Dihydrofolate reductase phosphorylation catalyzed by CK2α kinase and its influence on enzyme activity

Dihydrofolate reductase (DHFR) plays an essential role in cell growth and proliferation. In the cellular sole de novo thymidylate biosynthesis cycle, DHFR and thymidylate synthase (TS) catalyze sequential reactions, leading to formation of thymidine monophosphate — a substrate indispensable for DNA synthesis. Taking into account that posttranslational modifications, such as phosphorylation, can substantially change protein conformation and activity, studies of the enzyme’s regulation pathways are of great importance. It has been determined that thymidylate synthase (TS) catalytic activity is decreased due to CK2-catalyzed in vitro phosphorylation, whereas phosphorylation of dihydrofolate reductase (DHFR) is yet to be elucidated. CK2 is a constitutively active serine/ threonine kinase that phosphorylates even 20% of residues in eukaryotic cells. Considering the significance of the thymidylate synthesis cycle, we found it interesting to undertake studies on dihydrofolate reductase (DHFR) phosphorylation by CK2 and to examine possible influence of this modification on enzyme activity. Application of bioinformatics tools allowed in silico prediction of four amino acids on the polypeptide chain of human DHFR (hDHFR), which could be potentially phosphorylated by CK2. Performed experiments, involving far-Western techniques, indicated mutual interactions of human DHFR with the alpha subunit of human CK2 (CK2α). Kinetic parameters of CK2αcatalyzed phosphorylation of hDHFR were measured, using a quartz crystal microbalance with dissipation monitoring. Moreover, isotopic studies confirmed that hDHFR is a substrate for two variants of CK2α (the kinase with or without HisTag). However, linear time-dependence of hDHFR phosphorylation was observed only with CK2α without the HisTag variant, whereas the HisTag CK2α variant was inactivated in a time-dependent manner in the course of the reaction. P2