Multiple approaches for protein phosphorylation: a story of 14-3-3

NÁPLAVOVÁ, Alexandra, Aneta KOZELEKOVÁ a Jozef HRITZ. Multiple approaches for protein phosphorylation: a story of 14-3-3. In XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB. 2022.

Základní údaje

Originální název

Multiple approaches for protein phosphorylation: a story of 14-3-3

Autoři

NÁPLAVOVÁ, Alexandra, Aneta KOZELEKOVÁ a Jozef HRITZ

Vydání

XVIII Discussions in Structural Molecular Biology and 5th User Meeting of CIISB, 2022

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Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Organizační jednotka

Středoevropský technologický institut

Klíčová slova česky

14-3-3 protein; fosforylace; fosfomimikující mutace

Klíčová slova anglicky

14-3-3 protein; protein phosphorylation; phosphomimicking mutants

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Anotace

V originále

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.

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Návaznosti

GF20-05789L, projekt VaV	Název: Charakterizace přirozeně neuspořádaných proteinů Investor: Grantová agentura ČR, Characterization of intrinsically disordered proteins, Partnerská agentura (Rakousko)
LM2018127, velká výzkumná infrastruktura	Název: Czech Infrastructure for Integrative Structural Biology (Akronym: CIISB) Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Czech Infrastructure for Integrative Structural Biology
LM2018127, velká výzkumná infrastruktura	Název: Česká infrastruktura pro integrativní strukturní biologii (Akronym: CIISB) Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Czech Infrastructure for Integrative Structural Biology
MUNI/A/1419/2021, interní kód MU	Název: Specifický výzkum v oblasti „Life Sciences“ Investor: Masarykova univerzita, Specifický výzkum v oblasti „Life Sciences“