An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates

© 2024 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 (https://creativecommons.org/licenses/by/4.0/).

Detalles Bibliográficos
Autores: Cayuela, Andrés, Villasante-Fernández, Adela, Corbalán-Acedo, Antonio, Baena-González, Elena, Ferrando, Alejandro, Belda-Palazón, Borja
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2024
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/352829
Acceso en línea:http://hdl.handle.net/10261/352829
Access Level:acceso abierto
Palabra clave:Posttranslational modifications
Protein phosphorylation
Kinase
Escherichia coli phosphorylation system
Arabidopsis thaliana
SnRK1 signaling network
Mitogen-activated protein kinase cascades
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spelling An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase SubstratesCayuela, AndrésVillasante-Fernández, AdelaCorbalán-Acedo, AntonioBaena-González, ElenaFerrando, AlejandroBelda-Palazón, BorjaPosttranslational modificationsProtein phosphorylationKinaseEscherichia coli phosphorylation systemArabidopsis thalianaSnRK1 signaling networkMitogen-activated protein kinase cascades© 2024 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 (https://creativecommons.org/licenses/by/4.0/).Posttranslational modifications (PTMs), particularly phosphorylation, play a pivotal role in expanding the complexity of the proteome and regulating diverse cellular processes. In this study, we present an efficient Escherichia coli phosphorylation system designed to streamline the evaluation of potential substrates for Arabidopsis thaliana plant kinases, although the technology is amenable to any. The methodology involves the use of IPTG-inducible vectors for co-expressing kinases and substrates, eliminating the need for radioactive isotopes and prior protein purification. We validated the system’s efficacy by assessing the phosphorylation of well-established substrates of the plant kinase SnRK1, including the rat ACETYL-COA CARBOXYLASE 1 (ACC1) and FYVE1/FREE1 proteins. The results demonstrated the specificity and reliability of the system in studying kinase-substrate interactions. Furthermore, we applied the system to investigate the phosphorylation cascade involving the A. thaliana MKK3-MPK2 kinase module. The activation of MPK2 by MKK3 was demonstrated to phosphorylate the Myelin Basic Protein (MBP), confirming the system’s ability to unravel sequential enzymatic steps in phosphorylation cascades. Overall, this E. coli phosphorylation system offers a rapid, cost-effective, and reliable approach for screening potential kinase substrates, presenting a valuable tool to complement the current portfolio of molecular techniques for advancing our understanding of kinase functions and their roles in cellular signaling pathways.This research was funded by the CIDEGENT program of excellence of GVA, grant number CIDEXG/2022/27 (awarded to B.B.-P.), and Spanish Ministry of Science, Innovation and Universities (MCIU), grant number PID2022-142412NB-I00 (awarded to B.B.-P. and A.F.). A.C. was the beneficiary of a PRE2022-105204 contract funded by MCIN/AEI and FSE+. A.V.-F. was the beneficiary of a PhD contract CIDEXG/2022/27-02 funded by GVA. E.B.-G. was supported by FCT—Fundação para a Ciência e a Tecnologia (GREEN-IT—Bioresources for Sustainability R&D Unit UIDB/04551/2020, UIDP/04551/2020; LS4FUTURE Associated Laboratory LA/P/0087/2020; grant PTDC/BIA-FBT/4942/2020).Peer reviewedMultidisciplinary Digital Publishing InstituteMinisterio de Ciencia, Innovación y Universidades (España)Generalitat ValencianaAgencia Estatal de Investigación (España)Fundação para a Ciência e a Tecnologia (Portugal)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2024202420242024info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/352829reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2022-142412NB-I00https://doi.org/10.3390/ijms25073813Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3528292026-05-22T06:33:51Z
dc.title.none.fl_str_mv An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
title An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
spellingShingle An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
Cayuela, Andrés
Posttranslational modifications
Protein phosphorylation
Kinase
Escherichia coli phosphorylation system
Arabidopsis thaliana
SnRK1 signaling network
Mitogen-activated protein kinase cascades
title_short An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
title_full An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
title_fullStr An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
title_full_unstemmed An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
title_sort An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates
dc.creator.none.fl_str_mv Cayuela, Andrés
Villasante-Fernández, Adela
Corbalán-Acedo, Antonio
Baena-González, Elena
Ferrando, Alejandro
Belda-Palazón, Borja
author Cayuela, Andrés
author_facet Cayuela, Andrés
Villasante-Fernández, Adela
Corbalán-Acedo, Antonio
Baena-González, Elena
Ferrando, Alejandro
Belda-Palazón, Borja
author_role author
author2 Villasante-Fernández, Adela
Corbalán-Acedo, Antonio
Baena-González, Elena
Ferrando, Alejandro
Belda-Palazón, Borja
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia, Innovación y Universidades (España)
Generalitat Valenciana
Agencia Estatal de Investigación (España)
Fundação para a Ciência e a Tecnologia (Portugal)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Posttranslational modifications
Protein phosphorylation
Kinase
Escherichia coli phosphorylation system
Arabidopsis thaliana
SnRK1 signaling network
Mitogen-activated protein kinase cascades
topic Posttranslational modifications
Protein phosphorylation
Kinase
Escherichia coli phosphorylation system
Arabidopsis thaliana
SnRK1 signaling network
Mitogen-activated protein kinase cascades
description © 2024 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 (https://creativecommons.org/licenses/by/4.0/).
publishDate 2024
dc.date.none.fl_str_mv 2024
2024
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/352829
url http://hdl.handle.net/10261/352829
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2022-142412NB-I00
https://doi.org/10.3390/ijms25073813
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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