Advertisement

Metabolic Labeling of Inositol Phosphates and Phosphatidylinositols in Yeast and Mammalian Cells

  • Andrew T. Hale
  • Bradley P. Clarke
  • John D. YorkEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2091)

Abstract

Inositol phosphate (IP) and phosphatidylinositol (PI) signaling are critical signal transduction pathways responsible for generating numerous receptor-mediated cellular responses. Biochemical and genetic studies have revealed diverse roles of IP and PI signaling in eukaryotic signaling, but detailed characterization of unique IP and PI signaling profiles in response to different agonists and among cell types remains largely unexplored. Here, we outline steady-state inositol metabolic-labeling techniques that can be leveraged to assess the IP and PI signaling state in eukaryotic cells. This flexible technique can be amended and optimized to your cell line of interest, perturbed with biochemical, genetic, or pharmacological alteration, and used to provide comprehensive inositol profiling in various cellular systems.

Key words

Inositol phosphates Phosphatidylinositol Metabolic labeling Signal transduction HPLC Lipid signaling 

Notes

Acknowledgments

This work was supported by funds from the National Institutes of Health (5T32GM007347, A.T.H.) and (1R01GM12440401, J.D.Y).

References

  1. 1.
    Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341(6239):197–205.  https://doi.org/10.1038/341197a0CrossRefPubMedGoogle Scholar
  2. 2.
    Hatch AJ, York JD (2010) SnapShot: Inositol phosphates. Cell 143(6):1030–1030.e1031.  https://doi.org/10.1016/j.cell.2010.11.045CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Majerus PW (1992) Inositol phosphate biochemistry. Ann Rev Biochem 61:225–250.  https://doi.org/10.1146/annurev.bi.61.070192.001301CrossRefPubMedGoogle Scholar
  4. 4.
    Bennett M, Onnebo SM, Azevedo C, Saiardi A (2006) Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 63(5):552–564.  https://doi.org/10.1007/s00018-005-5446-zCrossRefPubMedGoogle Scholar
  5. 5.
    Chakraborty A, Kim S, Snyder SH (2011) Inositol pyrophosphates as mammalian cell signals. Sci Signal 4(188):re1.  https://doi.org/10.1126/scisignal.2001958CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lee YS, Mulugu S, York JD, O'Shea EK (2007) Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science 316(5821):109–112.  https://doi.org/10.1126/science.1139080CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pohlmann J, Fleig U (2010) Asp1, a conserved 1/3 inositol polyphosphate kinase, regulates the dimorphic switch in Schizosaccharomyces pombe. Mol Cell Biol 30(18):4535–4547.  https://doi.org/10.1128/MCB.00472-10CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Seeds AM, Sandquist JC, Spana EP, York JD (2004) A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster. J Biol Chem 279(45):47222–47232.  https://doi.org/10.1074/jbc.M408295200CrossRefPubMedGoogle Scholar
  9. 9.
    Tsui MM, York JD (2010) Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes. Adv Enzyme Regul 50(1):324–337.  https://doi.org/10.1016/j.advenzreg.2009.12.002CrossRefPubMedGoogle Scholar
  10. 10.
    York SJ, Armbruster BN, Greenwell P, Petes TD, York JD (2005) Inositol diphosphate signaling regulates telomere length. J Biol Chem 280(6):4264–4269.  https://doi.org/10.1074/jbc.M412070200CrossRefPubMedGoogle Scholar
  11. 11.
    Blind RD, Suzawa M, Ingraham HA (2012) Direct modification and activation of a nuclear receptor-PIP(2) complex by the inositol lipid kinase IPMK. Sci Signal 5(229):ra44.  https://doi.org/10.1126/scisignal.2003111CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cantley LC (2002) The phosphoinositide 3-kinase pathway. Science 296(5573):1655–1657.  https://doi.org/10.1126/science.296.5573.1655CrossRefPubMedGoogle Scholar
  13. 13.
    Dyson JM, Fedele CG, Davies EM, Becanovic J, Mitchell CA (2012) Phosphoinositide phosphatases: just as important as the kinases. Subcell Biochem 58:215–279.  https://doi.org/10.1007/978-94-007-3012-0_7CrossRefPubMedGoogle Scholar
  14. 14.
