title: Conversion of a tyrosine kinase protein substrate to a high affinity ligand by ATP linkage.
abstract: Protein kinases often show low affinity for their protein substrates, which makes it difficult to study kinase-substrate interactions. Here, we show using expressed protein ligation with the signaling protein Src that it is feasible to install a covalently linked ATP moiety into the tail of Src, generating a semisynthetic protein with a high affinity for its cognate tyrosine kinase, Csk. It is also established that this Src-ATP conjugate can be used to selectively pull down Csk from a complex protein mixture. This work outlines a general strategy for identifying an unknown kinase that is responsible for the phosphorylation of a protein substrate on a site of interest.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
authors.forename: Kui
authors.initials: K
authors.lastname: Shen
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D007700:Kinetics; D051379:Mice; D041681:NIH 3T3 Cells; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases
chemical_list: C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases