Han Z, Malik N, Carter T, Reeves W H, Wyche J H, Hendrickson E A
Department of Molecular Biology, Cell Biology, and Biochemistry, Box G, Brown University, Providence, Rhode Island 02912, USA.
J Biol Chem. 1996 Oct 4;271(40):25035-40. doi: 10.1074/jbc.271.40.25035.
We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PKcs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PKcs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PKcs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PKcs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PKcs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PKcs.
我们证明，在经星形孢菌素（STS）处理的HL-60细胞中，DNA依赖性蛋白激酶的催化亚基（DNA-PKcs）会被特异性地蛋白水解切割，STS是一种强效的凋亡诱导剂。DNA-PKcs的蛋白水解与凋亡性染色体DNA降解相关或先于其发生。用经STS处理的HL-60细胞制备的无细胞提取物，在以纯化的DNA-PK为底物的体外试验中重现了DNA-PKcs的蛋白水解过程。对凋亡细胞提取物的蛋白质印迹分析表明，CPP32的32 kDa前体在HL-60细胞中表达，并在STS处理后进行加工。此外，虽然DNA-PKcs蛋白酶活性不受许多传统蛋白酶抑制剂的抑制，但它可被一种高度选择性的CPP32肽衍生抑制剂抑制。这些数据强烈表明，CPP32或一种类似CPP32的蛋白酶负责DNA-PKcs的蛋白水解。最后，我们的结果表明，DNA-PKcs在体外的切割在存在Bcl-二的情况下进行，这表明Bcl-2提供的功能位于DNA-PKcs蛋白水解的上游。
