Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin |
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| Authors: | Ryo Sakasai Hirobumi Teraoka Randal S. Tibbetts |
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| Affiliation: | 1. Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan;2. Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, United States;1. Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium;2. Key Laboratory of Animal Diseases Diagnostic and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;3. Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium;1. Key Laboratory of Tropical Medicinal Plant Chemistry, the Ministry of Education of China, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, China;2. Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, the Ministry of Education of China, School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China;3. Hainan Bikai Pharmaceutical Co., Ltd., Haikou 570216, China;1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India;2. Department of Immunology, Indian Institute of Chemical Biology (IICB), 4, Raja S.C. Mullick Road, Jadavpur, Calcutta, 700032, WB, India;1. Scientific Research Center, Royal Scientific Society, Amman, Jordan;2. Mechanical Engineering Department, Jordan University of Science and Technology, Irbid, Jordan |
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| Abstract: | 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. |
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