Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells
- 967 Downloads
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.
Key words:Inositol phosphate G protein Phospholipase C HPLC Second messenger Signal transduction
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.
- 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.CrossRefPubMedGoogle 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.CrossRefPubMedGoogle 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