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Publication detail

Sequential phosphorylation of tau protein by kinase-loaded magnetic beads
Authors: Hromádková Lenka | Kupčík Rudolf | Šťovíčková Eliška | Slováková Marcela | Bílková Zuzana
Year: 2019
Type of publication: ostatní - přednáška nebo poster
Page from-to: nestránkováno
Titles:
Language Name Abstract Keywords
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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