Calcium influx-mediated translocation of m-calpain induces Ku80 cleavage and enhances the Ku80-related DNA repair pathway.
Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics.
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea.
Kyung Hye
KH
Baek
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea.
Han Vit
HV
Yu
Department of Psychiatry, Yonsei University College of Medicine, Seoul, Republic of Korea.
Eosu
E
Kim
College of Pharmacy, CHA University, Pocheon, Republic of Korea.
Younghwa
Y
Na
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea.
Youngjoo
Y
Kwon
D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000502:physiology*; D019008:Drug Resistance, Neoplasm; D004847:Epithelial Cells / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D000072200:Ku Autoantigen / Q000378:metabolism*; D058990:Molecular Targeted Therapy / Q000379:methods; D009369:Neoplasms / Q000188:drug therapy; D011485:Protein Binding; D000072417:Protein Domains / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D059748:Proteolysis; D040901:Proteomics; D034622:RNA Interference; D034741:RNA, Small Interfering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D061207:Calcium Ionophores; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D015759:Ionomycin; D002154:Calpain; C484806:CAPN1 protein, human; C569870:CAPN2 protein, human; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D002118:Calcium
DNA damage; Ku80-related DNA repair; adriamycin; m-calpain