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Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells

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Inositol Phosphates and Lipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 645))

Abstract

Lipid-derived inositol phosphates (InsPs) comprise a family of second messengers that arise through the action of six classes of InsP kinases, generally referred to as IPKs. Genetic studies have indicated that InsPs play critical roles in embryonic development, but the mechanisms of action for InsPs in mammalian cellular function are largely unknown. This chapter outlines a method for manipulating cellular InsP profiles through the coexpression of a constitutively active G protein and various IPKs. It provides a mechanism by which the metabolism of a variety of InsPs can be upregulated, enabling the evaluation of the effects of these InsPs on cellular functions.

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References

  1. Berridge, M. J., & Irvine, R. F. (1989) Inositol phosphates and cell signalling. Nature. 341, 197–205.

    Article  CAS  PubMed  Google Scholar 

  2. Rhee, S. G. (2001) Regulation of phosphoinositide-specific phospholipase C. Annu. Rev. Biochem. 70, 281–312.

    Article  CAS  PubMed  Google Scholar 

  3. Majerus, P. W. (1992) Inositol phosphate biochemistry. Annu. Rev. Biochem. 61, 225–250.

    Article  CAS  PubMed  Google Scholar 

  4. Irvine, R. F., Schell, M. J. (2001) Back in the water: the return of the inositol phosphates. Nat. Rev. Mol. Cell Biol. 2, 327–338.

    Article  CAS  PubMed  Google Scholar 

  5. Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. (2006) Inositol pyrophosphates: metabolism and signaling. Cell Mol. Life Sci. 63, 552–564.

    Article  CAS  PubMed  Google Scholar 

  6. Irvine, R. (2007) Cell signaling. The art of the soluble. Science. 316, 845–846.

    Article  CAS  PubMed  Google Scholar 

  7. Shears, S. B. (2004) How versatile are inositol phosphate kinases? Biochem. J. 377, 265–280.

    Article  CAS  PubMed  Google Scholar 

  8. York, J. D. (2006) Regulation of nuclear processes by inositol polyphosphates. Biochimica et biophysica acta. 1761, 552–559.

    CAS  PubMed  Google Scholar 

  9. Bhandari, R., Chakraborty, A., & Snyder, S. H. (2007) Inositol pyrophosphate pyrotechnics. Cell Metab. 5, 321–323.

    Article  CAS  PubMed  Google Scholar 

  10. Onnebo, S. M., Saiardi, A. (2007) Inositol pyrophosphates get the vip1 treatment. Cell. 129, 647–649.

    Article  CAS  PubMed  Google Scholar 

  11. Lee, Y. S., Mulugu, S., York, J. D., & O’Shea, E. K. (2007) Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science. 316, 109–112.

    Article  CAS  PubMed  Google Scholar 

  12. Frederick, J. P., Mattiske, D., Wofford, J. A., Megosh, L. C., Drake, L. Y., Chiou, S.-T., Hogan, B. L. M., & York, J. D. (2005) An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production. PNAS. 102, 8454–8459.

    Article  CAS  PubMed  Google Scholar 

  13. Verbsky, J., Lavine, K., & Majerus, P. W. (2005) Disruption of the mouse inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expression. PNAS. 102, 8448–8453.

    Article  CAS  PubMed  Google Scholar 

  14. Choi, K. Y., Kim, H. K., Lee, S. Y., Moon, K. H., Sim, S. S., Kim, J. W., Chung, H. K., & Rhee, S. G. (1990) Molecular cloning and expression of a complementary DNA for inositol 1,4,5-trisphosphate 3-kinase. Science. 248, 64–66.

    Article  CAS  PubMed  Google Scholar 

  15. Irvine, R. F., Letcher, A. J., Heslop, J. P., & Berridge, M. J. (1986) The inositol tris/tetrakisphosphate pathway – demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues. Nature. 320, 631–634.

    Article  CAS  PubMed  Google Scholar 

  16. Takazawa, K., Lemos, M., Delvaux, A., Lejeune, C., Dumont, J. E., & Erneux, C. (1990) Rat brain inositol 1,4,5-trisphosphate 3-kinase. Ca2(+)-sensitivity, purification and antibody production. Biochem. J. 268, 213–217.

    CAS  PubMed  Google Scholar 

  17. Wilson, M. P., & Majerus, P. W. (1996) Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme. J. Biol. Chem. 271, 11904–11910.

    Article  CAS  PubMed  Google Scholar 

  18. Yang, X., & Shears, S. B. (2000) Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P(4) 1-kinase/Ins(1,3,4)P(3) 5/6-kinase. Biochem. J. 351(Pt 3), 551–555.

    Article  CAS  PubMed  Google Scholar 

  19. Chang, S. C., Miller, A. L., Feng, Y., Wente, S. R., & Majerus, P. W. (2002) The human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase. J. Biol. Chem. 277, 43836–43843.

    Article  CAS  PubMed  Google Scholar 

  20. Nalaskowski, M. M., Deschermeier, C., Fanick, W., & Mayr, G. W. (2002) The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization. Biochem. J. 366, 549–556.

    Article  CAS  PubMed  Google Scholar 

  21. Odom, A. R., Stahlberg, A., Wente, S. R., & York, J. D. (2000) A role for nuclear Inositol 1,4,5-Trisphosphate Kinase in transcriptional control. Science. 287, 2026–2029.

    Article  CAS  PubMed  Google Scholar 

  22. Saiardi, A., Caffrey, J. J., Snyder, S. H., & Shears, S. B. (2000) Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae. FEBS Lett. 468, 28–32.

    Article  CAS  PubMed  Google Scholar 

  23. Verbsky, J. W., Wilson, M. P., Kisseleva, M. V., Majerus, P. W., & Wente, S. R. (2002) The synthesis of inositol hexakisphosphate. Characterization of human inositol 1,3,4,5,6-pentakisphosphate 2-kinase. J. Biol. Chem. 277, 31857–31862.

    Article  CAS  PubMed  Google Scholar 

  24. York, J. D., Odom, A. R., Murphy, R., Ives, E. B., & Wente, S. R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science. 285, 96–100.

    Article  CAS  PubMed  Google Scholar 

  25. Saiardi, A., Caffrey, J. J., Snyder, S. H., & Shears, S. B. (2000) The inositol hexakisphosphate kinase family. Catalytic flexibility and function in yeast vacuole biogenesis. J. Biol. Chem. 275, 24686–24692.

    Article  CAS  PubMed  Google Scholar 

  26. Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., & Snyder, S. H. (1999) Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases. Curr. Biol. 9, 1323–1326.

    Article  CAS  PubMed  Google Scholar 

  27. York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., & York, J. D. (2005) Inositol diphosphate signaling regulates telomere length. J. Biol. Chem. 280, 4264–4269.

    Article  CAS  PubMed  Google Scholar 

  28. Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., & Vogel, G. (1996) Structures of diphospho-myo-inositol pentakisphosphate and bisdiphospho-myo-inositol tetrakisphosphate from Dictyostelium resolved by NMR analysis. Biochem. J. 315, 715–720.

    CAS  PubMed  Google Scholar 

  29. Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., & York, J. D. (2007) A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science. 316, 106–109.

    Article  CAS  PubMed  Google Scholar 

  30. Choi, J. H., Williams, J., Cho, J., Falck, J. R., & Shears, S. B. (2007) Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress. J. Biol. Chem. 282, 30763–30775.

    Article  CAS  PubMed  Google Scholar 

  31. Fridy, P. C., Otto, J. C., Dollins, D. E., & York, J. D. (2007) Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakis-phosphate kinases. J. Biol. Chem. 282, 30754–30762.

    Article  CAS  PubMed  Google Scholar 

  32. Lin, H., Fridy, P. C., Ribeiro, A. A., Choi, J. H., Barma, D. K., Vogel, G., Falck, J. R., Shears, S. B., York, J. D., & Mayr, G. W. (2009) Structural ana­lysis and detection of biological inositol pyro­phosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases. J. Biol. Chem, 284, 1863–1872.

    Article  CAS  PubMed  Google Scholar 

  33. De Vivo, M., Chen, J., Codina, J., & Iyengar, R. (1992) Enhanced phospholipase C stimulation and transformation in NIH-3T3 cells expressing Q209LGq-alpha-subunits. J. Biol. Chem. 267, 18263–18266.

    PubMed  Google Scholar 

  34. Otto, J. C., Kelly, P., Chiou, S. T., & York, J. D. (2007) Alterations in an inositol phosphate code through synergistic activation of a G protein and inositol phosphate kinases. Proc. Natl. Acad. Sci. U S A. 104, 15653–15658.

    Article  CAS  PubMed  Google Scholar 

  35. Otto, J. C., Mulugu, S., Fridy, P. C., Chiou, S. T., Armbruster, B. N., Ribeiro, A. A., & York, J. D. (2007) Biochemical analysis of inositol phosphate kinases. Meth. Enzymol. 434, 171–185.

    Article  CAS  PubMed  Google Scholar 

  36. Stevenson-Paulik, J., Chiou, S.-T., Frederick, J. P., dela Cruz, J., Seeds, A. M., Otto, J. C., & York, J. D. (2006) Inositol phosphate metabolomics: Merging genetic perturbation with modernized radiolabeling methods. Methods. 39, 112–121.

    Google Scholar 

  37. Safrany1, S. T., Caffrey, J. J., Yang, X., Bembenek, M. E., Moyer, M. B., Burkhart, W. A., & Shears, S. B. (1998) A novel context for the ‘MutT’ module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase. EMBO J. 17, 6599–6607.

    Google Scholar 

  38. Fujii, M., & York, J. D. (2005) A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in Rat-1 Cells. J. Biol. Chem. 280, 1156–1164.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by funds from the Howard Hughes Medical Institute and from the National Institute of Health Grants HL-55672 and DK-070272. We thank members of the York laboratory for their contributions to the study of IPKs.

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Authors and Affiliations

  1. Department of Pharmacology and Cancer Biology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA

    James C. Otto & John D. York

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  1. James C. Otto
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  2. John D. York
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Editors and Affiliations

  1. Dept. Molecular Medicine & Surgery, Rolf Luft Research Ctr. for Diabetes, Karolinska University Hospital Solna, Stockholm, Sweden

    Christopher J. Barker

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Otto, J.C., York, J.D. (2010). Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells. In: Barker, C. (eds) Inositol Phosphates and Lipids. Methods in Molecular Biology, vol 645. Humana Press. https://doi.org/10.1007/978-1-60327-175-2_3

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  • DOI: https://doi.org/10.1007/978-1-60327-175-2_3

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-174-5

  • Online ISBN: 978-1-60327-175-2

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