Callus Bernard A, Moujallad Donia M, Silke John, Gerl Robert, Jabbour Anissa M, Ekert Paul G, Vaux David L
Department of Biochemistry, La Trobe University, Victoria 3086, Australia.
J Mol Biol. 2008 Dec 12;384(2):313-23. doi: 10.1016/j.jmb.2008.09.041. Epub 2008 Sep 25.
Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF(3) cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF(3) cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation.
彪马（p53上调凋亡调节因子）属于仅含BH3（Bcl-2同源结构域3）的凋亡调节蛋白家族。其表达由多种凋亡刺激诱导，包括辐射和细胞因子撤除。利用诱导系统表达彪马，我们研究了彪马诱导凋亡的性质。在BaF(3)细胞中，彪马的表达导致半胱天冬酶介导的ICAD（半胱天冬酶激活的脱氧核糖核酸酶抑制剂）和Mcl-1（髓样细胞白血病-1）快速裂解，导致细胞活力完全丧失。令人惊讶的是，彪马蛋白水平在诱导后2小时内达到峰值，随后降至基础水平。彪马的最大丰度与半胱天冬酶活性的开始相吻合。半胱天冬酶的抑制可防止随后彪马的丢失，表明其降解是半胱天冬酶依赖性的。在表达转染Bcl-2的细胞中，诱导的彪马达到显著更高的水平，但延迟后，半胱天冬酶变得活跃，细胞死亡发生。彪马与内源性Bcl-2和Mcl-1进行了共免疫沉淀，但未与Bax和Bak共沉淀，这表明在这些细胞中彪马不与Bax或Bak结合来启动细胞死亡。在小鼠胚胎成纤维细胞（MEF）中，彪马的量在诱导后4小时内达到峰值。相反，在bax/bak双敲除的MEF中，彪马在诱导后稳定表达，即使在非常高的水平也无法触发凋亡。野生型MEF中Bcl-2的过表达，与BaF(3)细胞一样，导致达到更高水平的彪马，但并未阻止细胞死亡的发生。这些结果表明，Bcl-2促生存家族的水平设定了一个阈值，在该阈值下彪马能够间接激活Bax或Bak，进而导致半胱天冬酶的激活，这不仅会导致细胞死亡，还会迅速诱导彪马降解。
