Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity

Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology <i>in vitro</i>, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both <i>in vitro</i> and <i>in cellulo</i>. The reporter contains a conformationally constrained phosphorylatable moiety (7-(<i>S</i>)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of l-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter