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Engineered kinases as a tool for phosphorylation of selected targets in vivo

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase–substrate interaction is that kinases form complex signaling networks and most of...

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Autores principales: Lepeta, Katarzyna, Roubinet, Chantal, Bauer, Milena, Vigano, M. Alessandra, Aguilar, Gustavo, Kanca, Oguz, Ochoa-Espinosa, Amanda, Bieli, Dimitri, Cabernard, Clemens, Caussinus, Emmanuel, Affolter, Markus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9477969/
https://www.ncbi.nlm.nih.gov/pubmed/36102907
http://dx.doi.org/10.1083/jcb.202106179
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author Lepeta, Katarzyna
Roubinet, Chantal
Bauer, Milena
Vigano, M. Alessandra
Aguilar, Gustavo
Kanca, Oguz
Ochoa-Espinosa, Amanda
Bieli, Dimitri
Cabernard, Clemens
Caussinus, Emmanuel
Affolter, Markus
author_facet Lepeta, Katarzyna
Roubinet, Chantal
Bauer, Milena
Vigano, M. Alessandra
Aguilar, Gustavo
Kanca, Oguz
Ochoa-Espinosa, Amanda
Bieli, Dimitri
Cabernard, Clemens
Caussinus, Emmanuel
Affolter, Markus
author_sort Lepeta, Katarzyna
collection PubMed
description Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase–substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase–substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.
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spelling pubmed-94779692022-09-27 Engineered kinases as a tool for phosphorylation of selected targets in vivo Lepeta, Katarzyna Roubinet, Chantal Bauer, Milena Vigano, M. Alessandra Aguilar, Gustavo Kanca, Oguz Ochoa-Espinosa, Amanda Bieli, Dimitri Cabernard, Clemens Caussinus, Emmanuel Affolter, Markus J Cell Biol Tools Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase–substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase–substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors. Rockefeller University Press 2022-09-14 /pmc/articles/PMC9477969/ /pubmed/36102907 http://dx.doi.org/10.1083/jcb.202106179 Text en © 2022 Lepeta et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
spellingShingle Tools
Lepeta, Katarzyna
Roubinet, Chantal
Bauer, Milena
Vigano, M. Alessandra
Aguilar, Gustavo
Kanca, Oguz
Ochoa-Espinosa, Amanda
Bieli, Dimitri
Cabernard, Clemens
Caussinus, Emmanuel
Affolter, Markus
Engineered kinases as a tool for phosphorylation of selected targets in vivo
title Engineered kinases as a tool for phosphorylation of selected targets in vivo
title_full Engineered kinases as a tool for phosphorylation of selected targets in vivo
title_fullStr Engineered kinases as a tool for phosphorylation of selected targets in vivo
title_full_unstemmed Engineered kinases as a tool for phosphorylation of selected targets in vivo
title_short Engineered kinases as a tool for phosphorylation of selected targets in vivo
title_sort engineered kinases as a tool for phosphorylation of selected targets in vivo
topic Tools
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9477969/
https://www.ncbi.nlm.nih.gov/pubmed/36102907
http://dx.doi.org/10.1083/jcb.202106179
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