Sugiura, F
Sugiura, F. 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. DNA-dependent protein kinase (DNA-PK), composed of a Ku70/80 heterodimer and a catalytic subunit (DNA-PKcs), is the key component of nonhomologous-end joining (NHEJ), the predominant DNA double-strand break (DSB) repair pathway in mammalian cells. The intrinsic kinase activity of DNA-PKcs is essential for radioresistance and NHEJ-mediated DSB repair (18), most likely through phosphorylation of NHEJ components, including DNA-PKcs itself. DNA-PKcs is rapidly autophosphorylated in vitro upon activation and is phosphorylated in vivo after IR. Many in vitro and in vivo phosphorylation sites of DNA-PKcs have been identified thus far, including the T2609 cluster (7, 11, 32), S2056 (8), and the recently identified C-terminal phosphorylation sites (21). The majority of these phosphorylation sites are the (S/T)Q motifs (serine or threonine followed by a glutamine residue) common in many DNA damage repair proteins and are the cognate substrates of phosphoinositide kinase-related protein kinases (PIKKs), including DNA-PKcs and ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related) kinases (17, 35). Similar to its kinase Gastrodenol activity, DNA-PKcs phosphorylation is also required for NHEJ-mediated DSB repair. Mutations at the T2609 cluster or S2056 severely compromise the ability of DNA-PKcs to restore the radioresistance and DSB repair defects in DNA-PKcs-deficient cells (3, 7, 8, 10). We demonstrated that among the many DNA-PKcs phosphorylation sites identified, phosphorylation at T2609, S2056, and two additional sites within the T2609 cluster, T2638 and T2647, can be detected in culture cells after IR, and the in vivo phosphorylation is involved with DNA-PKcs itself as well as ATM kinase (7-9). IR-induced S2056 phosphorylation is mediated by a DNA-PKcs autophosphorylation event and detected in cells expressing wild-type DNA-PKcs but diminished in cells expressing kinase-dead mutant DNA-PKcs (8). On the other hand, IR-induced Gastrodenol phosphorylation within the T2609 cluster (T2609 and T2647) remained detectible in cells expressing kinase-dead mutant DNA-PKcs but was much reduced Gastrodenol in ataxia-telangiectasia cells, suggesting that ATM is likely the main kinase responsible for IR-induced DNA-PKcs phosphorylation at the T2609 cluster (8, 9). In addition to IR induction, DNA-PKcs phosphorylation could be induced upon treatment with DNA replication-inhibiting agents, including camptothecin, UV light (UV), and hydroxyurea (8). The possible involvement of DNA-PKcs in replication stress responsiveness is also supported by evidence that DNA-PKcs is required for RPA2 (the p34 subunit of replication protein A [RPA]) hyperphosphorylation after SPP1 DNA damage (4, 6). RPA is a multisubunit single-stranded-DNA-binding protein that is essential for normal DNA replication as well as DNA damage repair. The N terminus of RPA2 becomes hyperphosphorylated upon DNA damage. Consequently, hyperphosphorylation of RPA2 leads to down-regulation in DNA replication but not DNA repair, suggesting that RPA2 hyperphosphorylation could function as Gastrodenol a molecular switch to direct RPA activity from DNA replication to DNA damage repair (2). Furthermore, DNA-PKcs is required for cellular resistance (24) and response to UV irradiation (27), which is known to cause replication stress. It was reported that UV-induced replication arrest is normal in DNA-PKcs-proficient M059K cells but attenuated in DNA-PKcs-deficient M059J cells, implying that DNA-PKcs is required for UV-induced replication arrest (27). In light of this evidence, we hypothesized that DNA-PKcs could play a significant role in the cellular response to replication stress. To test this hypothesis, we investigated whether UV irradiation-generated replication stress could.