• Home
  • Search Results
  • Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.

Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.

PLoS pathogens (2014-09-19)
Mathieu Cayla, Najma Rachidi, Olivier Leclercq, Dirk Schmidt-Arras, Heidi Rosenqvist, Martin Wiese, Gerald F Späth
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrogen peroxide solution, 30 % (w/w) in H2O, contains stabilizer
Sigma-Aldrich
Hydrogen peroxide solution, contains inhibitor, 30 wt. % in H2O, ACS reagent
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Urea, ACS reagent, 99.0-100.5%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, BioUltra, for molecular biology, pH 8.0, ~0.5 M in H2O
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
SAFC
BIS-TRIS
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Urea, powder, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
MES hydrate, ≥99.5% (titration)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Sigma-Aldrich
Hydrogen peroxide solution, 50 wt. % in H2O, stabilized
Sigma-Aldrich
Thiourea, ACS reagent, ≥99.0%
Millipore
Hydrogen peroxide solution, 3%, suitable for microbiology
Supelco
Hydrogen peroxide solution, ≥30%, for trace analysis
Sigma-Aldrich
Propidium iodide solution, solution (1.0 mg/ml in water)
Sigma-Aldrich
BIS-TRIS, ≥98.0% (titration)
Sigma-Aldrich
Urea, ReagentPlus®, ≥99.5%, pellets
Sigma-Aldrich
MES monohydrate, BioXtra, ≥99.0% (T)
Sigma-Aldrich
MES hydrate, BioPerformance Certified, suitable for cell culture, ≥99.5%
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, 0.02% in DPBS (0.5 mM), sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Thiourea, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid, purified grade, ≥98.5%, powder
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Hydrogen peroxide solution, contains ~200 ppm acetanilide as stabilizer, 3 wt. % in H2O
Supelco
Urea, 8 M (after reconstitution with 16 mL high purity water)
Supelco
Hydrogen peroxide solution, 30 % (w/w), for ultratrace analysis