A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the kinase reaction of a substrate peptide

Abstract

We recently reported a fluorescence resonance energy transfer (FRET) system for the analysis of the dephosphorylation of a 5-carboxyfluorescein (FAM)-labeled phosphopeptide by using a Phos-tag (1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) derivative attached to a (7-amino-4-methylcoumarin-3-yl)acetic acid (AMCA) moiety. This FRET system is based on the principle that the Phos-tag captures the phosphopeptide in preference to its nonphosphorylated counterpart. Binding of the phosphopeptide to the Phos-tag molecule brings the donor AMCA (λ_ex 345 nm) close to the acceptor FAM (λ_em 520 nm), resulting in an efficient FRET signal. Here we introduce an application of the FRET system to the reverse reaction, phosphorylation of a FAM-labeled peptide substrate by a kinase, such as epidermal growth factor receptor or c-Src, in the presence of 15 mM magnesium(II) ion, 20 μM ATP, and a hydrophilic AMCA-labeled Phos-tag molecule. Furthermore, the inhibition profiles of Abl and c-Src kinases were determined using specific inhibitors, Glivec and c-Src kinase inhibitor I, respectively. The dose-dependent inhibitions of the kinase reactions were determined from real-time changes in the FRET efficiency, which showed IC₅₀ values of 85 nM of Glivec and 0.13 μM of c-Src kinase inhibitor I.