Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [³H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P₃ levels 9-fold. By using fluorescent Ca²⁺ probes, we determined that dGBP initially mobilized Ca²⁺ from intracellular pools; the ensuing depletion of intracellular Ca²⁺ stores by dGBP subsequently activated a Ca²⁺ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P₃ receptor almost completely eliminated mobilization of intracellular Ca²⁺ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P₄, Ins(1,4,5,6)P₄ and Ins(1,3,4,5,6)P₅. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3^ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3^ITPK1 cells did not synthesize Ins(3,4,5,6)P₄, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P₅+Ins(1,3,4)P₃→Ins(3,4,5,6)P₄+Ins(1,3,4,6)P₄] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117–28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.