DNA ligase III is degraded by calpain during cell death induced by DNA-damaging agents.
A yeast two-hybrid screen identified the regulatory subunit of the calcium-dependent protease calpain as a putative DNA ligase III-binding protein. Calpain binds to the N-terminal region of DNA ligase III, which contains an acidic proline, aspartate, serine, and threonine (PEST) domain frequently present in proteins cleaved by calpain. Recombinant DNA ligase III was a substrate for calpain degradation in vitro. This calpain-mediated proteolysis was calcium-dependent and was blocked by the specific calpain inhibitor calpeptin. Western blot analysis revealed that DNA ligase III was degraded in human fibrosarcoma HT1080 cells following exposure to gamma-radiation. The degradation of DNA ligase III was prevented by pretreatment with calpeptin, which protected irradiated cells from death. Calpeptin treatment also blocked 9-amino camptothecin-induced DNA ligase III proteolysis and simultaneously protected the cells from death. HT1080 clones expressing a modified DNA ligase III that lacked a recognizable PEST domain were significantly more resistant to killing by gamma-radiation or 9- amino camptothecin than were cells that overexpressed the wild-type form of DNA ligase III. These data show that calpain-mediated proteolysis of DNA ligase III plays an essential role in DNA damage-induced cell death in human cells.
Bordone
Laura
L
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Campbell
Colin
C
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid; D002154:Calpain / Q000378:metabolism*; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D000075223:Poly-ADP-Ribose Binding Proteins; D011392:Proline; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D013912:Threonine; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D029867:Xenopus Proteins
D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D011392:Proline; D002154:Calpain; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human