Self-reporting fluorescent substrates of protein tyrosine kinases.
A new mechanistic principle by which protein tyrosine kinase substrates fluorescently report the introduction of a phosphate moiety has been developed. NMR was used to establish that tyrosine phosphorylation induces the disruption of pi-pi stacking interactions of the tyrosine moiety with a proximal fluorophore on the peptide substrate. We have demonstrated that (1) the peptide substrates described in this study are useful for a wide variety of different tyrosine kinases, (2) physiological concentrations of ATP can be employed (unlike the standard radioactive ATP kinase assays), thus providing a more realistic assessment of inhibitor potency, and (3) protein kinase self-activation can be observed in real-time.
Wang
Qunzhao
Q
Department of Biochemistry, The Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Cahill
Sean M
SM
Blumenstein
Michael
M
Lawrence
David S
DS
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D009842:Oligopeptides; D011505:Protein-Tyrosine Kinases