title: 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.
authors.affiliation: Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Cayla
authors.affiliation: Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Najma
authors.initials: N
authors.lastname: Rachidi
authors.affiliation: Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Leclercq
authors.affiliation: Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schmidt-Arras
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland; Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Heidi
authors.initials: H
authors.lastname: Rosenqvist
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland.
authors.forename: Martin
authors.initials: M
authors.lastname: Wiese
authors.affiliation: Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, Unité de Parasitologie Moléculaire et Signalisation, Paris, France.
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Späth
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D025461:Feedback, Physiological; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D007898:Leishmaniasis, Visceral / Q000201:enzymology / Q000469:parasitology* / Q000473:pathology; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid
chemical_list: D049452:Green Fluorescent Proteins; D020928:Mitogen-Activated Protein Kinases