Abstract
Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
Highlights
dihydrofolate reductase (DHFR) and thymidylate synthase (TS) protein levels were measured by means of western blot in A-549 and CCRF-CEM, whereas the levels of SHMT1 and
In this follow-up study, we show the interactions between the pairs TS and SHMT1, DHFR, and SHMT1 and the formation of the tri-molecular complex consisting of all three thymidylate synthesis proteins, TS, DHFR, and SHMT1
The only statistically significant results showing some changes are the increase in DHFR (Wińska et al, 2019) and SHMT1 transcripts in cells treated with 6 μM CX-4945 for 24 h, decrease in TS and SHMT2 in cells treated with 3 μM CX-4945 for 72 h, and decrease in DHFR in cells treated with 6 μM inhibitor for 72 h
Summary
CK2 (formerly known as casein kinase II) is a protein kinase involved in regulation of many processes such as transcription (Lüscher et al, 1990; Cabrejos et al, 2004) and translation (Riera et al, 1999; Szebeni et al, 2003; Borgo et al, 2015; Gandin et al, 2016), control of protein stability (Zhang et al, 2002; Patsoukis et al, 2013; Niechi et al, 2015) and degradation (Shen et al, 2001; Scaglioni et al, 2009), cell cycle progression (Homma and Homma, 2008), cell survival (Ahmed et al, 2002; Piazza et al, 2006; Duncan et al, 2011), and circadian rhythms (Tsuchiya et al, 2009) The role of this conserved, constitutively active serine–threonine kinase in a cell regulatory network is highly complex, and the extensive interplay between CK2-mediated phosphorylation and other posttranslational modifications has been suggested (Nuñez de Villavicencio–Diaz et al, 2017).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
