The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells.
The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells.
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DOI：
10.4049/jimmunol.158.5.2083
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发表时间：
1997-03
影响因子：
4.4
通讯作者：
K. McConnell;W. Dynan;J. Hardin
中科院分区：
文献类型：
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作者：
K. McConnell;W. Dynan;J. Hardin
DNA依赖性蛋白激酶（DNA-PK）是一种与DNA修复和V（D）J重组相关的丝氨酸/苏氨酸激酶。它由一个460 kDa的催化亚基（DNA-PKcs）和一个70/86 kDa的异源二聚体调节组分组成，该组分与人自身抗原Ku相同。调节亚基将催化亚基靶向到dsDNA断裂的游离末端。由于细胞凋亡与核小体间染色质断裂和双链DNA断裂的产生有关，我们研究了细胞表面受体Fas介导的细胞凋亡过程中DNA-PKcs或Ku的生化量是否发生变化。我们发现，催化亚基裂解成几个较小的多肽在细胞凋亡早期。与DNA-PKcs相反，Ku在凋亡过程中既不被切割也不减少。然后，我们将我们的体内结果扩展到无细胞系统。来自凋亡细胞的胞质提取物能够将DNA-PKcs切割成与体内所见大小相同的多肽，并且这种切割被半胱氨酸蛋白酶抑制剂碘乙酰胺和N-乙基马来酰亚胺抑制。此外，DNA-PKcs在体外被纯化的脱辅基蛋白酶（CPP 32）切割，但不被IL-1 β转化酶切割。切割也受到特异性四肽DEVD（DNA-PKcs序列的氨基酸2709-2712）的抑制，表明蛋白酶作用的候选位置。最后，我们发现在凋亡细胞中DNA-PKcs的催化活性降低。我们的结论是，DNA-PKcs是受选择性裂解的蛋白酶在凋亡过程中。DNA-PKcs的裂解可能代表在程序性细胞死亡期间调节DNA依赖性激酶功能的机制。
The DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase linked to DNA repair and V(D)J recombination. It is composed of a 460-kDa catalytic subunit (DNA-PKcs) and a 70/86-kDa heterodimeric regulatory component that is identical with the human autoantigen Ku. The regulatory subunit targets the catalytic subunit to the free ends of dsDNA breaks. Since apoptosis is associated with internucleosomal chromatin fragmentation and creation of dsDNA breaks, we examined whether the biochemical amounts of either DNA-PKcs or Ku changed during apoptosis mediated by the cell surface receptor Fas. We found that the catalytic subunit was cleaved into several smaller polypeptides early in apoptosis. In contrast to DNA-PKcs, Ku was neither cleaved nor decreased in amount during apoptosis. We then extended our in vivo results to a cellfree system. Cytosolic extracts derived from apoptotic cells were able to cleave DNA-PKcs into polypeptides of sizes identical with those seen in vivo, and this cleavage was inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. Furthermore, DNA-PKcs was cleaved in vitro by purified apopain (CPP32), but not IL-1beta-converting enzyme. Cleavage was also inhibited by the specific tetrapeptide DEVD (amino acids 2709-2712 of the DNA-PKcs sequence), suggesting a candidate position for protease action. Finally, we found that the catalytic activity of DNA-PKcs was decreased in apoptotic cells. We conclude that DNA-PKcs is subject to selective cleavage by proteases during apoptosis. Cleavage of DNA-PKcs may represent a mechanism for regulating the function of DNA-dependent kinase during programmed cell death.