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Updated: Jun 27, 2026
Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Molecular imaging of phosphorylation events for drug development
C T Chan1, R Paulmurugan, R E Reeves
1Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Purpose:
Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging.
Procedures:
An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA).
Results:
The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity.
Conclusion:
This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events.
Insights
A novel split firefly luciferase system enables noninvasive monitoring of protein phosphorylation and kinase inhibitor efficacy in cells and living subjects. This tool accelerates the discovery of new kinase inhibitors.
Area of Science:
- Biochemistry and Molecular Biology
- Cellular Signaling
- Biotechnology
Background:
- Protein phosphorylation is a critical regulator of cellular processes, controlled by protein kinases.
- Monitoring phosphorylation events and kinase inhibitor efficacy is essential for understanding cellular functions and developing therapeutics.
- Existing methods for monitoring phosphorylation can be invasive or lack generalizability.
Purpose of the Study:
- To develop a genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system for noninvasive monitoring of phosphorylation events.
- To assess the system's utility in evaluating the efficacies of kinase inhibitors in cell culture and small living subjects using optical bioluminescence imaging.
- To establish a tool for screening chemical libraries to identify novel modulators of kinase activity.
Main Methods:
- Construction of an Akt sensor (AST) utilizing split firefly luciferase complementation to monitor Akt phosphorylation.
- Validation of AST specificity using a non-phosphorylable mutant sensor (ASA).
- Application of the system in cell culture (293T cells) and in vivo (nude mice xenografts) to assess responses to PI-3K and Akt inhibitors (LY294002, perifosine).
Main Results:
- The AST system demonstrated temporal- and dose-dependent increases in complemented FL activity in response to inhibitors in AST-expressing cells, but not in ASA cells.
- Inhibition of endogenous Akt phosphorylation and kinase activity by perifosine correlated with increased FL activity in AST cells.
- In vivo studies showed a significant increase in FL activity in xenografts treated with perifosine, confirming the system's applicability in living subjects.
Conclusions:
- The developed split firefly luciferase-assisted complementation system provides a generalizable and noninvasive method for monitoring phosphorylation events.
- This approach significantly accelerates the discovery and validation of novel kinase inhibitors and modulators of phosphorylation.
- The system holds promise for advancing drug discovery and development in kinase-related diseases.
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