title: 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.
abstract: 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 (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 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.
authors.affiliation: Inositol Signaling Section, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
authors.forename: Yixing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Shilan
authors.initials: S
authors.lastname: Wu
authors.forename: Huanchen
authors.initials: H
authors.lastname: Wang
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Gary S
authors.initials: GS
authors.lastname: Bird
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D000818:Animals; D001483:Base Sequence; D020013:Calcium Signaling; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D008954:Models, Biological; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D016207:Cytokines; D017931:DNA Primers; D029721:Drosophila Proteins; D007295:Inositol Phosphates; D019476:Insect Proteins; D011994:Recombinant Proteins; C068690:growth blocking peptide, Apanteles kariyai; D017853:Phosphotransferases (Alcohol Group Acceptor); C103909:inositol-tetrakisphosphate 1-kinase; D010738:Type C Phospholipases