DnaK promotes autophosphorylation of DYRK1A and its family kinases in Escherichia coli-based cell-free protein expression.

Biochem Biophys Res Commun

Laboratory for Drug Target Research, Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304 Minami-Minowa, Kami-Ina, Nagano, 399-4598, Japan; Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minami-Minowa, Kami-ina, Nagano, 399-4598, Japan. Electronic address:

Published: December 2023

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.

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http://dx.doi.org/10.1016/j.bbrc.2023.149220DOI Listing

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