    Hansen SB (2015) Lipid agonism: The PIP2 paradigm of ligand-gated ion channels. Biochimica et Biophysica Acta 1851(5):620–628.  https://doi.org/10.1016/j.bbalip.2015.01.011CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kim YJ, Hernandez ML, Balla T (2013) Inositol lipid regulation of lipid transfer in specialized membrane domains. Trends Cell Biol 23(6):270–278.  https://doi.org/10.1016/j.tcb.2013.01.009CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Skwarek LC, Boulianne GL (2009) Great expectations for PIP: phosphoinositides as regulators of signaling during development and disease. Develop Cell 16(1):12–20.  https://doi.org/10.1016/j.devcel.2008.12.006CrossRefGoogle Scholar
  17. 17.
    Vanhaesebroeck B, Stephens L, Hawkins P (2012) PI3K signalling: the path to discovery and understanding. Nat Rev Mol Cell Biol 13(3):195–203.  https://doi.org/10.1038/nrm3290CrossRefPubMedGoogle Scholar
  18. 18.
    Billcliff PG, Lowe M (2014) Inositol lipid phosphatases in membrane trafficking and human disease. Biochem J 461(2):159–175.  https://doi.org/10.1042/BJ20140361CrossRefPubMedGoogle Scholar
  19. 19.
    Eramo MJ, Mitchell CA (2016) Regulation of PtdIns(3,4,5)P3/Akt signalling by inositol polyphosphate 5-phosphatases. Biochem Soc Trans 44(1):240–252.  https://doi.org/10.1042/BST20150214CrossRefPubMedGoogle Scholar
  20. 20.
    Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT (2017) The PI3K pathway in human disease. Cell 170(4):605–635.  https://doi.org/10.1016/j.cell.2017.07.029CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Liu Y, Bankaitis VA (2010) Phosphoinositide phosphatases in cell biology and disease. Prog Lipid Res 49(3):201–217.  https://doi.org/10.1016/j.plipres.2009.12.001CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Majerus PW, York JD (2009) Phosphoinositide phosphatases and disease. J Lipid Res 50(Suppl):S249–S254.  https://doi.org/10.1194/jlr.R800072-JLR200CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pirruccello M, De Camilli P (2012) Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL. Trends Biochem Sci 37(4):134–143.  https://doi.org/10.1016/j.tibs.2012.01.002CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Stevenson-Paulik J, Chiou ST, Frederick JP, dela Cruz J, Seeds AM, Otto JC, York JD (2006) Inositol phosphate metabolomics: merging genetic perturbation with modernized radiolabeling methods. Methods 39(2):112–121.  https://doi.org/10.1016/j.ymeth.2006.05.012CrossRefPubMedGoogle Scholar
  25. 25.
    Fujii M, York JD (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(2):1156–1164.  https://doi.org/10.1074/jbc.M412006200CrossRefPubMedGoogle Scholar
  26. 26.
    Otto JC, Kelly P, Chiou ST, York JD (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(40):15653–15658.  https://doi.org/10.1073/pnas.0705729104CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Otto JC, Mulugu S, Fridy PC, Chiou S-T, Armbruster BN, Ribeiro AA, York JD (2007) Biochemical analysis of inositol phosphate kinases. Methods Enzymol 434:171–185.  https://doi.org/10.1016/s0076-6879(07)34010-xCrossRefPubMedGoogle Scholar
  28. 28.
    Otto JC, York JD (2010) Molecular manipulation and analysis of inositol phosphate and pyrophosphate levels in Mammalian cells. Methods Mol Biol 645:47–60.  https://doi.org/10.1007/978-1-60327-175-2_3CrossRefPubMedGoogle Scholar
  29. 29.
    Seeds AM, Bastidas RJ, York JD (2005) Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae. J Biol Chem 280(30):27654–27661.  https://doi.org/10.1074/jbc.M505089200CrossRefPubMedGoogle Scholar
  30. 30.
    Stevenson-Paulik J, Odom AR, York JD (2002) Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J Biol Chem 277(45):42711–42718.  https://doi.org/10.1074/jbc.M209112200CrossRefPubMedGoogle Scholar
  31. 31.
    Stephens LR, Irvine RF (1990) Stepwise phosphorylation of myo-inositol leading to myo-inositol hexakisphosphate in Dictyostelium. Nature 346(6284):580–583.  https://doi.org/10.1038/346580a0CrossRefPubMedGoogle Scholar
  32. 32.
    Frederick JP, Mattiske D, Wofford JA, Megosh LC, Drake LY, Chiou ST, Hogan BL, York JD (2005) An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production. Proc Natl Acad Sci U S A 102(24):8454–8459.  https://doi.org/10.1073/pnas.0503706102CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Andrew T. Hale
    • 1
  • Bradley P. Clarke
    • 1
  • John D. York
    • 1
    Email author
  1. 1.Department of BiochemistryVanderbilt University School of MedicineNashvilleUSA

Personalised recommendations