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Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein

Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phos...

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Autores principales: Kozeleková, Aneta, Náplavová, Alexandra, Brom, Tomáš, Gašparik, Norbert, Šimek, Jan, Houser, Josef, Hritz, Jozef
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8935074/
https://www.ncbi.nlm.nih.gov/pubmed/35321476
http://dx.doi.org/10.3389/fchem.2022.835733
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author Kozeleková, Aneta
Náplavová, Alexandra
Brom, Tomáš
Gašparik, Norbert
Šimek, Jan
Houser, Josef
Hritz, Jozef
author_facet Kozeleková, Aneta
Náplavová, Alexandra
Brom, Tomáš
Gašparik, Norbert
Šimek, Jan
Houser, Josef
Hritz, Jozef
author_sort Kozeleková, Aneta
collection PubMed
description Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phosphomimicking mutations at the desired site of phosphorylation. 14-3-3 proteins are regulatory protein hubs that interact with hundreds of phosphorylated proteins and modulate their structure and activity. 14-3-3 protein function relies on its dimeric nature, which is controlled by Ser58 phosphorylation. However, incomplete Ser58 phosphorylation has obstructed the detailed study of its effect so far. In the present study, we describe the full and specific phosphorylation of 14-3-3ζ protein at Ser58 and we compare its characteristics with phosphomimicking mutants that have been used in the past (S58E/D). Our results show that in case of the 14-3-3 proteins, phosphomimicking mutations are not a sufficient replacement for phosphorylation. At physiological concentrations of 14-3-3ζ protein, the dimer-monomer equilibrium of phosphorylated protein is much more shifted towards monomers than that of the phosphomimicking mutants. The oligomeric state also influences protein properties such as thermodynamic stability and hydrophobicity. Moreover, phosphorylation changes the localization of 14-3-3ζ in HeLa and U251 human cancer cells. In summary, our study highlights that phosphomimicking mutations may not faithfully represent the effects of phosphorylation on the protein structure and function and that their use should be justified by comparing to the genuinely phosphorylated counterpart.
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spelling pubmed-89350742022-03-22 Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein Kozeleková, Aneta Náplavová, Alexandra Brom, Tomáš Gašparik, Norbert Šimek, Jan Houser, Josef Hritz, Jozef Front Chem Chemistry Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phosphomimicking mutations at the desired site of phosphorylation. 14-3-3 proteins are regulatory protein hubs that interact with hundreds of phosphorylated proteins and modulate their structure and activity. 14-3-3 protein function relies on its dimeric nature, which is controlled by Ser58 phosphorylation. However, incomplete Ser58 phosphorylation has obstructed the detailed study of its effect so far. In the present study, we describe the full and specific phosphorylation of 14-3-3ζ protein at Ser58 and we compare its characteristics with phosphomimicking mutants that have been used in the past (S58E/D). Our results show that in case of the 14-3-3 proteins, phosphomimicking mutations are not a sufficient replacement for phosphorylation. At physiological concentrations of 14-3-3ζ protein, the dimer-monomer equilibrium of phosphorylated protein is much more shifted towards monomers than that of the phosphomimicking mutants. The oligomeric state also influences protein properties such as thermodynamic stability and hydrophobicity. Moreover, phosphorylation changes the localization of 14-3-3ζ in HeLa and U251 human cancer cells. In summary, our study highlights that phosphomimicking mutations may not faithfully represent the effects of phosphorylation on the protein structure and function and that their use should be justified by comparing to the genuinely phosphorylated counterpart. Frontiers Media S.A. 2022-03-07 /pmc/articles/PMC8935074/ /pubmed/35321476 http://dx.doi.org/10.3389/fchem.2022.835733 Text en Copyright © 2022 Kozeleková, Náplavová, Brom, Gašparik, Šimek, Houser and Hritz. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Chemistry
Kozeleková, Aneta
Náplavová, Alexandra
Brom, Tomáš
Gašparik, Norbert
Šimek, Jan
Houser, Josef
Hritz, Jozef
Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title_full Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title_fullStr Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title_full_unstemmed Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title_short Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
title_sort phosphorylated and phosphomimicking variants may differ—a case study of 14-3-3 protein
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8935074/
https://www.ncbi.nlm.nih.gov/pubmed/35321476
http://dx.doi.org/10.3389/fchem.2022.835733
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