Marissen W E, Guo Y, Thomas A A, Matts R L, Lloyd R E
Department of Microbiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
J Biol Chem. 2000 Mar 31;275(13):9314-23. doi: 10.1074/jbc.275.13.9314.
Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.
多种细胞类型中凋亡的诱导会导致蛋白质合成的抑制。最近，半胱天冬酶3对真核生物翻译起始因子4G（eIF4G）的切割被描述为可能导致翻译抑制的一个事件。在此，我们报道凋亡细胞中另一种起始因子eIF2α的切割，这可能有助于凋亡过程中翻译的调控。用Z-VAD-fmk预处理HeLa细胞可完全抑制这一切割事件。使用纯化的eIF2和纯化的半胱天冬酶进行的体外分析表明，半胱天冬酶3、6、8和10可切割eIF2α，但半胱天冬酶9不能。半胱天冬酶3最有效地切割eIF2α，并且在体外添加Ac-DEVD-CHO可抑制这一切割。磷酸化与非磷酸化eIF2α切割的比较显示，半胱天冬酶对非磷酸化形式有一定程度的偏好。当eIF2·2B复合物用作底物时，只有半胱天冬酶3能够切割eIF2α，这不受α亚基磷酸化的影响。eIF2·GDP二元复合物被半胱天冬酶3切割的效率要低得多。切割片段的序列分析表明切割位点位于该蛋白质的C末端部分。分析表明，半胱天冬酶切割后，与eIF2结合的GDP交换非常迅速，并且不再依赖于eIF2B。此外，体外翻译实验表明，eIF2α的切割导致eIF2复合物的功能改变，该复合物不再刺激含有病毒内部核糖体进入位点的mRNA上的上游AUG选择，并且不再能够刺激整体翻译。总之，我们在此描述了一种翻译起始因子eIF2α的切割，这可能有助于凋亡后期蛋白质合成的抑制或改变。
