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

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.