Publikace detail

Sequential phosphorylation of tau protein by kinase-loaded magnetic beads

Autoři: Hromádková Lenka | Kupčík Rudolf | Šťovíčková Eliška | Slováková Marcela | Bílková Zuzana

Rok: 2019

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	Sequential phosphorylation of tau protein by kinase-loaded magnetic beads	Objectives: Tau is a multiply phosphorylated protein. In mature neurons, tau phosphorylation is likewise dynamically processed and participates in tau proper functions. In Alzheimer's disease, the occurrence of altered tau protein hyperphosphorylated in a site-specific manner results in tau missorting and aggregation. Thus, we introduce the preparation of tau forms with specifically phosphorylated residues in high purity as a defined product for further in vitro applications. Methods: Three kinases - GSK-3β, PKA and ERK2 - were covalently immobilized to magnetic beads. The residual activities as well as storage and operational stabilities were examined by phosphorylation of kinase-specific low molecular weight substrates. The kinase-loaded MPs were applied for sequential phosphorylation of tau. PKA and ERK2 loaded to the beads served as priming kinases before phosphorylation by GSK-3β-loaded magnetic beads. The phosphorylated residues of tau were determined by Q Exactive Plus (Thermo Fisher Scientific). Results: Kinase-loaded magnetic beads in their active forms were successfully prepared. Tau protein was phosphorylated at 35 residues by ERK2/GSK-3β immobilized system and at 32 residues by PKA/GSK-3β immobilized kinases. Pre-phosphorylation of tau by priming kinases has a great impact on tau phosphorylation profile, since 22 residues phosphorylated by these two systems differ. Conclusion: We successfully applied kinase-loaded magnetic beads for sequential phosphorylation of tau. By advanced MS analysis, we are able to define phosphorylated residues. The immobilized kinases may serve as a tool for preparation tau in defined phosphorylated form and in high purity for further sensitive immunochemical applications.	Sequential; phosphorylation; tau protein; kinase-loaded; magnetic; beads

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