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Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3β Recombinant Kinases: Analysis of Phosphorylation Patterns by Nuclear Magnetic Resonance Spectroscopy

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Tau Protein

Abstract

Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein–protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer’s disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3β, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.

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Acknowledgments

The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille I. We acknowledge support from the TGE RMN THC (FR-3050, France) and the Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, “Structural & Functional Biochemistry of Biomolecular Assemblies”) for providing the scientific and technical environment. This study was supported by a grant from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer’s disease), and in part by the French government funding agency (Agence Nationale de la Recherche; ANR-21-CE29-0024 MAGNETAU).

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El Hajjar, L., Bridot, C., Nguyen, M., Cantrelle, FX., Landrieu, I., Smet-Nocca, C. (2024). Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3β Recombinant Kinases: Analysis of Phosphorylation Patterns by Nuclear Magnetic Resonance Spectroscopy. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_15

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