Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives

Abstract

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.

Figure 1

Schematic illustration of the PI3K reaction. A. the endogenous PIP₂ is phosphorylated at the 3′-OH to form PIP₃; B, C. Chemical structures of fluorescent PIP₂ derivatives BODIPY-PIP₂ (B) and FL-PIP₂ (C).

Figure 2

Biophysical characterization of FL-PIP₂ and BODIPY-PIP₂. A. Measurement of critical micelle concentrations (CMCs) of FL-PIP₂ and BODIPY-PIP₂. B. Fluorescence excitation and emission spectra of FL-PIP₂ and BODIPY-PIP₂.

Figure 3

Separation of the PI3K reaction mixtures with fluorescent PIP₂ derivatives through TLC or CE analysis. A. the separation of the reaction mixture of FL-PIP₂ on TLC. lane 1: FL-PIP₂; lane 2: the reaction mixture was extracted with CHCl₃/MeOH (v/v = 2:1) before TLC separation; lane 3: the reaction mixture was diluted with CHCl₃/MeOH (v/v = 1:1) and separated on TLC; lane 4: FL-PIP₃. B. the separation of the reaction mixture of BODIPY-PIP₂ on TLC. lane 1: BODIPY-PIP₂; lane 2: the reaction mixture was extracted with CHCl₃/MeOH (v/v = 2:1) before TLC separation; lane 3: the reaction mixture was diluted with CHCl₃/MeOH (v/v = 1:1) and separated on TLC; lane 4: BODIPY-PIP₃. C. The separation of a mixture of FL-PIP₂, FL-PIP₃, BODIPY-PIP₂, and BODIPY-PIP₃ by CE; D. A 1:1 mixture of FL-PIP₂ and BODIPY-PIP₂ were used for the PI3K reaction, and the reaction mixture was separated by CE.

Figure 4

Kinetic analysis of PI3K reaction with fluorescent PIP₂ derivatives. A. The reaction progress curves with different amounts of PI3K were used. B. Kinetic studies on PI3K reaction with FL-PIP₂. C. Kinetic studies on PI3K reaction with BODIPY-PIP₂. Each experiment was carried out in duplicate and repeated three times.

Figure 5

Measurement of K_M for ATP for the PI3K reactions with FL-PIP₂ (A) or BODIPY-PIP₂ (B) as the lipid substrate. Each experiment was carried out in duplicate and repeated three times.

Figure 6

Dose response curves of PI3K inhibitors LY294002 and wortmannin. A. IC₅₀ values of LY294002 and wortmannin were measured with FL-PIP₂ as the PI3K substrate; B. IC₅₀ values of LY294002 and wortmannin were measured with BODIPY-PIP₂ as the PI3K substrate. Each experiment was carried out in duplicate and repeated three times.