Song Q, Lees-Miller S P, Kumar S, Zhang Z, Chan D W, Smith G C, Jackson S P, Alnemri E S, Litwack G, Khanna K K, Lavin M F
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Australia.
EMBO J. 1996 Jul 1;15(13):3238-46.
Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis.
源自X射线交叉互补组5（XRCC5）的放射敏感细胞系、重症联合免疫缺陷（SCID）小鼠以及一种人类胶质瘤细胞系缺乏DNA依赖性蛋白激酶（DNA-PK）的组分，这表明DNA-PK在DNA双链断裂修复中起重要作用。另一种与DNA修复有关的酶，聚（ADP-核糖）聚合酶，在细胞凋亡过程中被切割并失活，这表明一些DNA修复蛋白可能在细胞凋亡期间被选择性地靶向破坏。在此我们证明，DNA-PK的催化亚基DNA-PKcs在不同细胞类型暴露于各种已知可导致细胞凋亡的试剂后被优先降解。然而，该酶的DNA结合组分Ku保持完整。DNA-PKcs的降解伴随着DNA-PK活性的丧失。一种对依托泊苷诱导的细胞凋亡具有抗性的细胞系未显示出DNA-PKcs的降解。蛋白酶抑制剂数据表明一种类ICE蛋白酶参与了DNA-PKcs的切割，随后发现半胱氨酸蛋白酶CPP32而非Mch2α、ICE或TX将纯化的DNA-PKcs切割成三个大小与在经历细胞凋亡的细胞中观察到的片段相当的片段。通过抗体定位和微量测序确定的DNA-PKcs中的切割位点显示对于CPP32切割和由经历细胞凋亡的细胞提取物催化的切割是相同的。这些观察结果表明DNA-PKcs是细胞凋亡期间类ICE蛋白酶进行蛋白水解的关键靶点。
