| Abstrakt: |
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, eIF2α, 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 vitroanalysis using purified eIF2 and purified caspases showed cleavage of eIF2α by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2α and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versusnonphosphorylated eIF2α 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 eIF2α cleavage, which was not affected by phosphorylation of the α 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 vitrotranslation experiments indicated that cleavage of eIF2α 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, eIF2α that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis. |
