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Sensitive and Selective Next-Generation FRET-based PKA Biosensors.

Jin-Fan Zhang1,2,3,4, Wei Lin1,5,4, Leyi Huang1

  • 1Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.

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|February 23, 2026
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Summary
This summary is machine-generated.

New FRET sensors (AKAR6) offer enhanced sensitivity for visualizing protein kinase A (PKA) activity. These tools reveal compartmentalized PKA signaling, differentiating responses to growth factors and GPCRs at sub-organelle levels.

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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.