Jänicke R U, Walker P A, Lin X Y, Porter A G
Institute of Molecular and Cell Biology, National University of Singapore, Republic of Singapore.
EMBO J. 1996 Dec 16;15(24):6969-78.
Interleukin 1beta-converting enzyme-like (ICE-like) proteases are important mediators of apoptosis in diverse cell types and organisms. However, the role of these proteases in apoptosis cannot be satisfactorily explained on the basis of the physiological functions of their known substrates. Here we show that the C-terminal 42 amino acid peptide of the retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti-apoptotic function, is specifically cleaved off by an ICE-like protease in tumour necrosis factor (TNF)- and staurosporine-induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of ICE-like proteases, and in vitro by a point mutation (Asp886 to Ala) within the ICE-like protease cleavage site of Rb, (883)DEAD(886). An antibody raised against the C-terminal 15 amino acid peptide of Rb recognized the full-length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF-induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F-1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C-terminus has important regulatory functions, our results suggest that Rb cleavage is an important event in apoptosis.
白细胞介素1β转换酶样（ICE样）蛋白酶是多种细胞类型和生物体中凋亡的重要介质。然而，基于其已知底物的生理功能，这些蛋白酶在凋亡中的作用尚不能得到令人满意的解释。在此我们表明，视网膜母细胞瘤（Rb）蛋白的C末端42个氨基酸肽，一种具有已知抗凋亡功能的重要细胞周期调节因子，在肿瘤坏死因子（TNF）和星形孢菌素诱导的凋亡中被一种ICE样蛋白酶特异性切割。TNF诱导的Rb切割在体内和体外被两种ICE样蛋白酶的特异性抑制剂阻断，在体外被Rb的ICE样蛋白酶切割位点（883）DEAD（886）内的点突变（Asp886突变为Ala）阻断。针对Rb的C末端15个氨基酸肽产生的抗体识别全长Rb而非切割形式的Rb。在所检测的所有肿瘤细胞系中，Rb切割程度与TNF诱导的凋亡直接相关。切割后的Rb结合细胞周期蛋白D3并抑制E2F-1的转录活性，但不能结合与凋亡有关的调节蛋白MDM2。由于Rb抑制细胞死亡且其C末端具有重要的调节功能，我们的结果表明Rb切割是凋亡中的一个重要事件。
