title: Profiling the substrate specificity of protein kinases by on-bead screening of peptide libraries.
abstract: A robust, high-throughput method has been developed to screen one-bead-one-compound peptide libraries to systematically profile the sequence specificity of protein kinases. Its ability to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and nonpermissive residues. Application of the library method to kinases Pim1, MKK6, and Csk revealed that Pim1 and Csk are highly active toward peptide substrates and recognize specific sequence motifs, whereas MKK6 has little activity or sequence selectivity against peptide substrates. Pim1 recognizes peptide substrates of the consensus RXR(H/R)X(S/T); it accepts essentially any amino acid at the S/T-2 and S/T+1 positions, but strongly disfavors acidic residues (Asp or Glu) at the S/T-2 position and a proline residue at the S/T+1 position. The selected Csk substrates show strong sequence covariance and fall into two classes with the consensus sequences of (D/E)EPIYϕXϕ and (D/E)(E/D)S(E/D/I)YϕXϕ (where X is any amino acid and ϕ is a hydrophobic amino acid). Database searches and in vitro kinase assays identified phosphatase PTP-PEST as a Pim1 substrate and phosphatase SHP-1 as a potential Csk substrate. Our results demonstrate that the sequence specificity of protein kinases is defined not only by favorable interactions between permissive residue(s) on the substrate and their cognate binding site(s) on the kinase but also by repulsive interactions between the kinase and nonpermissive residue(s).
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210, USA.
authors.forename: Thi B
authors.initials: TB
authors.lastname: Trinh
authors.forename: Qing
authors.initials: Q
authors.lastname: Xiao
authors.forename: Dehua
authors.initials: D
authors.lastname: Pei
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins; D048669:MAP Kinase Kinase 6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12 / Q000737:chemistry / Q000378:metabolism; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D005656:Fungal Proteins; D019151:Peptide Library; D010455:Peptides; D012235:Rhodamines; C005358:tetramethylrhodamine; D011494:Protein Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; C432694:Pim1 protein, Pichia pastoris; D048669:MAP Kinase Kinase 6; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6