DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response

Abstract

The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1-Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs-deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.

Figure 1.

DNA-PKcs deficiency attenuates hydroxyurea-induced Chk1 phosphorylation. (A) The parental human HCT116 and DNA-PKcs^−/− cells were treated with 5 mM hydroxyurea (HU) and harvested at the indicated time points. Whole-cell lysates were analyzed by western blotting with the indicated antibodies. (B) HeLa cells transfected with siRNA against green fluorescent protein (siGFP) or DNA-PKcs (siPKcs) were western blot analyzed for HU-induced Chk1 phosphorylation. (C) HCT116 and DNA-PKcs^−/− cells were treated with 5 mM HU for 30 min and were immuno-stained for anti-γH2AX (red) and anti-phospho-S317 Chk1 (green) antibodies. (D) Cells were treated with HU for the indicated time durations. Percentages of Chk1 pS317 positive cells were scanned and quantified using In Cell 2000 Analyzer imaging system. The result was represented by mean values from three independent experiments. Statistical analyses were performed using two-way ANOVA. *P < 0.05; ***P < 0.001.

Figure 2.

Decreased Claspin expression in DNA-PKcs–deficient cells. (A) HCT116 and DNA-PKcs^−/− cells were exposed to 5 mM HU for the indicated time points. Whole-cell lysates were analyzed with the indicated antibodies. (B) HeLa cells transfected with siRNA against GFP or DNA-PKcs were subjected to HU treatment and were analyzed as described above. (C) DNA-PKcs^−/− cells were complemented with a flag-tagged full-length DNA-PKcs (PKcs⁺). Expression of Chk1 and Claspin proteins as well as HU-induced Chk1 pS317 in PKcs⁺ cells were examined and compared with that in HCT116 and DNA-PKcs^−/− cells.

Figure 3.

DNA-PKcs is required for CLSPN gene expression and Chk1 protein stability. (A) HCT116 and DNA-PKcs^−/− cells were incubated with protein synthesis inhibitor cycloheximide (CHX, 0.1 mg/ml) and were harvested at the indicated time points. Whole-cell lysates were subjected to western blotting analysis with anti-Claspin, anti-Chk1 and anti-Ku80 antibodies. (B) Total protein levels of Chk1 and Claspin before and after CHX treatment were quantified using ImageJ, and were normalized with Ku 80 protein levels. Relative Chk1 (left panel) and Claspin (right panel) protein stabilities were represented by mean values from at least three independent experiments. Statistical analyses were performed using two-way ANOVA. **P < 0.01; ***P < 0.001. (C) Steady-state mRNA levels of CHEK1 and CLSPN genes in HCT116 and DNA-PKcs^−/− cells were measured by quantitative real-time PCR and normalized by 18S rRNA level. Each bar represents the mean values from the three independent experiments. Statistical analyses were performed by Student’s t-test. ****P < 0.0001.

Figure 4.

DNA-PKcs enhances Chk1 and Claspin association with chromatin on hydroxyurea treatment. (A) HCT116 and DNA-PKcs^−/− cells were subjected to 5 mM HU for 1 h. Soluble nuclear protein fraction (S3) and chromatin-nuclear matrix fraction (P3) were prepared for western blot analysis using antibodies against Claspin, Chk1, RPA2 and Ku80. (B) Increase of chromatin-bound RPA2 in DNA-PKcs^−/− cells. HCT116 and DNA-PKcs^−/− cells were pulse-labeled with 50 μM EdU for 1 h. Cells were preextracted with 0.1% TX-100 followed by immuno-staining with anti-RPA2 antibody (green) and EdU staining for S-phase cells (red). (C) Percentages of RPA2 positive cells among EdU-labeled S-phase cell population in the presence or absence of hydroxyurea. The result was generated from two independent experiments. Statistical analyses were performed using t-test. *P < 0.05.

Figure 5.

Attenuation of intra-S checkpoint in DNA-PKcs–deficient cells. (A) HCT116 and DNA-PKcs^−/− cells were pulse-labeled with EdU (50 µM) for 1 h, treated with 5 mM HU for 1 h and harvested at 4 and 8 h after HU treatment. Cell cycle progression of EdU-labeled cells were monitored for phospho-histone H3 (p-H3) by FACS analysis. The gating heptagons in the upper panel represent EdU(+) cells, which were further analyzed for p-H3(+) in the lower panel. (B) Representative images of p-H3 analysis. The gating ovals indicate the percentages of p-H3(+) among the EdU(+) cells at 4 and 8 h after HU treatment. (C) Percentages of EdU(+) cells migrated into mitosis (p-H3+) at 4 and 8 h. The result was generated from three independent experiments. (D) Proportions of pH3(+) in HU-treated samples compared with p-H3(+) in mock-treated samples. Statistical analyses were performed using t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6.

Defect in Chk1-mediated replication checkpoint in DNA-PKcs–deficient cells. (A) HCT116 and DNA-PKcs^−/− cells were pulse-labeled with iododeoxyuridine (IdU, 100 µM) for 10 min followed by chlorodeoxyuridine (CldU,100 µM) for 20 min. DNA tracks labeled with IdU (red) and CldU (green) were detected using monoclonal mouse and rat anti-BrdU antibodies, respectively. (B) HCT116 and DNA-PKcs^−/− cells were pulse-labeled with IdU for 10 min and followed by CldU labeling in the presence of 1 mM HU for 1 h. (C and D) The ratios of CldU to IdU in length were calculated from ongoing replication tracks (red-green tracks, N > 100). The profiles in the absence or presence of HU were analyzed in HCT116 cells (C) and DNA-PKcs^−/− cells (D).

Figure 7.

Defect of Chk1–Claspin stabilities and replication checkpoint in human DNA-PKcs hypomorphic fibroblasts. (A) Whole-cell lysates of control human fibroblasts (WT), ATR hypomorphic (ATR*) and DNA-PKcs hypomorphic (PKcs*) fibroblasts were western blot analyzed with the indicated antibodies. (B) Human wild type, ATR*, PKcs* fibroblasts were pulse-labeled with IdU (red) for 10 min and followed by CldU (green) labeling in the presence of HU (0.5 mM) for 1 h. (C) The ratios of CldU to IdU in length were calculated from ongoing replication tracks in WT, ATR* and PKcs* fibroblasts in the absence or presence of HU. The results were generated from >100 ongoing DNA replication tracks from each cell line.