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. 2006 Oct;26(20):7520-8.
doi: 10.1128/MCB.00048-06. Epub 2006 Aug 14.

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress

Hirohiko Yajima  1 , Kyung-Jong Lee, Benjamin P C Chen
Affiliations

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress

Hirohiko Yajima et al. Mol Cell Biol. 2006 Oct.

Abstract

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

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Figures

FIG. 1.
FIG. 1.
UV irradiation induces DNA-PKcs phosphorylation at T2609 and T2647 but not S2056. (A) HeLa cells were mock treated or subjected to either UV irradiation (10 J/m2, 2-h recovery) or IR (10 Gy, 1-h recovery). Nuclear extract from treated and untreated HeLa cells was prepared and analyzed by Western blot for DNA-PKcs phosphorylation at T2609, T2647, and S2056. (B) HeLa cells were mock treated or subjected to 10 J/m2 of UV irradiation and harvested at the indicated time points. Nuclear extract was prepared for analysis of the kinetics of DNA-PKcs phosphorylation at T2609 and T2647 after UV irradiation.
FIG. 2.
FIG. 2.
UV exposure correlates to DNA-PKcs phosphorylation at T2609 and T2647. (A) Monolayer HeLa cells were irradiated with 30 J/m2 of UV in the presence of a 3-μm Isopore polycarbonate membrane. One hour after UV irradiation, cells were fixed and were subjected to immunofluorescent staining against anti-CPD and anti-DNA-PKcs phosphospecific antibodies as indicated. (B) HeLa cells were UV irradiated in the presence of a 3-μm Isopore polycarbonate membrane followed by IF staining against anti-γH2AX and anti-DNA-PKcs phosphospecific antibodies as indicated. (C) HeLa cells were UV irradiated in the absence of an Isopore polycarbonate membrane followed by IF staining against anti-γH2AX and anti-DNA-PKcs phosphospecific antibodies.
FIG. 3.
FIG. 3.
UV-induced DNA-PKcs phosphorylation occurs at S phase of the cell cycle progression. (A) HSFs were synchronized at G1 or S phase via serum starvation and aphidicolin treatment (for S phase only) and were subjected to FACS analysis for cell cycle synchronization. (B) Asynchronous, G1-phase-synchronized, and S-phase-synchronized HSFs were subjected to 20 J/m2 of UV irradiation. Two hours after UV irradiation, the cells were harvested and Western blotted for DNA-PKcs phosphorylation at T2647. (C, D) Exponentially growing HeLa cells were pulse-labeled with 10 μM of bromodeoxyuridine (BrdU) in the culture medium for 10 min followed by 10 J/m2 of UV irradiation. One hour after UV irradiation, the cells were fixed and IF stained with (C) anti-BrdU and anti-pT2647 antibodies or (D) anti-BrdU and anti-pT2609 antibodies.
FIG. 4.
FIG. 4.
ATR kinase but not DNA-PKcs or ATM kinase is required for UV-induced DNA-PKcs phosphorylation. (A) HeLa cells were mock treated or subjected to increasing concentrations of wortmannin for 30 min prior to UV irradiation (10 J/m2). Two hours after UV irradiation, the cells were harvested for Western blot analyses of DNA-PKcs phosphorylation at T2609 and T2647, CHK1 phosphorylation at S345, and CHK2 phosphorylation at T68. (B) DNA-PKcs-deficient CHO V3 cells expressing wild-type human DNA-PKcs (V3-wt) or kinase-dead mutant DNA-PKcs (V3-kd) were UV irradiated and were harvested at 2 h for analysis of DNA-PKcs phosphorylation. (C) HeLa cells were mock-treated or pretreated with 2 mM caffeine for 1 h and were subjected to UV irradiation. Two hours after UV irradiation, the cells were harvested for Western blot analysis.
FIG. 5.
FIG. 5.
