- Research article
- Open Access
Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage
© Olsen et al; licensee BioMed Central Ltd. 2012
Received: 29 November 2011
Accepted: 9 March 2012
Published: 9 March 2012
The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks.
In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks.
Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.
A wide variety of lesion types can affect the DNA requiring the intervention of distinct and lesion-specific DNA-repair mechanisms. However, it is known that repair mechanisms may complement each other in some respects by sharing many protein components . DNA double-strand breaks (DSBs) induced, for instance, by ionizing radiation (IR) and radiomimetic drugs are difficult to repair and extremely toxic although they do not occur as frequently as other types of DNA lesion [1, 2]. DSBs are repaired by two main mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is the major repair mechanism in mammalian cells whereas HR is the predominant repair mechanism in budding yeast . In NHEJ, DNA lesions are recognized by the Ku70/80 heterodimer. Localization of Ku to sites of DSB serves to recruit other NHEJ proteins such as the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), ligases and polymerases [3, 4]. The convergence of so many proteins to sites of DNA lesion is thought to protect at first the DNA ends from nucleases attack and later to facilitate the repair process. DNA-PKcs is a Ser/Thr kinase characterized by a weak activity that is significantly enhanced in the presence of double-strand DNA and Ku . Modulation of its activity is not exclusively regulated by the interaction with Ku on DNA ends. In this respect, it has been reported that DNA-PKcs undergoes autophosphorylation at multiple sites, which is followed by DNA-PKcs release from DSBs in vivo and loss of protein kinase activity in vitro. The significance of these events is not fully understood, however, it has been suggested that autophosphorylation of DNA-PKcs favors a conformational change that may serve to facilitate subsequent repair steps by making the DNA ends more accessible for damage-responsive proteins to sites of DNA damage [5–7].
Protein kinase CK2 is an evolutionary highly conserved Ser/Thr kinase composed of two catalytic subunits α and/or α' and two regulatory β-subunits [8, 9]. CK2 is often described as a tetrameric enzyme but evidence has suggested that the individual subunits do not exist exclusively within the tetrameric complex but also as free proteins [10–12]. CK2 is invariably elevated in tumors and highly proliferating tissues and the de-regulated expression of the catalytic subunits is causative of transformation especially in combination with the altered expression of oncogenes and tumor suppressor genes [13, 14]. CK2 appears to be involved in a plethora of cellular processes including regulation of cell cycle progression, survival, proliferation and those associated with various diseases, particularly cancer, neurodegenerative- and inflammatory disorders [15, 16]. Current data suggest that CK2 plays a role in DNA sensing and repair. CK2 has been shown to phosphorylate the scaffold protein XRCC1 thereby enabling the assembly and activity of DNA single-strand breaks repair at sites of chromosome breakage . Moreover, this kinase has been reported to phosphorylate the N-terminal domain of the MDC1 adaptor protein enabling its interaction with the NBS1 subunit of MRN protein complex which is involved in the initial processing of DNA repair [18, 19]. Recently, by employing DNA-PKcs-proficient glioblastoma cells, we have reported evidence that siRNA-mediated down-regulation of the CK2 catalytic subunits results in significant cell death, decreased DNA-PKcs autophosphorylation at S2056 and delayed DNA repair following induction of DSBs .
In this study, given the importance of DNA-PK in DSBs repair, we have further elucidated the role of CK2 with respect to the molecular mechanisms activated in response to DNA damage. We report for the first time evidence that CK2 co-localizes with phospho-histone H2AX (γ-H2AX) to sites of DSB. We show that CK2 associates exclusively with DNA-PKcs and that the binding increases upon treatment of cells with radiomimetic drugs. Furthermore, depletion of the CK2 catalytic subunits destabilizes the interaction of DNA-PKcs with Ku80, which indicates that the observed decreased DNA-PKcs autophosphorylation might be the consequence of lack of interaction between DNA-PKcs and DNA on sites of genetic lesions.
Taken together, the reported observations strongly suggest that CK2 is required for the NHEJ-dependent cellular response to DSBs for an efficient repair process by stabilizing NHEJ proteins at sites of DNA damage.
M059K, M059J, HCT116 and Cos-1 cell lines were cultured in Dulbecco's Modified Eagle's Medium (Invitrogen, Denmark) supplemented with 10% FBS (Biochrom AG, Germany). The H1299 cell line was cultured in Roswell Park Memorial Institute medium (Invitrogen) supplemented with 10% FBS. All cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained at 37°C under a 5% CO2 atmosphere.
