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 (λex 345 nm) close to the acceptor FAM (λem 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.