Design of an encodable tyrosine kinase-inducible domain: detection of tyrosine kinase activity by terbium luminescence.
Tyrosine kinases are critical mediators of intracellular signaling and of intracellular responses to extracellular signaling. Changes in tyrosine kinase activity are implicated in numerous human diseases, including cancers, diabetes, and pathogen infectivity. To address questions in tyrosine phosphorylation, we have designed a protein tyrosine kinase-inducible domain, a small, genetically encodable protein motif whose structure is dependent on its tyrosine phosphorylation state. Tyrosine kinase-inducible domain peptides are based on EF-hand loops in which a structurally critical Glu12 residue is replaced by tyrosine at residue 11 or at residue 15 of the protein. Tyrosine kinase-inducible domain peptides bind terbium(III) in a phosphorylation-dependent manner, showing strong terbium luminescence when phosphorylated but weak terbium luminescence when not phosphorylated. Lanthanide binding was confirmed by NMR. A tyrosine kinase-inducible domain peptide, pKID-Abl, was designed to incorporate a recognition sequence of the Abl kinase. Incubation of pKID-Abl with Abl kinase resulted in a large increase in terbium luminescence. This increase in luminescence was abolished when pKID-Abl and Abl kinase were incubated with the Abl kinase inhibitor Gleevec. In addition, incubation of phosphorylated pKID-Abl with the tyrosine phosphatase YOP resulted in a large reduction in terbium luminescence. pKID-Abl was employed as a fluorescent sensor of Abl tyrosine kinase activity in HeLa cell extracts, exhibiting low luminescence with extracts from serum-starved cells and increased luminescence using extracts from EGF-treated cells. These results indicate that tyrosine kinase-inducible domains may be used as sensors of tyrosine kinase and tyrosine phosphatase activity and in the detection of tyrosine kinase inhibitors.
Zondlo
Susan Carr
SC
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Gao
Feng
F
Zondlo
Neal J
NJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D006801:Humans; D008163:Luminescent Measurements; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013725:Terbium / Q000378:metabolism*
D013725:Terbium; D011505:Protein-Tyrosine Kinases