Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis.
Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.
Gama
Vivian
V
Case Comprehensive Cancer Center, Department of Pharmacology and Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Yoshida
Tomoyuki
T
Gomez
Jose A
JA
Basile
David P
DP
Mayo
Lindsey D
LD
Haas
Arthur L
AL
Matsuyama
Shigemi
S
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D061945:Caspase Inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D015398:Signal Transduction / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D034961:Antigens, Nuclear; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C089486:carbobenzoxy-leucyl-leucyl-norvalinal; C067713:lactacystin; D004317:Doxorubicin; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen; D019311:Staurosporine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine