Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Feb 3;24(3):3023.
doi: 10.3390/ijms24033023.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing

Patrycja Wińska  1 , Anna Sobiepanek  1 , Katarzyna Pawlak  2 , Monika Staniszewska  3 , Joanna Cieśla  1
Affiliations

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing

Patrycja Wińska et al. Int J Mol Sci. .

Abstract

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

Keywords: acute lymphoblastic leukaemia; dihydrofolate reductase DHFR; in-gel kinase assay; lung adenocarcinoma; protein kinase CK2; protein phosphorylation; serine hydroxymethyltransferase SHMT; thymidylate synthase TS.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Thymidylate synthesis cycle. TS—thymidylate synthase; DHFR—dihydrofolate reductase; SHMT—serine hydroxymethyltransferase; dUMP—2′-deoxyuridine-5′-monophosphate; TMP, TDP, TTP—2′-deoxythymidine-5′-mono-, di-, and triphosphate, respectively; DHF—dihydrofolate; THF—tetrahydrofolate; meTHF—N5,10-methylenetetrahydrofolate.
Figure 2
Figure 2
Immunodetection of CK2α, TS, and DHFR proteins in A-549 treated with 25 nM, 50 nM, and 100 nM CSNK2A1 siRNA for 48 h and 72 h. (a) A representative Western blot. (b–e) Densitometry analysis data for the tested proteins in A-549 treated with CSNK2A1 siRNA for 48 h (b), and for TS (c), DHFR (d), and CK2α (e) after 72 h of treatment with CSNK2A1 siRNA. Tubulin was used as a loading control for each sample. Section 4 describes the preparation of cell extracts and protein detection. Densitometry quantifications for each tested protein, given under each cell line panel, were calculated with untreated cells (CTRL) as the reference point. Graphs represent mean values ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, relative to control; ns—not significant.
Figure 3
Figure 3
The relative gene expression of thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) in A-549 treated with 50 nM and 100 nM CSNK2A1 siRNA for 48 h (a) and with 25 nM, 50 nM, and 100 nM CSNK2A1 siRNA for 72 h (b). * p < 0.05, ** p < 0.01, *** p < 0.001, relative to control; ns—not significant.
Figure 4
Figure 4
The effect of silencing of CSNK2A1 on localisation of DHFR (e) and TS (d) in A-549 cells. Detection of TS, DHFR and CK2α in control cells are shown in (a), (b), (c), respectively. The cells were treated with 100 nM CSNK2A1 siRNA for 72 h (d–f), fixed, and after blocking, probed with the primary anti-DHFR or anti-TS antibodies. Subsequently, the cells were treated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (green fluorescence) and Hoechst 33, 342 (nuclei, blue fluorescence).
Figure 5
Figure 5
In-gel kinase assay results for TS (a) and DHFR (c). CCRF-CEM and A-549 lysates were prepared in NP-40 buffer (20 mM Tris-HCl pH 8.0; 137 mM NaCl; 1% Nonidet P-40; 2 mM EDTA). The left parts of (a) and (c) represent a fragment of gel stained with CBB with visible protein markers (M, PageRuler Prestained Protein Ladder, Thermo Scientific) and recombinant forms of CK2α and CK2α’. Human recombinant catalytic subunits of CK2, i.e., CK2α (29 µg) and CK2α’ (8 µg), were used as positive controls. Lysates containing proteins at three different levels were loaded in the range of 89–22 µg/well. Co-polymerisation of TS and DHFR was at acrylamide:bis-acrylamide ratio 37.5:1 and 19:1, respectively. Autoradiograms shown in (a,b), and (c,d) were obtained after 18 h and 6 h of X-ray films exposure, respectively.
Figure 6
Figure 6
The molecular weight distribution of the proteins in the excised gel band against the Score describes the probability of matching experimental data with proteins in the database. The bubble size corresponds to emPAI value—the parameter dependent on the protein concentration. Most abundant proteins are indicated with numbers: 1. Actin cytoplasmic 1/2, 2. Fructose-bisphosphate aldolase A, 3. Phosphoglycerate kinase 1, 4. POTE ankyrin domain family member E, 5. Actin, alpha cardiac muscle.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Pang K., Wang W., Qin J., Shi Z., Hao L., Ma Y., Xu H., Wu Z., Pan D., Chen Z., et al. Role of protein phosphorylation in cell signaling, disease, and the intervention therapy. Medcomm. 2022;3:175. doi: 10.1002/mco2.175. - DOI - PMC - PubMed
    1. Mori Y., Kawamura H., Sato T., Fujita T., Nagata R., Fujihashi M., Miki K., Atomi H. Identification and enzymatic analysis of an archaeal ATP-dependent serine kinase from the hyperthermophilic archaeon Staphylothermus marinus. J. Bacteriol. 2021;203:e00025-21. doi: 10.1128/JB.00025-21. - DOI - PMC - PubMed
    1. Tavernier N., Sicheri F., Pintard L. Aurora A kinase activation: Different means to different ends. J. Cell Biol. 2021;220:e202106128. doi: 10.1083/jcb.202106128. - DOI - PMC - PubMed
    1. Boulos J.C., Idres M.R.Y., Efferth T. Investigation of cancer drug resistance mechanisms by phosphoproteomics. Pharmacol. Res. 2020;160:105091. doi: 10.1016/j.phrs.2020.105091. - DOI - PubMed
    1. Nuñez de Villavicencio-Diaz T., Rabalski A.J., Litchfield D.W. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks. Pharmaceuticals. 2017;10:27. doi: 10.3390/ph10010027. - DOI - PMC - PubMed

Substances

Grants and funding

This research was funded by the Warsaw University of Technology.

LinkOut - more resources