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

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 whethe...

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Autores principales: Wińska, Patrycja, Sobiepanek, Anna, Pawlak, Katarzyna, Staniszewska, Monika, Cieśla, Joanna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917831/
https://www.ncbi.nlm.nih.gov/pubmed/36769342
http://dx.doi.org/10.3390/ijms24033023
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author Wińska, Patrycja
Sobiepanek, Anna
Pawlak, Katarzyna
Staniszewska, Monika
Cieśla, Joanna
author_facet Wińska, Patrycja
Sobiepanek, Anna
Pawlak, Katarzyna
Staniszewska, Monika
Cieśla, Joanna
author_sort Wińska, Patrycja
collection PubMed
description 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.
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spelling pubmed-99178312023-02-11 Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing Wińska, Patrycja Sobiepanek, Anna Pawlak, Katarzyna Staniszewska, Monika Cieśla, Joanna Int J Mol Sci Article 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. MDPI 2023-02-03 /pmc/articles/PMC9917831/ /pubmed/36769342 http://dx.doi.org/10.3390/ijms24033023 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wińska, Patrycja
Sobiepanek, Anna
Pawlak, Katarzyna
Staniszewska, Monika
Cieśla, Joanna
Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title_full Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title_fullStr Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title_full_unstemmed Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title_short Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing
title_sort phosphorylation of thymidylate synthase and dihydrofolate reductase in cancer cells and the effect of ck2α silencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917831/
https://www.ncbi.nlm.nih.gov/pubmed/36769342
http://dx.doi.org/10.3390/ijms24033023
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