Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland.
J Cell Sci. 2010 May 1;123(Pt 9):1401-6. doi: 10.1242/jcs.061143. Epub 2010 Mar 31.
In the present study, we quantitatively analysed the interface between apoptosis initiation and execution by determining caspase-8 activation, Bid cleavage and mitochondrial engagement (onset of mitochondrial depolarisation) in individual HeLa cervical cancer cells following exposure to tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL). Employing resonance-energy-transfer probes containing either the caspase-8 recognition site IETD or full-length Bid, we observed a significant delay between the times of caspase-8 activation and Bid cleavage, suggesting the existence of control steps separating these two processes. Subsequent analyses suggested that the divergence of caspase-8 activation and Bid cleavage are critically controlled by kinase signalling: inhibiting protein kinase CK2 by using 5,6-dichloro-l-(beta-D-ribofuranosyl-1)-benzimidazole (DRB) or by overexpression of a dominant-negative CK2alpha catalytic subunit largely eliminated the lag time between caspase-8 activation and Bid cleavage. We conclude that caspase-8 activation and Bid cleavage are temporally uncoupled events, providing transient tolerance to caspase-8 activities.
在本研究中，我们通过测定肿瘤坏死因子相关凋亡诱导配体（TRAIL）作用后单个宫颈癌 HeLa 细胞中 caspase-8 的激活、Bid 的切割和线粒体的结合（线粒体去极化的开始），定量分析了凋亡起始与执行之间的界面。采用含有 caspase-8 识别位点 IETD 或全长 Bid 的共振能量转移探针，我们观察到 caspase-8 的激活和 Bid 的切割之间存在显著的时间延迟，表明这两个过程之间存在控制步骤。进一步的分析表明，caspase-8 的激活和 Bid 的切割的发散受到激酶信号的严格控制：用 5,6-二氯-l-（β-D-核糖呋喃基）-1-苯并咪唑（DRB）抑制蛋白激酶 CK2 或过表达显性失活 CK2alpha 催化亚基，可大大消除 caspase-8 激活和 Bid 切割之间的时间延迟。我们的结论是，caspase-8 的激活和 Bid 的切割是时间上不偶联的事件，为 caspase-8 活性提供了短暂的耐受。
