. Epub 2010 Feb 21.
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.
墨西哥利什曼原虫的必需丝裂原活化蛋白激酶（MAPK）LmxMPK4 在重组表达于大肠杆菌后进行纯化时活性很低，因此不适合用于药物筛选，直到现在。我们使用大肠杆菌蛋白共表达系统鉴定了 LmxMKK5，这是一种来自墨西哥利什曼原虫的 STE7 样蛋白激酶，它可以在体外磷酸化并激活重组 LmxMPK4。LmxMKK5 由 525 个氨基酸组成，计算分子量为 55.9kDa。共表达和纯化的 LmxMPK4 在放射性激酶测定中表现出强烈的磷酸转移酶活性，并通过免疫印迹和串联质谱分析证实其在典型的 TXY MAP 激酶激活基序的苏氨酸 190 和酪氨酸 192 上磷酸化。通用蛋白激酶抑制剂星形孢菌素以剂量依赖的方式降低共表达和激活的 LmxMPK4 的磷酸转移酶活性。据我们所知，这是首次鉴定出一种必需的利什曼原虫 MAP 激酶的体外激活剂，我们的发现为开发药物筛选测定奠定了基础，以寻找针对利什曼病的新的治疗性药物小分子抑制剂。
