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@proceedings{1855578,
author = {Náplavová, Alexandra and Kozeleková, Aneta and Hritz, Jozef},
booktitle = {XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB},
keywords = {14-3-3 protein; protein phosphorylation; phosphomimicking mutants},
language = {eng},
title = {Multiple approaches for protein phosphorylation: a story of 14-3-3},
url = {https://www.xray.cz/setkani/abst2022/442.htm},
year = {2022}
}
TY - CONF
ID - 1855578
AU - Náplavová, Alexandra - Kozeleková, Aneta - Hritz, Jozef
PY - 2022
TI - Multiple approaches for protein phosphorylation: a story of 14-3-3
KW - 14-3-3 protein
KW - protein phosphorylation
KW - phosphomimicking mutants
UR - https://www.xray.cz/setkani/abst2022/442.htm
N2 - Protein phosphorylation is a key regulatory mechanism involved in majority of biological processes [1]. In eukaryotes the dominantly phosphorylated residue is serine [2]. The phosphorylation of Ser58 has been observed for ubiquitous dimeric 14-3-3 proteins [3]. The 14-3-3 protein family represents a signalling hub, and its involvement has been confirmed in cancer progression and neurodegenerative diseases [4,5]. Since the Ser58 phosphorylation has been shown to induce monomerization, it has become a target of numerous studies to explore the properties of such monomer [3]. Unfortunately, the study of phosphorylation is often hindered by complicated sample preparation. This was the case of 14-3-3ζ as low or no phosphorylation has been achieved in pilot experiments, leading to the usage of so-called phosphomimicking mutants [6]. Since the reliability of phosphomimicking mutants is disputable [7], the goal of our work was to find a phosphorylation approach applicable for 14-3-3. Here we present four distinct methods for preparation of 14-3-3ζ phosphorylated at Ser58: in vitro phosphorylation by catalytic subunit of protein kinase A (PKA), co-expression of 14-3-3ζ and PKA in E. coli, in vivo phosphorylation by PKA covalently linked to the 14-3-3ζ (i.e., chimeric construct) and a direct incorporation of phosphoserine via expanded genetic code. We have tested and compared the methods based on their efficiency, yield and simplicity. Moreover, we offer direct comparison with the phosphomimicking mutants and show the shortcomings of their employment.
ER -
NÁPLAVOVÁ, Alexandra, Aneta KOZELEKOVÁ and Jozef HRITZ. Multiple approaches for protein phosphorylation: a story of 14-3-3. In \textit{XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB}. 2022.
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