title: Engineering a "methionine clamp" into Src family kinases enhances specificity toward unnatural ATP analogues.
abstract: A single alanine or glycine mutation in the ATP binding site of a protein kinase allows unique use of an unnatural analogue of ATP (N(6)-(benzyl) ATP) as a phosphodonor, which is not accepted by wild-type kinases. Addition of [gamma(32)P] N(6)-(benzyl) ATP to a cell lysate containing an ATP analog-specific kinase allele (as1 allele) results in the exclusive radiolabeling of bona fide substrates of the mutant kinase. Here we report efforts to engineer kinase alleles that have enhanced selectivity for ATP analogues and decreased catalytic activity with ATP, thus increasing the signal-to-noise ratio of substrate labeling. Two conserved leucine residues that contact each face of the adenine ring of ATP were mutated to methionine. The introduction of this "methionine clamp" resulted in Src and Fyn kinase alleles that have markedly improved specificity for unnatural N(6)-substituted ATP analogues over the natural substrate, ATP. This preference for unnatural nucleotides is reflected in more efficient labeling of protein substrates in cell extracts using the new analogue-specific v-Src allele. Kinase alleles with enhanced selectivity for unnatural ATP analogues should greatly facilitate the ultimate goal of labeling kinase substrates in intact cells, where concentrations of ATP and other competing nucleotides are high.
authors.affiliation: , Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94107-2280, USA.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Ulrich
authors.forename: Denise M
authors.initials: DM
authors.lastname: Kenski
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D008715:Methionine; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D015202:Protein Engineering; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D008715:Methionine; C494960:Fyn protein, mouse; D015688:Oncogene Protein pp60(v-src); D051076:Proto-Oncogene Proteins c-fyn