Competitive Fluorescence Polarization Assays for the Detection of Phosphoinositide Kinase and Phosphatase Activity

Abstract

We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domain-containing inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kalpha and SHIP2 with diC(8)-PI(4,5)P(2) and diC(8)-PI(3,4,5)P(3) as respective substrates. IC(50) values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development.

Immobilized Metal Ion Affinity-Based Fluorescence Polarization (IMAP): Advances in Kinase Screening

“…[4][5][6][7] In the context of the current issue of this journal, it is useful to look at these two FP techniques in light of the continuing interest in comparisons of assay technologies to underscore the contrasts between FPIAs for kinases and the IMAP technique. Figure 2 illustrates the steps in each type of assay as typically practiced.…”

A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

“…FP is often used to detect the binding of fluorescentlylabeled small ligands to larger binding partners (e.g., refs. [27][28][29][30][31][32]). FP is based on the physical principle that fluorescein and other fluorophores are only excited by incident light that is polarized parallel to their axis.…”

“…Fluorescence polarization assays have been developed to detect various biological events such as phosphorylation, proteolytic cleavage, single nucleotide polymorphism detection, cAMP production, protein-protein interactions, and protein-DNA interactions [28,29,31,32,[37][38][39][40][41]. This article focuses on our development and validation of a ligand displacement assay to screen for inhibitors of RGS12/G i1 and GPSM2/G i1 interactions (Fig.…”

A High-Throughput Microfluidic Assay for SH2 Domain-Containing Inositol 5-Phosphatase 2

“…In addition to such TLC methods, a competitive fluorescence polarization assay for phosphoinositide phosphatases has also been reported. 15 Herein we describe the development of a homogeneous SHIP2 assay using recombinant enzyme allowing direct detection of fluorophore-labeled PIP3 substrate and PIP2 product that does not require the use of radioactivity or specific phosphoinositide binding proteins, and that may be readily applied to other phosphoinositide phosphatases (e.g., SH2 domain-containing inositol 5-phosphatase 1 [SHIP1] or PTEN) or phosphoinositide kinases (e.g., PI3K). This assay, using LabChip ® technology from Caliper Life Sciences (Mountainview, CA), relies on a shift in mobility within an electric field between a fluorophore-labeled PIP3 substrate and the corresponding PIP2 product during pressure-driven mass flow on a microfluidic chip, allowing quantitation of the extent of substrate conversion to product.…”