Cell transfection and treatments
Cells were transfected with a set of four small interfering RNA duplexes directed against CK2α and CK2α' (ON-TARGET plus SMARTpools, Dharmacon, CO, USA), respectively, using Dharmafect I transfection reagent (Dharmacon) for 72 hours according to the manufacturer's instructions. Where indicated, control experiments were performed employing cells treated with transfection reagent alone or in combination with scramble-siRNA (Dharmacon). DNA damage was induced by cell treatment with the radiomimetic drug NCS (0.5 μg/ml, a gift from Dr. Hiroshi Maeda, Kumamoto University, Japan) and 33 μM Cisplatin (Sigma, Denmark), respectively, or exposure to 20 J/m2 UV irradiation (Stratalinker UV Crosslinker, Stratagene, CA, USA) and 10 Gy ionizing radiation (6 MV energy from an Elekta Synergy Accelerator, Elekta, Sweden), respectively. For transient overexpression of the indicated proteins, cells were electroporated with the corresponding expression plasmids by using the NEON™ Tranfection system (Invitrogen) according to the manufacturer's instructions.
The coding region of CK2α' cloned into pcDNA3.1-MycHis  was excised with HindIII/XhoI restriction enzymes and inserted into pDsRed-Monomer N1 or pEGFP-N1 (both from Clontech-Takara Bio Europe, France), digested with HindIII/SalI, for the expression of DsRed- and GFP-tagged CK2α', respectively. The following DNA-PKcs fragments: Fragment A (aa 515-830), Fragment B (aa 1079-1533), Fragment C (aa 2005-2555), Fragment D (aa 2768-3258) and Fragment E (aa 3414-4123) were all cloned into the mammalian expression vector pcDNA3.1-MycHis (Invitrogen) using reverse transcription PCR and total RNA extracted from HCT116 cells used as template. The following primer pairs were employed: for fragment A, forward primer 5'-CGGGATCCACCATGGGAAGAACTGGCAAATGGAAGGTGC-3' and reverse primer 5'-CGCTCGAGCACCACTTTATTAAATCCTTTCTGG-3'; for fragment B, forward primer 5'-CGGGATCCACCATGGGATCGCTTGCCTTTAATAATATCTACAG-3' and reverse primer 5'-CGCTCGAGCAGGAGAAGACTCACAAGGCG-3'; for fragment C, forward primer 5'-CGGGATCCACCATGGGAATCGAAATTAGGAAAGAAGCC-3' and reverse primer 5'-CGCTCGAGGGTATTTGAAGGTAACCTAG-3'; for fragment D, forward primer 5'-CGGAATTCACCATGGGACAGGTCGTTCTGTACAG-3' and reverse primer 5'-CGCTCGAG TAGTTTCATAGCAAGTGAG-3'; for fragment E, forward primer 5'-CGGAATTCACCATGGGAACGCTGGCAGATTTCTGTG-3' and reverse primer 5'-CGCTCGAGTCCTTCCCAGGTTCTGCC-3'. PCR products were inserted into BamHI-XhoI (Fragments A, B and C) or EcoRI/XhoI (Fragments D and E) restriction sites. The correctness of the sequences was verified by DNA sequencing analysis of the cloned fragments.
Purification of recombinant proteins
Human recombinant CK2α was expressed and purified essentially as described .
Clonogenic survival assay
M059K cells were transfected with siRNAs against CK2α or CK2α' and 48 hours after transfection, cells were incubated with 0.5 μg/ml NCS for additional 24 hours. Afterwards, cells were trypsinized and seeded in six-well plates and colonies were allowed to grow for 14 days. Subsequently, cells were washed once with PBS and stained with a solution containing 6% glutaraldehyde/0.5% crystal violet and de-stained with tap water. Colonies containing > 50 cells were quantified using the UVIdoc software (Uvitec Ltd., UK).
Western blot analysis
Whole cell extracts and Western blots were as previously described [21, 23]. The antibodies used were as follows: mouse monoclonal anti-CK2α (#218703, Calbiochem, UK); mouse monoclonal anti-β-actin (#A3853, Sigma); mouse monoclonal anti-DNA-PKcs, -Myc and -Ku80 (#sc-5282, #sc-40, #sc-33653, all from Santa Cruz Biotechnology, CA, USA); rabbit polyclonal anti-53BP1 (#4937, Cell Signaling Technology, MA, USA) and rabbit polyclonal anti-phospho-DNA-PKcs (p-S2056) (#18192, Abcam, UK). Rabbit polyclonal anti-CK2α' was obtained by immunizing rabbits with a specific peptide (SQPCADNAVLSSGTAAR) of human CK2α'. Rabbit polyclonal anti-phospho-DNA-PKcs (p-T2609) and -(p-T2647) were as described [23, 24].
