Influence of Phosphorylation of THR-3, SER-11, and SER-15 on Deoxycytidine Kinase Activity and Stability

Edited by Zhijie Chang Keywords:Deoxycytidine kinase Nucleoside analog Ser-74 phosphorylation Protein phosphatase Ser/Thr protein phosphatase 2A a b s t r a c t Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn 2+ -activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.

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“…Representative immunoblots are shown. When indicated, fluorescence intensities have been used to quantify dCK phosphorylation or expression [22].…”

Section: Immunoblot Analysismentioning

confidence: 99%

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser‐74 dephosphorylation

et al. 2014

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“…Phosphorylation and conformational change of dCK have been suggested to lead dCK activation [24], [33]–[35]. On one hand, phosphorylation and dephosphorylation of dCK were important in the regulation of dCK activity [22].…”

Section: Resultsmentioning

confidence: 99%

“…On one hand, phosphorylation and dephosphorylation of dCK were important in the regulation of dCK activity [22]. The Ser74 residue in dCK was identified to be the only phosphorylation site that influences intracellular activity [33], [34]. To clarify whether the enhancement of dCK activity by TSN is the result of auto-phosphorylation of the enzyme, site-directed mutagenesis of Ser74 was carried out to generate three mutants of S74E, S74A and S74Q.…”

Section: Resultsmentioning

confidence: 99%

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The Apoptotic Effects of Toosendanin Are Partially Mediated by Activation of Deoxycytidine Kinase in HL-60 Cells

Cai

et al. 2012

PLoS ONE

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Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

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“…The phosphorylation of dCK and post-translational modification is crucial for its enzymatic activity (19). dCK protein has four phosphorylation sites, Thr-3, Ser-11, Ser-15 and Ser74 (20)(21)(22)(23). dCK activity can be increased by Ser-74 phosphorylation (24,25) and this phosphorylation is required for the initiation of the G2/M checkpoint (26).…”

Section: Introductionmentioning

confidence: 99%

Deoxycytidine kinase participates in the regulation of radiation-induced autophagy and apoptosis in breast cancer cells

et al. 2018

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Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for the phosphorylation of endogenous deoxynucleosides and for the anti‑tumor activity of many nucleoside analogs. dCK is activated in response to ionizing radiation (IR) and it is required for the G2/M checkpoint induced by IR. However, whether dCK plays a role in radiation-induced autophagy and apoptosis is less clear. In this study, we reported that dCK decreased IR-induced total cell death and apoptosis, and increased IR-induced autophagy in SKBR3 and MDA‑MB‑231 breast cancer cell lines. A molecular switch exists between apoptosis and autophagy. We further demonstrated that serine 74 phosphorylation was required for the regulation of autophagy. In dCK wild‑type (WT) or dCK S74E (mutant) MDA‑MB‑231 cell models, the expression levels of phospho-Akt, phospho-mammalian target of rapamycin (mTOR) and phospho-P70S6K significantly decreased following exposure to IR. Moreover, the ratio of Bcl‑2/Beclin1 (BECN1) significantly decreased in the S74E mutant cells; however, no change was observed in the ratio of Bcl‑2/BAX. Taken together, our findings indicate that phosphorylated and activated dCK inhibits IR-induced total cell death and apoptosis, and promotes IR-induced autophagy through the mTOR pathway and by inhibiting the binding of Bcl‑2 protein to BECN1.

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Influence of Phosphorylation of THR-3, SER-11, and SER-15 on Deoxycytidine Kinase Activity and Stability

Cited by 7 publications

References 11 publications

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser‐74 dephosphorylation

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser‐74 dephosphorylation

The Apoptotic Effects of Toosendanin Are Partially Mediated by Activation of Deoxycytidine Kinase in HL-60 Cells

Deoxycytidine kinase participates in the regulation of radiation-induced autophagy and apoptosis in breast cancer cells

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