Phos-tag-based fluorescence resonance energy transfer system for the analysis of protein kinase and phosphatase reactions

Abstract

We introduce a novel fluorescence resonance energy transfer (FRET) system for the detection of a phosphorylated molecule such as a phosphopeptide using a phosphate-binding tag molecule, Zn(II)-Phos-tag (1,3-bis[bis (pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). 5-Carboxyfluorescein (FAM)-labeled phosphopeptides and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, emission at 520 nm) and AMCA (a fluorescent donor, excitation at 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn(II)-Phos-tag captured specifically the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent reversible phosphorylation of the FAM-labeled substrate peptide. The Phos-tag-based FRET system has the following major advantages: i) The real-time analysis of the reversible phosphorylation reaction is possible without multiple samplings, ii) the analysis requires a simple procedure just using two solutions of AMCA-labeled Phos-tag and a FAM-labeled compound, and iii) the system would be useful for the reliable and comprehensive phosphorylation assays for various phosphopeptides containing phosphoserine, phosphothreonine, or phosphotyrosine, in vitro. Thus, the principle of this system would be applied to high-throughput kinase/phosphatase profiling, measurement of enzyme activity, and determination of an activator or an inhibitor.