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.
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Research Article|
November 07 2012
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
Yixing Zhou;
Yixing Zhou
*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, U.S.A.
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Shilan Wu;
Shilan Wu
†Calcium Regulation 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, U.S.A.
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Huanchen Wang;
Huanchen Wang
*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, U.S.A.
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Yoichi Hayakawa;
Yoichi Hayakawa
‡Department of Applied Biological Sciences, Saga University, Honjo-1, Saga 840-8502, Japan
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Gary S. Bird;
Gary S. Bird
†Calcium Regulation 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, U.S.A.
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Stephen B. Shears
Stephen B. Shears
1
*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, U.S.A.
1To whom correspondence should be addressed (email shears@niehs.nih.gov).
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Publisher: Portland Press Ltd
Received:
April 30 2012
Revision Received:
August 21 2012
Accepted:
August 29 2012
Accepted Manuscript online:
August 29 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 448 (2): 273–283.
Article history
Received:
April 30 2012
Revision Received:
August 21 2012
Accepted:
August 29 2012
Accepted Manuscript online:
August 29 2012
Citation
Yixing Zhou, Shilan Wu, Huanchen Wang, Yoichi Hayakawa, Gary S. Bird, Stephen B. Shears; 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. Biochem J 1 December 2012; 448 (2): 273–283. doi: https://doi.org/10.1042/BJ20120730
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