Sensitive and Selective Next-Generation FRET-based PKA Biosensors

Area of Science:

• Cellular Biology
• Molecular Signaling
• Biotechnology

Background:

• The cyclic AMP (cAMP)/protein kinase A (PKA) pathway is crucial for cellular processes, requiring precise spatiotemporal control.
• Förster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) visualize PKA activity but have limitations in dynamic range and specificity.
• Detecting subtle or compartment-specific PKA signaling events remains challenging with existing tools.

Purpose of the Study:

• To develop and validate a new generation of FRET-based PKA sensors (AKAR6) with improved sensitivity and selectivity.
• To enable more accurate visualization of PKA activity across subcellular compartments and diverse experimental settings.
• To investigate the compartmentalization and temporal dynamics of PKA signaling in response to different stimuli.

Main Methods:

• Engineering and systematic optimization of cyan/yellow FRET-based PKA sensors (AKAR6 series).
• Leveraging kinome atlas data to enhance sensor selectivity for nuclear PKA activity.
• Application of AKAR6 sensors in flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices.
• Utilizing PC12 cells to compare PKA activity induced by GPCRs versus growth factors (NGF, EGF).

Main Results:

• The AKAR6 series demonstrates substantially enhanced sensitivity and selectivity compared to previous AKARs.
• AKAR6 sensors enable robust detection of subtle PKA activity changes across various experimental modalities.
• Growth factors induce significant PKA activity at the trans-Golgi network but not cis-Golgi, highlighting sub-organelle compartmentalization.
• NGF and EGF elicit sustained and transient PKA activity, respectively, across multiple intracellular compartments, including the nucleus.

Conclusions:

• The AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling.
• These advanced sensors allow for a more nuanced understanding of PKA activity dynamics across subcellular locations.
• The findings reveal sophisticated spatial and temporal regulation of PKA signaling by different stimuli.