A fluorescent indicator for tyrosine phosphorylation based insulin signaling pathways

A fluorescent indicator for tyrosine phosphorylation-based insulin signalin g is described. Upon binding of insulin to cell-surface insulin receptor, t he receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthe tic phosphopeptide pY939 derived from the tyrosine phosphorylation domain o f IRS-1 and its target protein SH2N containing an N-terminal SH2 domain of PI 3-kinase. The SH2N protein and pY939 phosphopeptide were labeled with fl uorescein (F-SH2N) and tetramethylrhodamine (T-pY939), respectively. Format ion of a F-SH2N-T-pY939 complex (termed a fluorescence resonance energy-tra nsfer (FRET) pair) was evaluated from a change in a fluorescence emission s pectrum based on FRET between the two fluorophores. The FRET pair was forme d to dissociate in competition with the unlabeled pY939 phosphopeptide, res ulting in a decrease in a pY939 phosphopeptide-dependent FRET emission at 5 80 nm and causing an increase in emission at 520 nm. Tyrosine phosphorylati on by the partially purified insulin receptor of substrate peptide Y939 was detected with this formed FRET pair, and resulting changes in fluorescence emission spectra were observed for insulin concentration from about 1.0 x 10(-9) to 1.0 x 10(-6) M. These results indicated that the FRET pair served as a competitive fluorescent indicator for tyrosine phosphorylation-based insulin signaling.