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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-based sensors has made it poss...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068576/ https://www.ncbi.nlm.nih.gov/pubmed/29976855 http://dx.doi.org/10.3390/s18072164 |
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author | Musheshe, Nshunge Lobo, Miguel J. Schmidt, Martina Zaccolo, Manuela |
author_facet | Musheshe, Nshunge Lobo, Miguel J. Schmidt, Martina Zaccolo, Manuela |
author_sort | Musheshe, Nshunge |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-6068576 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-60685762018-08-07 Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 Musheshe, Nshunge Lobo, Miguel J. Schmidt, Martina Zaccolo, Manuela Sensors (Basel) Article 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. MDPI 2018-07-05 /pmc/articles/PMC6068576/ /pubmed/29976855 http://dx.doi.org/10.3390/s18072164 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Musheshe, Nshunge Lobo, Miguel J. Schmidt, Martina Zaccolo, Manuela Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title | 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_short | Targeting FRET-Based Reporters for cAMP and PKA Activity Using AKAP79 |
title_sort | targeting fret-based reporters for camp and pka activity using akap79 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068576/ https://www.ncbi.nlm.nih.gov/pubmed/29976855 http://dx.doi.org/10.3390/s18072164 |
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