Kei Takiyama

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).

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.

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.