title: Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity.
abstract: Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, 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 in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-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.
authors.forename: Abigail H
authors.initials: AH
authors.lastname: Turner
authors.affiliation: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599 and North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Lebhar
authors.forename: Angela
authors.initials: A
authors.lastname: Proctor
authors.forename: Qunzhao
authors.initials: Q
authors.lastname: Wang
authors.forename: David S
authors.initials: DS
authors.lastname: Lawrence
authors.affiliation: Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599 and North Carolina State University, Raleigh, North Carolina 27695, United States.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D045744:Cell Line, Tumor; D057075:Enzyme Assays / Q000379:methods; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D059010:Single-Cell Analysis / Q000379:methods*; D044005:Tetrahydroisoquinolines / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D044005:Tetrahydroisoquinolines; C078986:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; D014443:Tyrosine; C512478:EGFR protein, human; D066246:ErbB Receptors