Human dihydrofolate reductase is a substrate of protein kinase CK2α

Katarzyna Skierka , Paweł Wilamowski , Monika Wielechowska , Dominik Cysewski , Elżbieta Senkara-Barwijuk , Patrycja Wińska , Maria Bretner , Joanna Cieśla

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 x 104 M-1s-1 and koff = 1.40 x 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.
Author Katarzyna Skierka (FC / CDSB)
Katarzyna Skierka,,
- Chair of Drug and Cosmetics Biotechnology
, Paweł Wilamowski (FC / CDSB)
Paweł Wilamowski,,
- Chair of Drug and Cosmetics Biotechnology
, Monika Wielechowska (FC / CDSB)
Monika Wielechowska,,
- Chair of Drug and Cosmetics Biotechnology
, Dominik Cysewski
Dominik Cysewski,,
-
, Elżbieta Senkara-Barwijuk (FC / CDSB)
Elżbieta Senkara-Barwijuk,,
- Chair of Drug and Cosmetics Biotechnology
, Patrycja Wińska (FC / CDSB)
Patrycja Wińska,,
- Chair of Drug and Cosmetics Biotechnology
, Maria Bretner (FC / CDSB)
Maria Bretner,,
- Chair of Drug and Cosmetics Biotechnology
, Joanna Cieśla (FC / CDSB)
Joanna Cieśla,,
- Chair of Drug and Cosmetics Biotechnology
Journal seriesBiochemical and Biophysical Research Communications, ISSN 0006-291X, (A 25 pkt)
Issue year2019
Vol513
No2
Pages368-373
Publication size in sheets0.5
Keywords in Englishprotein phosphorylation, site-directed mutagenesis, dihydrofolate reductase, CK2, quartz crystal microbalance, mass spectrometry
ASJC Classification1307 Cell Biology; 1312 Molecular Biology; 1303 Biochemistry; 1304 Biophysics
DOIDOI:10.1016/j.bbrc.2019.03.186
URL https://www.sciencedirect.com/science/article/pii/S0006291X19305935?via%3Dihub
Languageen angielski
File
wdpb_publikacje_pliki_plik_publikacja_3686_org.pdf 496.51 KB
Score (nominal)25
ScoreMinisterial score = 25.0, 13-08-2019, ArticleFromJournal
Publication indicators Scopus SNIP (Source Normalised Impact per Paper): 2016 = 0.688; WoS Impact Factor: 2017 = 2.559 (2) - 2017=2.455 (5)
Citation count*
Cite
Share Share

Get link to the record


* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
Back