A cell-based screening method for specifically detecting kinase activity.
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.
Suda
Mikiya
M
GlaxoSmithKline K.K., 43 Wadai, Tsukuba-shi, Ibaraki, 300-4247, Japan.
Ishii
Tsuyoshi
T
Sootome
Hiroshi
H
King
Alastair J
AJ
Shibahara
Megumi
M
Noro
Nobuhiro
N
Yamashita
Keizo
K
Noumi
Takato
T
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
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