title: Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase.
abstract: Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK-substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Lee
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lin
authors.forename: Nguyen Hai
authors.initials: NH
authors.lastname: Nam
authors.forename: Keykavous
authors.initials: K
authors.lastname: Parang
authors.forename: Gongqin
authors.initials: G
authors.lastname: Sun
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D018909:src Homology Domains; D019061:src-Family Kinases
chemical_list: D010455:Peptides; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D000409:Alanine