Experiments were performed essentially as described previously  employing 1 mg cell lysate and monoclonal anti-Myc (#2278, Cell Signaling Technology), rabbit polyclonal anti-DNA-PKcs (#300-516A, Bethyl Laboratories, TX, USA) or anti-CK2α serum antibodies as indicated in the figure legends. Anti-CK2α serum was obtained by immunizing rabbits against the full-length protein. 500 μg crude extracts from cells expressing various DNA-PKcs deletion mutants and 0.5 μg human recombinant CK2α were employed in co-immunprecipitation experiments for mapping the DNA-PKcs interaction domains.
Immunofluorescence and in situ PLA
M059K cells grown on cover slips were incubated with rabbit polyclonal anti-γ-H2AX (p-S139) antibody (#2577, Cell Signaling Technology, Denmark) followed by incubation with biotinylated swine anti-rabbit IgG (Dako, Denmark) and streptavidin-conjugated fluorescein-isothiocyanate (Dako) as previously described . Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) or Hoechst dye (Sigma), analyzed on a DMRBE microscope (400× magnification) equipped with a Leica DFC420C camera (Leica, Denmark) and processed using ImageJ software (NIH, MD, USA). Threshold adjustments were set to ensure quantification of true positively immunostained particles. For the analysis of γ-H2AX/CK2α' interaction by in situ PLA, cells grown on cover slips were incubated with mouse monoclonal anti-γ-H2AX (p-Ser139) antibody (#11174, Abcam) and a rabbit polyclonal antibody against CK2α'. Visualization of protein-protein interaction was performed following the manufacturer's instructions (Olink Biosciences, Sweden) and as reported earlier (20). Similarly, interaction between DNA-PKcs and Ku80 or histone H3 was analyzed labeling the cells with a mouse monoclonal antibody against DNA-PKcs and a rabbit polyclonal anti-Ku80 antibody (#sc-5282, #sc-9034, both from Santa Cruz Biotechnology) or a rabbit monoclonal anti-histone H3 (#4499, Cell Signaling Technology). Interactions between CK2α'/DNA-PKcs or CK2α'/Ku80 were analyzed using a rabbit polyclonal antibody against CK2α' and a mouse monoclonal anti-Ku80 or anti-DNA-PKcs antibodies (#sc-33653, #sc-9051, both from Santa Cruz Biotechnology). The number of in situ proximity ligation signals was determined using the freeware software BlobFinder http://www.cb.uu.se/~amin/BlobFinder.
Statistical and densitometric analysis
The statistical significance of differences between means of two groups was evaluated by the two-tailed t-test (Student's t-test). The levels of significance are indicated in the figure legends.
Down-regulation of CK2 significantly impairs cell survival and results in persistent phosphorylation of histone H2AX
CK2 is recruited to sites of DNA double-strand break
Down-regulation of CK2 regulates DNA-PKcs phosphorylation in response to DNA double-strand break induction
Lack of CK2 destabilizes the association of DNA-PKcs with Ku80
NHEJ is the predominant repair pathway in mammalian cells that is activated in the presence of DSBs and requires the intervention of the DNA-PK complex for an adequate damage response. Although the importance of DNA-PKcs in DSB repair is well established and has been extensively studied, the precise mechanism by which this enzyme localizes to sites of DSB, phosphorylates itself and other repair proteins to promote NHEJ is still elusive. In this study, we report evidence that protein kinase CK2 contributes significantly to the DNA-PKcs-mediated cellular response to DNA damage. Lack of CK2 leads to marked cell death, persistent DNA damage and reduced survival rate in cells treated with radiomimetic drugs. We show that endogenous CK2 co-localizes with γ-H2AX to sites of DNA damage and its overexpression results in rapid decrease in the number of detected nuclear foci. This suggests that CK2 plays an important role in the DNA damage response. Experiments of co-immunoprecipitation and in situ PLA led to the observation that lack of CK2 destabilizes the Ku80/DNA-PKcs complex formation. These findings might explain the delayed DNA repair observed in cells lacking CK2 and, because survival of cells exposed to genotoxic stress is largely dependent on efficient repair of DSBs, the low clonogenic survival observed in CK2-depleted cells. The reported lack of association between Ku80 and DNA-PKcs in CK2-knockdown cells suggested that CK2 might mediate their interaction. However, we showed that endogenous CK2 binds to DNA-PKcs and the association is enhanced in response to DNA damage but we could not detect the presence of Ku80 in the complex formation. This has led to the hypothesis that CK2 interacts exclusively with DNA-PKcs facilitating and/or stabilizing the binding of the latter to DNA which has been reported to be weak . In support of this notion, results obtained by in situ PLA where the binding of DNA-PK to chromatin components (i.e. histone H3) was tested in cells exposed to NCS, show