Human dihydrofolate reductase is a substrate of protein kinase CK2α

Skierka, Katarzyna; Wilamowski, Paweł; Wielechowska, Monika; Cysewski, Dominik; Senkara, Elżbieta; Wińska, Patrycja; Bretner, Maria; Cieśla, Joanna

doi:10.1016/j.bbrc.2019.03.186

Cited by 7 publications

(6 citation statements)

References 28 publications

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“…However, in the case of cells treated with CX-4945, such an increase is new evidence that may indicate the important role of phosphorylation in the regulation of DHFR protein expression. Taking into account that DHFR was found to be phosphorylated by CK2 in vitro (27), our results seem to confirm the physiological role of such phosphorylation. The level of phosphorylation of Ser529-p65, reflecting CK2 activity, was highest in control cells and lowest in cells treated with the drug combination.…”

Section: Induction Of Apoptosis and Caspase 3/7 Activation In Ccrf-cesupporting

confidence: 79%

“…When methotrexate binds to DHFR protein, this suppressive effect may be lost, allowing the synthesis of new enzyme molecules (35,38). Moreover, in the case of the observed increase in DHFR protein level after CX-4945 treatment, the regulatory role of CK2 should also be taken into account, particularly in the light of our most recent study (27) showing the phosphorylation of DHFR by CK2 in vitro. Taking into account that DHFR-CK2 interaction is strong and specific, shown by means of quartz crystal microbalance (27), it is likely that such phosphorylation may also occur in vivo.…”

Section: Discussionmentioning

confidence: 99%

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Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells

et al. 2019

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Background/Aim: Recently, we demonstrated the ability of inhibitors of protein kinase 2 (casein kinase II; CK2) to enhance the efficacy of 5-fluorouracil, a thymidylate synthase (TYMS)-directed drug for anticancer treatment. The present study aimed to investigate the antileukemic effect of simultaneous inhibition of dihydrofolate reductase (DHFR), another enzyme involved in the thymidylate biosynthesis cycle, and CK2 in CCRF-CEM acute lymphoblastic leukemia cells. Materials and Methods: The influence of combined treatment on apoptosis and cell-cycle progression, as well as the endocellular level of DHFR protein and inhibition of CK2 were determined using flow cytometry and western blot analysis, respectively. Real-time quantitative polymerase chain reaction was used to examine the influence of silmitasertib (CX-4945), a selective inhibitor of CK2 on the expression of DHFR and TYMS genes. Results: The synergistic effect was correlated with the increase of annexin Vbinding cell fraction, caspase 3/7 activation and a significant reduce in the activity of CK2. An increase of DHFR protein level was observed in CCRF-CEM cells after CX-4945 treatment, with the mRNA level remaining relatively constant. Conclusion: The obtained results demonstrate a possibility to improve methotrexate-based anti-leukemia therapy by simultaneous inhibition of CK2. The effect of CK2 inhibition on DHFR expression suggests the important regulatory role of CK2mediated phosphorylation of DHFR inside cells.

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Section: Induction Of Apoptosis and Caspase 3/7 Activation In Ccrf-cesupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells

et al. 2019

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“…There are reports indicating the occurrence of CK2-mediated phosphorylation of TS, DHFR, and SHMT (Frączyk et al, 2010;Frączyk et al, 2015;Ludwiczak et al, 2016;Rusin et al, 2017;Skierka et al, 2019); however, to this date, there have been no studies showing the physiological role of CK2-mediated phosphorylation of thymidylate synthesis cycle enzymes. Our previous (Wińska et al, 2019) and present qPCR results indicate that inhibition of CK2 in CCRF-CEM cells treated with a specific CX-4945 inhibitor does not significantly influence the mRNA levels.…”

Section: Discussionmentioning

confidence: 99%

“…This may indicate the physiological relevance for the conformational switching of TS activity with possible stabilization of the inactive form by phosphorylation. The in vitro phosphorylation by CK2 of the second enzyme of the thymidylate synthesis cycle, DHFR, has been demonstrated recently by us (Skierka et al, 2019), while the third enzyme of the cycle, SHMT, has been indicated as the CK2 substrate in phosphoproteomics studies on mitotic HEK 293T cells (Rusin et al, 2017).…”

Section: Introductionmentioning

confidence: 92%

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Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells

Wińska

Widło

Senkara

et al. 2022

Front. Mol. Biosci.

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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.

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Synthesis, antibacterial evaluation and molecular docking studies of novel series of acridone- 1,2,3-triazole derivatives

et al. 2020

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Human dihydrofolate reductase is a substrate of protein kinase CK2α

Cited by 7 publications

References 28 publications

Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells

Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells

Inhibition of Protein Kinase CK2 Affects Thymidylate Synthesis Cycle Enzyme Level and Distribution in Human Cancer Cells

Synthesis, antibacterial evaluation and molecular docking studies of novel series of acridone- 1,2,3-triazole derivatives

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