Abstract
Phosphatidylinositol 3,4,5 trisphosphate [PtdIns(3,4,5)P 3] is a potent membrane-bound signaling molecule transiently synthesized by phosphoinositide 3-kinase (PI3-kinase) in response to extracellular agonists. PtdIns(3,4,5)P 3 signals need to be strictly controlled. PtdIns(3,4,5)P 3 recruits and binds effectors that function in oncogenic signaling pathways. PtdIns(3,4,5)P 3 activates cell proliferation, growth, and migration as well as regulating insulin signaling. The inositol polyphosphate 5-phosphatase family of enzymes dephosphorylate and thereby modulate PtdIns(3,4,5)P 3 levels, attenuating PI3-kinase-dependent signaling. PtdIns(3,4,5)P 3 5-phosphatase enzyme activity can be assessed in vitro by analysis of the hydrolysis of radiolabeled or fluorescently labeled PtdIns(3,4,5)P 3 and in vivo by visualization of the recruitment and turnover of the PtdIns(3,4,5)P 3-specific biosensor GFP-PH/ARNO or other PtdIns(3,4,5)P 3 binding proteins at the plasma membrane.
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Abbreviations
- 5-phosphatase: Inositol:
-
polyphosphate 5-phosphatase;
- DMSO: Dimethyl:
-
sulfoxide;
- PI3-kinase: Phosphoinositide:
-
3-kinase;
- PH: Pleckstrin:
-
homology;
- PtdIns(4,5)P 2: Phosphatidylinositol:
-
4,5 bisphosphate;
- PtdIns(3,4,5)P 3: Phosphatidylinositol:
-
3,4,5 trisphosphate;
- PtdSer::
-
Phosphatidylserine;
- TLC::
-
Thin layer chromatography.
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Acknowledgments
The authors would like to thank Dr. Tamas Balla for the GFP-PH/ARNO construct, Dr. Meredith Layton for the recombinant PI3-kinase, and Drs. Absorn Sriratana and Richard Huysmans for technical assistance.
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Ooms, L.M., Dyson, J.M., Kong, A.M., Mitchell, C.A. (2009). Analysis of Phosphatidylinositol 3,4,5 Trisphosphate 5-Phosphatase Activity by in vitro and in vivo 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_15
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DOI: https://doi.org/10.1007/978-1-60327-115-8_15
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