Human dihydrofolate reductase is a substrate of protein kinase CK2α

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 transformati...

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Bibliographic Details
Published in:	Biochemical and biophysical research communications Vol. 513; no. 2; pp. 368 - 373
Main Authors:	Skierka, Katarzyna, Wilamowski, Paweł, Wielechowska, Monika, Cysewski, Dominik, Senkara, Elżbieta, Wińska, Patrycja, Bretner, Maria, Cieśla, Joanna
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 28-05-2019
Subjects:	Casein Kinase II - chemistry Casein Kinase II - metabolism Catalytic Domain CK2 Dihydrofolate reductase Humans Kinetics Mass spectrometry Phosphorylation Protein Binding Protein Interaction Maps Protein phosphorylation Quartz crystal microbalance Site-directed mutagenesis Substrate Specificity Tetrahydrofolate Dehydrogenase - metabolism Dihydrofolate reductase CK2 Quartz crystal microbalance Protein phosphorylation Mass spectrometry Site-directed mutagenesis
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Summary:	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. •Dihydrofolate reductase and CK2 kinase are molecular targets in chemotherapies.•CK2 strongly interacts with and phosphorylates dihydrofolate reductase.•Serines 168 and 145 were identified as CK2 phosphorylation sites.
ISSN:	0006-291X 1090-2104
DOI:	10.1016/j.bbrc.2019.03.186