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Open AccessArticle

Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79

1
Department of Molecular Pharmacology, University of Groningen, PO Box 72, 9700 AB Groningen, The Netherlands
2
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 2JD, UK
3
Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen, University of Groningen, PO Box 72, 9700 AB Groningen, The Netherlands
*
Author to whom correspondence should be addressed.
Sensors 2018, 18(7), 2164; https://doi.org/10.3390/s18072164
Received: 29 May 2018 / Revised: 28 June 2018 / Accepted: 3 July 2018 / Published: 5 July 2018
(This article belongs to the Special Issue Luminescence and Chemiluminescence Sensors)
Fluorescence resonance energy transfer (FRET)-based sensors for 3′–5′cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) allow real-time imaging of cAMP levels and kinase activity in intact cells with high spatiotemporal resolution. The development of FRET-based sensors has made it possible to directly demonstrate that cAMP and PKA signals are compartmentalized. These sensors are currently widely used to dissect the organization and physiological function of local cAMP/PKA signaling events in a variety of cell systems. Fusion to targeting domains has been used to direct the sensors to a specific subcellular nanodomain and to monitor cAMP and PKA activity at specific subcellular sites. Here, we investigate the effects of using the A-kinase anchoring protein 79 (AKAP79) as a targeting domain for cAMP and PKA FRET-based reporters. As AKAP79 interacts with PKA itself, when used as a targeting domain, it can potentially impact on the amplitude and kinetics of the signals recorded locally. By using as the targeting domain wild type AKAP79 or a mutant that cannot interact with PKA, we establish that AKAP79 does not affect the amplitude and kinetics of cAMP changes or the level of PKA activity detected by the sensor. View Full-Text
Keywords: fluorescence resonance energy transfer (FRET); AKAP79; cAMP; protein kinase A (PKA); phosphatases; adrenergic signaling; real-time imaging fluorescence resonance energy transfer (FRET); AKAP79; cAMP; protein kinase A (PKA); phosphatases; adrenergic signaling; real-time imaging
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Musheshe, N.; Lobo, M.J.; Schmidt, M.; Zaccolo, M. Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79. Sensors 2018, 18, 2164.

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