Quantitative measurement of phosphatidylinositol 3,4,5-trisphosphate.
The activation of class I phosphoinositide 3-kinases (PI(3)Ks) by cell surface receptors represents the initiation of a large and complex signaling network that couples many growth factors, antigens, and inflammatory stimuli to important cellular responses, such as cell growth, survival, and movement. The most direct measurement of class I PI(3)K activity in cells is the rate of production of its lipid product, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. This chapter describes in detail two approaches used to estimate the levels of PtdIns(3,4,5)P(3) in cells. One approach uses radiotracer labeling of cells, lipid extraction, deacylation, and subsequent quantitation of phosphoinositides by anion-exchange high-performance liquid chromatography. The second approach uses a novel, nonradioactive assay in which the cellular lipids are extracted, phosphoinositides are enriched through binding to a neomycin matrix, dried onto a nitrocellulose membrane, and PtdIns(3,4,5)P(3) quantified by a protein-lipid overlay approach using a GRP(1) PH domain probe.
Guillou
Hervé
H
The Inositide Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.
Stephens
Len R
LR
Hawkins
Phillip T
PT
D019870:1-Phosphatidylinositol 4-Kinase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002468:Cell Physiological Phenomena; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D008055:Lipids / Q000302:isolation & purification; D009504:Neutrophils / Q000502:physiology; D010710:Phosphates / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000032:analysis*; D010716:Phosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000302:isolation & purification; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000302:isolation & purification
D007202:Indicators and Reagents; D008055:Lipids; D010710:Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; D019870:1-Phosphatidylinositol 4-Kinase