Luminescent kinase activity biosensors based on a versatile bimolecular switch

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Real-time kinase activity tracking is crucial for understanding cellular signaling.
• Current methods, often using FRET, face sensitivity limitations.
• New tools are needed for sensitive and versatile kinase activity detection.

Purpose of the Study:

• To design a novel kinase-inducible bimolecular switch for enhanced sensitivity and versatility in detecting kinase activity.
• To couple this switch to FRET and bioluminescence readouts.
• To demonstrate its application in sensing specific kinase activities like PKA and PKC.

Main Methods:

• Development of a kinase-inducible bimolecular switch comprising a kinase substrate and a phosphoamino acid binding domain.
• Coupling the switch to Förster Resonance Energy Transfer (FRET) and bioluminescence imaging.
• Design and testing of sensors for protein kinase A (PKA) and protein kinase C (PKC).

Main Results:

• Bimolecular switches successfully detected PKA and PKC activities using both FRET and bioluminescence.
• FRET-based sensors exhibited larger dynamic ranges compared to unimolecular sensors.
• Bioluminescence reporters demonstrated high sensitivity, detecting basal kinase activities.

Conclusions:

• The generalizable bimolecular switch design significantly advances kinase activity detection tools.
• This approach offers versatile and sensitive detection for various biological systems.
• The novel reporters enable new applications in in vivo imaging and high-throughput screening.