Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

Abstract

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

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

“…Several studies indicate that there is a complex interplay between the stress responses and drug resistance in the Leishmania parasites [57][58][59]. Other potentially important therapeutically relevant proteins include the mitogen activated protein kinase 10 (Gene ID: LmxM.10.0200) and the cell division protein kinase 2 (Gene ID: LmxM.21.1080); the former has been reported to be stage-specifically regulated in Leishmania with its kinase activity increasing from promastigote to amastigote conversion [60] and the latter is a protein kinase that acts as a crucial regulator of cell division cycle in the parasite [61].…”

Identification and characterization of PDC1, a novel protein involved in the epigenetic cell degeneration Crippled Growth in Podospora anserina

“…This strongly suggests that activation of PaMpk1 requires additional modification(s). This is not unprecedented as some MAPK are known not to be regulated by phosphorylation of the TxY motif (Cayla et al, ). Previously, nuclear accumulation was shown to be correlated with stationary phase and CG (Kicka and Silar, ).…”

A touch of Zen: post-translational regulation of theLeishmaniastress response

“…Two other Leishmania MAPKs, MPK10 and MPK7, have been linked to stress‐regulated parasite differentiation and growth. Transgenic expression of MPK10 in L. donovani revealed a transient increase in kinase activity during the first 48 h of axenic amastigote differentiation, which is regulated by an auto‐inhibitory domain that is essential for axenic amastigote viability (Cayla et al ., ). Transgenic expression of MPK7, on the other hand, presents a defect in intracellular and axenic amastigote growth likely due to an enhanced endogenous stress response as judged by over‐phosphorylation of the translation initiation factor eIF2α and substantial reduction in de novo protein biosynthesis (Morales et al ., ; Lahav et al ., ).…”