Inhibition of ATR kinase attenuates UV-induced DNA-PKcs phosphorylation. (A) Human osteosarcoma U2OS-derived GW33 cells expressing tetracycline-inducible FLAG-tagged ATR-wt were cultured in the absence or presence of doxycycline (Dox) for 2 days prior to UV irradiation (10 J/m2). One hour after UV irradiation, the cells were fixed and were immunostained against anti-FLAG antibody for the expression of ATR-wt and anti-pT2647 for UV-induced DNA-PKcs phosphorylation. (B) U2OS derived GK41 cells expressing tetracycline-inducible FLAG-tagged ATR-kd were cultured in the absence or presence of doxycycline for 2 days prior to UV irradiation (10 J/m2) followed by immunostaining against anti-FLAG (for ATR-kd) and anti-pT2647 antibodies. (C) GK41 cells with or without induction of ATR-kd were subjected to UV irradiation (10 J/m2) followed by immunostaining against anti-pT2609 monoclonal and anti-pT2647 polyclonal antibodies.
FIG. 6.
FIG. 6.
Inhibition of ATR kinase attenuates UV-induced DNA-PKcs phosphorylation. (A) Human osteosarcoma U2OS cell lines expressing inducible ATR-wt (GW33) or dominant-negative ATR-kd (GK41) were mock treated or subjected to either 10 J/m2 of UV irradiation or 10 Gy of IR. Two hours after UV irradiation or 1 h after IR, the cells were harvested for Western blot analysis of DNA-PKcs phosphorylation at T2647. (B) HeLa cells were mock treated or transfected with small inhibitory RNA against ATR or ATM kinase. Three days after transfection, the cells were irradiated with 10 J/m2 of UV. Two hours after UV irradiation, the cells were harvested for Western blot analysis of DNA-PKcs phosphorylation at T2609 and T2647.
FIG. 7.
FIG. 7.
ATR complexes with DNA-PKcs in vivo and phosphorylates the T2609 cluster in vitro. (A) Unirradiated or UV-irradiated HeLa nuclear extracts were immunoprecipitated (IP) with mouse IgG or 25-4 anti-DNA-PKcs mouse MAb. The precipitated protein complex was analyzed by Western blotting for the presence of DNA-PKcs and ATR. (B) HeLa nuclear extracts were immunoprecipitated with rabbit IgG or anti-ATR rabbit polyclonal antibody followed by Western blot analysis. (C) ATR immunoprecipitated from HeLa nuclear extract was subjected to an in vitro kinase assay with [γ-32P]ATP and recombinant GST fusion proteins (Coomassie blue staining, left panel) GST-Xrcc4, GST-p53, and GST-DNA-PKcs (GST-PKcs), covering the T2609 cluster region. As a control, IgG precipitation was subjected to the kinase reaction. The positions of 32P-labeled substrates are indicated by the arrows. (D) GST-PKcs fusion proteins with the wild-type sequence (WT) or alanine substitutions at the T2609 cluster (6A mutant) were subjected to an ATR in vitro kinase assay (top). The bottom panel shows Coomassie blue staining of the same gel. (E) A wild-type GST-PKcs fusion protein was subjected to ATR in vitro kinase assay with cold ATP and was Western blot analyzed with anti-pT2647 (top) or anti-GST (bottom) antibodies.
FIG. 8.
FIG. 8.
DNA-PKcs phosphorylation at the T2609 cluster is required for cellular response to UV damage. (A) DNA-PKcs protein level in wild-type CHO AA8 cells, DNA-PKcs-deficient CHO V3 cells, and V3 cells complemented with wild-type human DNA-PKcs (V3-wt) or mutant DNA-PKcs lacking the entire the T2609 cluster of phosphorylation sites (V3-6A). Forty micrograms of nuclear extract from each cell line was analyzed. (B) Clonogenic survival of the above cell lines after UV irradiation. Mean values from three independent experiments are shown. Bars indicate standard errors. (C) Kinetics of histone H2AX S139 phosphorylation in CHO V3, V3-wt, and V3-6A cells after 10 J/m2 of UV irradiation. The cells were harvested at the indicated time points after UV irradiation and were Western blotted for H2AX phosphorylation.
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