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Biochem Biophys Res Commun. 2019 Apr 5. pii: S0006-291X(19)30593-5. doi: 10.1016/j.bbrc.2019.03.186. [Epub ahead of print]

Human dihydrofolate reductase is a substrate of protein kinase CK2α.

Author information

1
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664, Warsaw, Poland.
2
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664, Warsaw, Poland. Electronic address: pwilamowski92@gmail.com.
3
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.
4
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664, Warsaw, Poland. Electronic address: esenkara@senkara.pl.
5
Warsaw University of Technology, Faculty of Chemistry, Noakowskiego 3, 00-664, Warsaw, Poland. Electronic address: jciesla@ch.pw.edu.pl.

Abstract

Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (Kd below 0.5 μM, kon = 10.31 × 104 M-1s-1 and koff = 1.40 × 10-3s-1) and calculated Gibbs free energy (-36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.

KEYWORDS:

CK2; Dihydrofolate reductase; Mass spectrometry; Protein phosphorylation; Quartz crystal microbalance; Site-directed mutagenesis

PMID:
30961929
DOI:
10.1016/j.bbrc.2019.03.186

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