Abstract
The regulation of phosphoinositide (PI) 3-kinase activities has been linked to many normal and disease-related processes, including cell survival, cell growth and proliferation, cell differentiation, cell motility, and intracellular vesicle trafficking. However, as the family of enzymes has now grown to include eight true members, in three functional classes, plus several related protein kinases that are also inhibited by the widely used PI 3-kinase selective inhibitors, wortmannin and LY294002, extended methodologies are required to identify which type of kinase is involved in a particular cellular process, or protein complex, under study. A robust in vitro PI 3-kinase assay, suitable for use with immunoprecipitates, or purified proteins, is described here together with a series of modifications of substrate and assay conditions that will aid researchers in the identification of the particular class and isoform of PI 3-kinase that is involved in a signaling process under investigation.
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Abbreviations
- CDTA::
-
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetra-acetic acid;
- DMSO::
-
dimethylsulphoxide;
- EDTA::
-
diaminoethanetetra-acetic acid;
- PBS::
-
phosphate buffered saline;
- PI::
-
phosphoinositide;
- PMSF::
-
phenylmethylsulfonyl fluoride;
- PtdCho::
-
phosphatidylcholine;
- PtdEth::
-
phosphatidylethanolamine;
- PtdIns::
-
phosphatidylinositol;
- PtdSer::
-
phosphatidylserine;
- TLC::
-
thin layer chromatography.
References
Sugimoto Y, Whitman M, Cantley LC, Erikson RL. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci USA 1984;81:2117–21.
Macara IG, Marinetti GV, Balduzzi PC. Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: Possible role in tumorigenesis. Proc Natl Acad Sci USA 1984;81:2728–32.
Whitman M, Kaplan DR, Schaffhausen B, Cantley L, Roberts TM. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 1985;315:239–342.
Fry MJ, Gebhardt A, Parker PJ, Foulkes JG. PI turnover and transformation of cells by Abelson murine leukaemia virus. EMBO J 1985;4:3173–8.
Whitman M, Downes CP, Keeler M, Keller T, Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 1988;332:644–6.
Fry MJ. Structure, regulation and functional of phosphoinositide 3-kinases. Biochim Biophys Acta 1994;1226:237–68.
Rameh LE, Cantley LC. The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem 1999;274:8347–50.
Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular functions of phosphoinositide 3-kinases: Implications for development, homeostasis, and cancer. Ann Rev Cell Dev Biol 2001;17:615–75.
Foster FM, Traer CJ, Abraham SM, Fry MJ. The phosphoinositide (PI) 3-kinase family. J Cell Sci 2003;116:3037–40.
Arcaro A, Wymann MP. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: The role of phosphatidylinositol3,4,5-trisphosphate in neutrophil responses. Biochem J 1993;296:297–301.
Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-1 (LY294002). J Biol Chem 1994;269:5241–8.
Domin J, Waterfield MD. Using structure to define the function of phosphoinositide 3-kinase family members. FEBS Lett 1997;410:91–5.
Kastan MB, Lim DS. The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000;1:179–86.
Dann SG, Thomas G. The amino acid sensitive TOR pathway from yeast to mammals. FEBS Lett 2006;580:2821–9.
Traer CJ, Foster FM, Abraham SM, Fry MJ. Are class II phosphoinositide 3-kinases potential targets for anticancer therapies? Bull Cancer 2006;93:E53–8.
Maffucci T, Cooke FT, Foster FM, Traer CJ, Fry MJ, Falasca M. Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration. J Cell Biol 2005;169:789–99.
Stack JH, Emr SD. Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities. J Biol Chem 1994;269:31552–62.
Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD, Williams O, Loewith R, Stokoe D, Balla A, Toth B, Balla T, Weiss WA, Williams RL, Shokat KM. A pharmacological map of the PI3-kinase family defines a role for p110α in insulin signalling. Cell 2006;125:733–47.
Domin J, Pages F, Volinia S, Rittenhouse SE, Zvelebil MJ, Stein RC, Waterfield MD. Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin. Biochem J 1997;326:139–47.
Ward S, Sotsios Y, Dowden J, Bruce I, Finan P. Therapeutic potential of phosphoinositide 3-kinase inhibitors. Chem Biol 2003;10:207–13.
Workman P. Inhibiting the phosphoinositide 3-kinase pathway for cancer treatment. Biochem Soc Trans 2004;32:393–6.
Whitman M, Kaplan D, Roberts T, Cantley L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J 1987;247:165–74.
Woscholski R, Kodaki T, Palmer RH, Waterfield MD, Parker PJ. Modulation of the substrate specificity of the mammalian phosphatidylinositol 3-kinase by cholesterol sulfate and sulfatide. Biochemistry 1995;34:11489–93.
Lau MR. Characterisation of the class II phosphoinositide 3-kinase, PI3 K-C2β. Ph.D. thesis, University of London; 2000.
Arcaro A, Volinia S, Zvelebil MJ, Stein R, Watton SJ, Layton MJ, Gout I, Ahmadi K, Downward J, Waterfield MD. Human phosphoinositide 3-kinase C2β, the role of calcium and the C2 domain in enzyme activity. J Biol Chem 1998;273:33082–90.
Woscholski R, Kodaki T, McKinnom M, Waterfield MD, Parker PJ. A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase. FEBS Lett 1994;342:109–14.
Clarke NG, Dawson RMC. Alkaline O -> N-transacylation. A new method for the quantitative deacylation of phospholipids. Biochem J 1981;195:301–6.
Walsh JP, Caldwell KK, Majerus PW. Formation of phosphatidylinositol 3-phosphate by isomerisation from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci 1991; 88:9164–87.
Acknowledgments
The author would like to thank both former colleagues and members of his laboratory for discussions over many years that have led to the formulation of these assays. Thanks also to Mike Lau for assistance in preparing the figures.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Fry, M.J. (2009). Phosphoinositide (PI) 3-Kinase Assays. In: Larijani, B., Woscholski, R., Rosser, C. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 462. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-115-8_22
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DOI: https://doi.org/10.1007/978-1-60327-115-8_22
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