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

• Published in: International journal of molecular sciences Vol. 24; no. 3; p. 3023
• Main Authors: Wińska, Patrycja, Sobiepanek, Anna, Pawlak, Katarzyna, Staniszewska, Monika, Cieśla, Joanna
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 03-02-2023; MDPI
• Subjects: acute lymphoblastic leukaemia; Adenocarcinoma; Adenocarcinoma of Lung; Biosynthesis; Cancer; Dihydrofolate reductase; dihydrofolate reductase DHFR; Enzymes; Gene expression; Humans; Immunofluorescence; Kinases; Leukemia; Localization; lung adenocarcinoma; Lung cancer; Lungs; Mass spectrometry; Mass spectroscopy; Phosphorylation; Physiology; Polymerase chain reaction; protein phosphorylation; Proteins; Reductases; Reverse transcription; serine hydroxymethyltransferase SHMT; Signal transduction; Tetrahydrofolate Dehydrogenase - chemistry; Thymidylate synthase; Thymidylate Synthase - metabolism; thymidylate synthase TS; protein kinase CK2; thymidylate synthase TS; acute lymphoblastic leukaemia; dihydrofolate reductase DHFR; lung adenocarcinoma; in-gel kinase assay; serine hydroxymethyltransferase SHMT; protein phosphorylation
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24033023

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.