Development of tyrosine kinase peptide biosensors and methods for detection
New methods to monitor tyrosine kinase activity are critical for studying kinases in cell biology, drug discovery and the clinic. Peptide-based biosensors for detection of kinase activity utilitize a kinase specific artificial peptide substrate, which can report intercellular kinase activity through the incorporation of phosphate. An artificial Syk substrate peptide was developed and incorporated with other functional modules to produce a Syk biosensor. These modules included a biotin-tag for affinity capture, a photo-cleavable amino acid to allow release of the substrate from the delivery module and the cell penetrating peptides TAT. A live cell kinase assay utilizing this biosensor was developed to monitor intercellular Syk activity in response to physiologically relevant stimulation and inhibition and using chemifluorescent ELISA-based detection. Terbium (Tb3+) chelation has been used to achieve phosphorylation-dependent luminescence with kinase substrate peptides. Terbium is a hard acid giving it preferred coordination with negatively charge species such as phosphate and carboxylate groups. Tb3+ as a luminescent probe for detection of phosphorylation offers several advantages over organic fluorophores including large stokes shifts, long luminescent life times and narrow emission bands. The Syk substrate described above consist of an acidic motif with a central tyrosine providing the appropriate coordination environment and antenna for sensitization of Tb3+. The phosphopeptide displayed several phosphorylation-dependent photophysical changes resulting in enhanced Tb3+ luminescence. The use of Tb3+ and this sensor produced robust detection of Syk kinase activity and inhibition in vitro. An algorithm that combines the prediction and identification of kinase specific substrates with the identification of Tb3+ binding motif was developed to produce novel Tb3+ sensitizing peptide biosensors. Sensors were developed, validated, characterized and applied for the detection of Abl, Jak2 and Src-family kinase activities. The development of assays that can provide additional information in single assay thus increasing the throughput are needed. Using specific substrates for Lyn and Syk capable of phosphorylation-dependent sensitization of Tb simultaneous detection of both kinases in heterogeneous samples was accomplished using time resolved-lanthanide-based resonance energy transfer (TR-LRET) between the terbium bound phosphopeptides and the orthogonal fluorophore labels.
Parker, Purdue University.
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