Abstract
Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.
Highlights
Protein kinases C (PKCs) and extracellular signal-regulated kinases/mitogen-activated protein kinases (ERKs/MAPKs) are signalling enzymes that play a critical role in regulating cellular processes, such as gene expression, the cell cycle, growth, development and differentiation, cellular motility, survival and apoptosis [1,2]
PKC/ERK signalling occurs in response to various stimuli, including ligands that bind receptor tyrosine kinases (RTKs) and G-protein coupled receptors (GPCRs) [1,2]
The p44/p42 MAPK (ERK1/2) immunoprecipitation/kinase assay kit, phorbol 12myristate 13-acetate (PMA), cell lysis buffer, RIPA buffer, lambda phosphatase, and anti-rabbit horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signalling Technology
Summary
Protein kinases C (PKCs) and extracellular signal-regulated kinases/mitogen-activated protein kinases (ERKs/MAPKs) are signalling enzymes that play a critical role in regulating cellular processes, such as gene expression, the cell cycle, growth, development and differentiation, cellular motility, survival and apoptosis [1,2]. PKC/ERK signalling occurs in response to various stimuli, including ligands that bind receptor tyrosine kinases (RTKs) and G-protein coupled receptors (GPCRs) [1,2]. Putative PKCs and ERKs exist in kinomes of the blood flukes Schistosoma mansoni [3,4], S. japonicum [5] and S. haematobium [6]. These parasites cause human schistosomiasis, a neglected tropical disease (NTD) characterised by inflammatory granulomatous reactions in the host organs that occur in response to entrapped eggs from adult female worms [7].
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