that the association between DNA-PKcs and histone H3 is attenuated in cells lacking CK2 (Additional file 3: Figure S3). Data derived from the co-immunoprecipitation experiments and in situ PLA (Figure 7) suggest that CK2 binds exclusively DNA-PK. In this respect, the high level of fluorescent signal detected in M059K cells, which would indicate that CK2α' is also associated with Ku80, is almost completely suppressed in DNA-PKcs-deficient M059J cells. The association between CK2 and DNA-PKcs does not seem to be strictly dependent upon DSBs induction since we detected association also in the absence of DNA damage although to a lesser extent. This has been previously reported in interaction studies between mediator of DNA damage checkpoint protein 1 (MDC1) and NBS1 subunit of MRN comlex [18, 19]. Here, the authors showed that the CK2-mediated phosphorylation of the STD-repeats of MDC1 enhances the binding to NBS1 thereby promoting the local concentration and/or stability of DNA damage regulators at sites of genetic lesions. MDC1 has also been shown to regulate DNA-PK autophosphorylation . Our studies  indicate that it is not the catalytic activity of CK2 but rather the protein itself that regulates the interaction of DNA-PKcs with DNA at sites of damage underlining the multiplicity of the effects exerted by CK2. In light of the fact that CK2 might facilitate the binding of DNA-PKcs to DNA upon induction of DSBs, we believe that the apparent low phosphorylation levels of DNA-PKcs at S2056, T2609 and T2647 in CK2-depleted cells (Figures 4 and 5) might be caused by defective recruitment of DNA-PKcs to sites of DNA damage.
DNA-PKcs phosphorylation at T2609 in cells lacking CK2α or -α' and exposed to ionizing radiation did not lead to results similar to those obtained with cells treated with NCS (Figure 4). Currently, the reason for this discrepancy is not clear as both types of treatment generate DSB. However, T2609 is reported to be phosphorylated by all three members of the phosphoinositide 3-kinase-like family of protein kinases (PIKKs) and possibly other kinases , whose kinetic of phosphorylation might be differently regulated upon exposure of cells to IR or treatment with radiomimetic drugs. However, one cannot exclude that lack of chromatin remodeling may occur in CK2 deficient cells and lead to defective DNA-PK phosphorylation and other effects. In this respect, it has been shown that CK2 is implicated in chromatin remodeling by controlling the mobilization of HP1-beta through modulation of its phosphorylation at Thr51 .
Through experiments of mapping protein-protein interactions in vitro we defined two regions in DNA-PKcs responsible for the association with CK2α comprised between amino acid residues 2005-2555 (fragment C) and 2768-3258 (fragment D). It is conceivable that amino acids between these two regions which comprise the so-called ABCDE cluster of in vivo phosphorylation sites [35, 36] are also involved in the interaction with CK2. It remains to be addressed whether the phosphorylation of these amino acids are affected upon binding of CK2 to DNA-PKcs. Fragment D comprises part of the so-called FAT domain, which is conserved in PIKKs and seems to regulate PIKKs kinase activity . Part of the FAT domain comprises a high-affinity Ku binding (aa 3400-3420). Fragment E includes part of this Ku binding sequence. However, no interaction was found with CK2 suggesting that the association of DNA-PKcs with Ku and CK2, respectively, involves different domains.
In summary, we show for the first time that protein kinase CK2 co-localizes with γ-H2AX to sites of genetic lesions and modulation of its expression levels affects the cellular DNA damage response. CK2 interacts with DNA-PKcs in mammalian cells in the absence of DNA damage and the association increases upon cell treatment with radiomimetic drugs. In vitro studies employing a panel of DNA-PKcs fragments and human recombinant CK2α show that the interaction is direct although one cannot exclude that the complex formation between CK2 and native, full length DNA-PKcs might occur in the presence of other DNA repair proteins and DNA. Evidence suggests that CK2 might facilitate and/or stabilize the binding of DNA-PKcs and possibly other proteins of NHEJ, to DNA ends contributing to efficient DNA damage repair. Additional experiments are necessary to further dissect the molecular mechanisms involved in NHEJ, nevertheless, the present findings point to CK2 as a prominent novel signaling kinase regulating DNA-PKcs fate in cells exposed to ionizing radiation or radiomimetic drugs.
We thank Christian Rønn Hansen (Odense University Hospital) for technical assistance with the cell irradiation experiments. This work was supported by grants from the Danish Cancer Society (DP08152) and the Danish Natural Science Research Council (272-07-0258) to BG.
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