Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

Abstract Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosen...

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Autores principales:	Allen K. Kim, Helen D. Wu, Takanari Inoue
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bc7bba5871394f5b9099e2ddb367c9bb
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spelling	oai:doaj.org-article:bc7bba5871394f5b9099e2ddb367c9bb2021-12-02T17:52:13ZRational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter10.1038/s41598-020-66349-32045-2322https://doaj.org/article/bc7bba5871394f5b9099e2ddb367c9bb2020-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-66349-3https://doaj.org/toc/2045-2322Abstract Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.Allen K. KimHelen D. WuTakanari InoueNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-9 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Allen K. Kim Helen D. Wu Takanari Inoue Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
description	Abstract Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.
format	article
author	Allen K. Kim Helen D. Wu Takanari Inoue
author_facet	Allen K. Kim Helen D. Wu Takanari Inoue
author_sort	Allen K. Kim
title	Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
title_short	Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
title_full	Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
title_fullStr	Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
title_full_unstemmed	Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter
title_sort	rational design of a protein kinase a nuclear-cytosol translocation reporter
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/bc7bba5871394f5b9099e2ddb367c9bb
work_keys_str_mv	AT allenkkim rationaldesignofaproteinkinaseanuclearcytosoltranslocationreporter AT helendwu rationaldesignofaproteinkinaseanuclearcytosoltranslocationreporter AT takanariinoue rationaldesignofaproteinkinaseanuclearcytosoltranslocationreporter
_version_	1718379250688458752

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

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