Authors
M Mckenzie1; R S Kirk1; T J Walker1; 1 Kingston University Discussion
Protein kinases are intracellular signalling enzymes, fundamental to cellular function. We know little about the activation and downstream functional responses of the 252 protein kinases in Schistosoma mansoni. Our research focuses on the Akt signalling pathway in S. mansoni, which in humans is regulated by phosphoinositide 3-kinase (PI3K) and plays a role in insulin signalling and transcriptional regulation. Initial investigations led to the development of several tools and methods for detecting Akt in S. mansoni. Two antibodies were found to recognise conserved phosphorylation motifs, anti-phospho Akt (mThr-308 and pTyr-315) in addition to an antibody against total Akt. Akt in schistosomules and adult worms was activated by human skin and serum components, L-arginine and insulin, and was inhibited by Akt inhibitor X and Herbimycin A. Immunoprecipitation of phosphorylated Akt from protein extracts of insulin treated adult worms and schistosomules with anti-phospho Akt (mThr-308) antibodies, demonstrated that the immunoreactive Akt protein possessed kinase activity towards the Akt substrate glycogen synthase kinase 3 (GSK-3) in both life stages. Immunohistochemistry of adult worms, using the validated anti-phospho Akt antibodies, revealed that activated Akt is located in the tegument, gynaecophoric canal and oesophagus. RNAi of adult worms has been successful in knocking down 84% of total Akt and 25% of phosphorylated Akt. 
