Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, 226031, Uttar Pradesh, India.
Bernhard Nocht Institute for Tropical Medicine, D-20359, Hamburg, Germany.
Sci Rep. 2017 Aug 31;7(1):10202. doi: 10.1038/s41598-017-09725-w.
MAP kinases (MAPK) are the most downstream kinases in signal transduction cascades and regulate critical cellular activities such as cell proliferation, differentiation, mortality, stress response, and apoptosis. The Leishmania donovani MAPK1 (LdMAPK1) is involved in parasite viability and drug resistance, but its substrates have not been identified yet. Aiming to identify the possible targets(s) of LdMAPK1, we sought to isolate interacting partners by co-immunoprecipitation, gel electrophoresis and mass spectrometry. Out of fifteen analyzed protein bands, four were identified as subunits of the HSP90 foldosome complex, namely HSP 90, HSP70, STI and SGT. Western blot analysis not only confirmed that LdMAPK1 interacts with HSP70 and HSP90 but also demonstrated that MAPK1 abundance modulates their expression. The interaction is sensitive to treatment with AMTZD, a competitive ERK inhibitor. MAPK1 also displayed kinase activity with HSP90 or HSP70 as substrates. By phosphorylating HSPs in the foldosome complex, MAPK1 may regulate the stability and activity of the foldosome which in turn plays a pivotal role in the parasitic life cycle of L. donovani. Our study therefore implicates LdMAPK1 in the post-translational modification and possibly the regulation of heat shock proteins. Conversely, HSP90 and HSP70 are identified as the first substrates of LdMAPK1.
丝裂原活化蛋白激酶（MAPK）是信号转导级联反应中的最下游激酶，调节着细胞增殖、分化、死亡、应激反应和细胞凋亡等关键细胞活动。利什曼原虫 MAPK1（LdMAPK1）参与寄生虫活力和耐药性，但尚未鉴定其底物。为了鉴定 LdMAPK1 的可能靶标，我们试图通过免疫共沉淀、凝胶电泳和质谱分析来分离相互作用的伴侣。在分析的十五个蛋白质条带中，有四个被鉴定为 HSP90 折叠酶复合物的亚基，即 HSP90、HSP70、STI 和 SGT。Western blot 分析不仅证实了 LdMAPK1 与 HSP70 和 HSP90 相互作用，还表明 MAPK1 丰度调节它们的表达。这种相互作用对 ERK 抑制剂 AMTZD 的处理很敏感。MAPK1 还以 HSP90 或 HSP70 为底物显示出激酶活性。通过磷酸化折叠酶复合物中的 HSPs，MAPK1 可能调节折叠酶的稳定性和活性，而折叠酶在利什曼原虫的寄生虫生命周期中起着至关重要的作用。因此，我们的研究表明 LdMAPK1 参与了翻译后修饰，并可能调节热休克蛋白。相反，HSP90 和 HSP70 被鉴定为 LdMAPK1 的第一个底物。
