MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex

Abstract

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.

Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani

“…Such natural modulation pathways may include protein kinases, since it was shown that HSP90 and several associated chaperones and cochaperones are the subjects of amastigote stage-specific protein phosphorylation ( 87 ). The recent finding that HSP90 and HSP70 are both substrates for MAP kinase 1 ( 88 ) supports this idea, since MAP kinase 1 or LmxMPK1 is crucial for the intracellular survival of Leishmania ( 89 ). Another kinase recently shown (A. Hombach-Barrigah, K. Bartsch, D. Smirlis, H. Rosenqvist, A. MacDonald, F. Dingli, D. Loew, G. F. Späth, N. Rachidi, M. Wiese, and J. Clos, unpublished data) to catalyze HSP90 phosphorylation is casein kinase 1.2 ( 90 , 91 ), which is crucial for promastigote growth ( 92 ) and is also found in the HSP90-containing exosome-like vesicles that are shed by Leishmania as a means for host cell immune modulation ( 42 , 93 ).…”

Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants

“…The escape variants display wild-type level temperature tolerance and in vitro proliferation rates and chemoresistance, due to a massive amplification of the casein kinase 1.2 (CK1.2) gene. We further show that HSP23, but also P23, are substrates of CK1.2, establishing yet another link 44,45 between protein kinase-mediated signalling and the chaperone machinery of Leishmania.…”

A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

“…HSP90 has been recently shown to act as a downstream client of phosphorylation-mediated cellular signalling in L. donovani [45]. Similarly, HSPs in the HSP90 foldosome complex in L. donovani were recently shown to be phosphorylated by mitogen-activated protein kinase-1 [46]. Beyond their well-established role in protein folding, HSPs and many components of the cellular protein folding machinery are found to be associated with signalling molecules including protein kinases, transcription factors and receptors and mediate their activity [47].…”