A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4.
The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.
Xu
Kui
K
Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
Stern
Alvin S
AS
Levin
Wayne
W
Chua
Anne
A
Vassilev
Lyubomir T
LT
D000098:Acetophenones / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D001578:Benzopyrans / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007700:Kinetics; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000098:Acetophenones; D000906:Antibodies; D001578:Benzopyrans; D018797:Cell Cycle Proteins; C088096:Dbf4 protein, S cerevisiae; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D010769:Phosphothreonine; D000255:Adenosine Triphosphate; C085746:rottlerin; C489404:CDC7 protein, S cerevisiae; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D064111:Minichromosome Maintenance Complex Component 2