Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79
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-...
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doaj-0eb5239a5cc8498a9d42b3e32384ed152020-11-24T21:00:39ZengMDPI AGSensors1424-82202018-07-01187216410.3390/s18072164s18072164Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79Nshunge Musheshe0Miguel J. Lobo1Martina Schmidt2Manuela Zaccolo3Department of Molecular Pharmacology, University of Groningen, PO Box 72, 9700 AB Groningen, The NetherlandsDepartment of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 2JD, UKDepartment of Molecular Pharmacology, University of Groningen, PO Box 72, 9700 AB Groningen, The NetherlandsDepartment of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 2JD, UKFluorescence 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.http://www.mdpi.com/1424-8220/18/7/2164fluorescence resonance energy transfer (FRET)AKAP79cAMPprotein kinase A (PKA)phosphatasesadrenergic signalingreal-time imaging |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Nshunge Musheshe Miguel J. Lobo Martina Schmidt Manuela Zaccolo |
spellingShingle |
Nshunge Musheshe Miguel J. Lobo Martina Schmidt Manuela Zaccolo Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 Sensors fluorescence resonance energy transfer (FRET) AKAP79 cAMP protein kinase A (PKA) phosphatases adrenergic signaling real-time imaging |
author_facet |
Nshunge Musheshe Miguel J. Lobo Martina Schmidt Manuela Zaccolo |
author_sort |
Nshunge Musheshe |
title |
Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_short |
Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_full |
Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_fullStr |
Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_full_unstemmed |
Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_sort |
targeting fret-based reporters for camp and pka activity using akap79 |
publisher |
MDPI AG |
series |
Sensors |
issn |
1424-8220 |
publishDate |
2018-07-01 |
description |
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. |
topic |
fluorescence resonance energy transfer (FRET) AKAP79 cAMP protein kinase A (PKA) phosphatases adrenergic signaling real-time imaging |
url |
http://www.mdpi.com/1424-8220/18/7/2164 |
work_keys_str_mv |
AT nshungemusheshe targetingfretbasedreportersforcampandpkaactivityusingakap79 AT migueljlobo targetingfretbasedreportersforcampandpkaactivityusingakap79 AT martinaschmidt targetingfretbasedreportersforcampandpkaactivityusingakap79 AT manuelazaccolo targetingfretbasedreportersforcampandpkaactivityusingakap79 |
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1716779038314332160 |