title: A cell-based screening method for specifically detecting kinase activity.
abstract: No universal approach has been reported for specific monitoring of the catalytic activity of a wide range of kinases in cells. The present study describes an original platform for detecting the autonomous activity of serine/threonine kinases in cells through the introduction of expression vectors encoding modified substrate kinase fusion proteins. The surrogate substrate used consists of the p53 tumor suppressor protein fused with individual kinase domains (Chk1, DYRK3, and Cdk5) at its carboxy-terminal through four tandem Gly-Gly-Gly-Gly-Ser repeats. After transfection into cells, phosphorylation of the p53 moiety could be specifically induced by the catalytic activity of kinases contained in the fusion protein. Moreover, p53 phosphorylation was significantly blocked when a kinase-inactive mutant was used as the fusion partner instead of the active kinase. Using this system, the cell-based evaluation of several Cdk5 inhibitors was demonstrated. Thus, this approach provides a novel platform for the specific, cell-based screening of inhibitors of a wide prospective range of protein kinases and is of tremendous potential for drug discovery efforts.
authors.affiliation: GlaxoSmithKline K.K., 43 Wadai, Tsukuba-shi, Ibaraki, 300-4247, Japan.
authors.forename: Mikiya
authors.initials: M
authors.lastname: Suda
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ishii
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sootome
authors.forename: Alastair J
authors.initials: AJ
authors.lastname: King
authors.forename: Megumi
authors.initials: M
authors.lastname: Shibahara
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Noro
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamashita
authors.forename: Takato
authors.initials: T
authors.lastname: Noumi
mesh_terms: D000818:Animals; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002522:Chlorocebus aethiops; D051360:Cyclin-Dependent Kinase 5 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C408131:DYRK3 protein, human; D011505:Protein-Tyrosine Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases