Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase-mediated apoptosis of HeLa cells.
We show that caspase-3 cleaves Cdc6 at D(290)/S and D(442)/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-alpha-induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-alpha-induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-alpha- or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53-Bax-mediated apoptosis.
Yim
Hyungshin
H
Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Hwang
In Sun
IS
Choi
Joon-Seok
JS
Chun
Kwang-Hoon
KH
Jin
Ying Hua
YH
Ham
Young-Mi
YM
Lee
Kwang Youl
KY
Lee
Seung Ki
SK
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; D005047:Etoposide; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases