Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani

Dirk Schmidt-Arras; Olivier Leclercq; Pier Federico Gherardini; Manuela Helmer-Citterich; Wolfgang Faigle; Damarys Loew; Gerald F Späth

Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani

Dirk Schmidt-Arras ¹ Olivier Leclercq ¹ Pier Federico Gherardini ² Manuela Helmer-Citterich ² Wolfgang Faigle ³ Damarys Loew ³ Gerald F Späth ^{1, *}

* Corresponding author

1 Virulence Parasitaire

2 Centre for Molecular Bioinformatics

3 Laboratoire de Spectrométrie de Masse Protéomique

Abstract : The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase-substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development.

Keywords : Proteomics Life Cycle Stages Phosphotransferases Adenosine Triphosphate Animals Electrophoresis Gel Two-Dimensional Enzyme Assays Leishmania donovani Protozoan Proteins Signal Transduction

Document type :

Journal articles

Domain :

Life Sciences [q-bio] / Microbiology and Parasitology

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https://hal-pasteur.archives-ouvertes.fr/pasteur-01433562
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Submitted on : Thursday, January 12, 2017 - 5:09:53 PM
Last modification on : Thursday, January 11, 2018 - 6:21:35 AM

Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani

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Adaptation of a 2D in-gel kinase assay to trace phosphotransferase activities in the human pathogen Leishmania donovani

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