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@article{1839679,
author = {Kozeleková, Aneta and Náplavová, Alexandra and Brom, Tomáš and Gašparik, Norbert and Šimek, Jan and Houser, Josef and Hritz, Jozef},
article_number = {March},
doi = {http://dx.doi.org/10.3389/fchem.2022.835733},
keywords = {14-3-3; phosphorylation; phosphomimicking mutation; oligomeric state; dissociation constant},
language = {eng},
issn = {2296-2646},
journal = {Frontiers in Chemistry},
title = {Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein},
url = {https://www.frontiersin.org/articles/10.3389/fchem.2022.835733/full},
volume = {10},
year = {2022}
}
TY - JOUR
ID - 1839679
AU - Kozeleková, Aneta - Náplavová, Alexandra - Brom, Tomáš - Gašparik, Norbert - Šimek, Jan - Houser, Josef - Hritz, Jozef
PY - 2022
TI - Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein
JF - Frontiers in Chemistry
VL - 10
IS - March
SP - 1-17
EP - 1-17
PB - Frontiers Media SA
SN - 22962646
KW - 14-3-3
KW - phosphorylation
KW - phosphomimicking mutation
KW - oligomeric state
KW - dissociation constant
UR - https://www.frontiersin.org/articles/10.3389/fchem.2022.835733/full
N2 - 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.
ER -
KOZELEKOVÁ, Aneta, Alexandra NÁPLAVOVÁ, Tomáš BROM, Norbert GAŠPARIK, Jan ŠIMEK, Josef HOUSER and Jozef HRITZ. Phosphorylated and Phosphomimicking Variants May Differ—A Case Study of 14-3-3 Protein. \textit{Frontiers in Chemistry}. Frontiers Media SA, 2022, vol.~10, March, p.~1-17. ISSN~2296-2646. Available from: https://dx.doi.org/10.3389/fchem.2022.835733.
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