Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo, Japan.
DNA Repair (Amst). 2010 Jan 2;9(1):76-82. doi: 10.1016/j.dnarep.2009.10.008. Epub 2009 Dec 3.
The ubiquitin-proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.
泛素-蛋白酶体途径通过促进受损染色质部位 DNA 修复因子和信号蛋白的募集和激活，在 DNA 损伤信号转导和修复中发挥重要作用。一般认为，蛋白酶体活性对于顶端信号激酶（包括 ATM、ATR 和 DNA-PK 等与各种遗传毒性刺激物反应下游信号级联的 PI3K 相关激酶）的激活不是必需的。在之前的一项工作中，我们表明，蛋白酶体抑制剂 MG-132 抑制拓扑异构酶 I（TopI）毒物喜树碱（CPT）引起的 53BP1（p53 结合蛋白 1）磷酸化和 RPA2（复制蛋白 A2）磷酸化。为了研究依赖蛋白酶体的 RPA2 磷酸化的机制，我们研究了蛋白酶体抑制剂对上游 PIKKs 的影响。MG-132 强烈抑制 CPT 诱导的 DNA-PKcs 自动磷酸化，这是激活的标志物，而 MG-132 仅轻度抑制 ATM 和 ATR 底物的磷酸化，表明 PIKKs 中的 DNA-PK 是通过蛋白酶体对 CPT 特异性调节的。另一方面，MG-132 不会抑制 UV 或 IR 引起的 DNA-PK 激活。MG-132 阻断了 CPT 增强的 DNA-PKcs 和 Ku 异二聚体之间的相互作用，而羟基脲预处理完全消除了 CPT 诱导的 DNA-PKcs 自动磷酸化，表明需要进行持续的 DNA 复制。CPT 诱导的 TopI 降解独立于 DNA-PK 激活发生，表明 DNA-PK 激活不需要被捕获的 TopI 复合物的降解。综合结果表明，CPT 依赖性复制叉崩溃通过依赖蛋白酶体的、TopI 降解非依赖性途径激活 DNA-PK 信号转导。讨论了 DNA-PK 激活在 TopI 毒物为基础的治疗中的意义。
