A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Serine/threonine protein kinases regulate crucial cellular pathways.
• Dysregulated kinase activity is linked to various diseases, driving the need for targeted inhibitors.
• Existing high-throughput screening assays often require specific phospho-substrate antibodies, limiting their broad applicability.

Purpose of the Study:

• To develop a versatile, non-radioactive assay platform for screening kinase inhibitors.
• To establish a method utilizing a generic anti-phospho-threonine antibody for broad kinase activity assessment.
• To characterize the Cdc7/Dbf4 kinase and identify potential inhibitors.

Main Methods:

• Development of a time-resolved fluorescence assay.
• Utilizing a commercially available generic anti-phospho-threonine antibody.
• Assaying serine/threonine kinases capable of phosphorylating threonine residues on protein substrates.
• Characterization of Cdc7/Dbf4 kinase kinetics and inhibitor screening.

Main Results:

• A robust fluorescence assay platform was established for threonine-phosphorylating kinases.
• The assay was successfully applied to determine the ATP Michaelis constant (K(m)) for Cdc7/Dbf4 kinase.
• Rottlerin was identified as a non-ATP competitive inhibitor of Cdc7/Dbf4 kinase.

Conclusions:

• The developed assay platform offers a broadly applicable, high-throughput method for kinase inhibitor screening.
• This approach overcomes the limitation of generating specific antibodies for each substrate.
• The findings provide a valuable tool for drug discovery targeting kinases involved in disease.