Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts.
We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.
Gray
Alexander
A
Division of Signal Transduction Therapy, MSI/WTB Complex, University of Dundee, Dow Street, Scotland, Dundee DD1 5EH, UK. a.z.gray@dundee.ac.uk
Olsson
Henric
H
Batty
Ian H
IH
Priganica
Larisa
L
Peter Downes
C
C
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry; D002457:Cell Extracts / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis* / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism; D010716:Phosphatidylinositols / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D020358:Sodium Cholate / Q000494:pharmacology; D014020:Tissue Extracts / Q000032:analysis / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000378:metabolism
D001710:Biotin; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D011994:Recombinant Proteins; D020358:Sodium Cholate; D014020:Tissue Extracts; D025521:Tumor Suppressor Proteins