Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Cyclic AMP-dependent protein kinase (PKA) is crucial in cellular signaling pathways.
• Monitoring PKA activity in real-time within live cells is challenging.
• Firefly luciferase is a versatile reporter protein for biological processes.

Purpose of the Study:

• To engineer firefly luciferase to incorporate PKA phosphorylation sites.
• To create a novel biosensor for detecting PKA-mediated phosphorylation.
• To characterize the properties of the engineered luciferase variants.

Main Methods:

• In vitro amplification of Photinus pyralis luciferase cDNA.
• Site-directed mutagenesis to introduce RRFS phosphorylation site.
• Addition of kemptide sequence to N- or C-terminus.
• In vitro transcription and translation of engineered genes.
• Characterization of recombinant proteins: specific activity, pI, pH-dependent spectral shift, and kinase/phosphatase assays.

Main Results:

• Engineered luciferase variants were successfully produced.
• The RRFS mutant exhibited significantly altered properties compared to wild-type luciferase.
• RRFS mutant showed reduced specific activity, a lower pH optimum, and a greener light emission at low pH.
• Phosphorylation of RRFS mutant by PKA catalytic subunit decreased its activity by up to 80%, reversible by phosphatase.

Conclusions:

• The RRFS mutant firefly luciferase is a sensitive reporter for PKA activity.
• This engineered protein enables the measurement of cyclic AMP-dependent phosphorylation in live cells for the first time.
• The developed biosensor holds potential for advancing research in cellular signaling and drug discovery.