Identification of caspase 3-mediated cleavage and functional alteration ofeukaryotic initiation factor 2 alpha in apoptosis

Induction of apoptosis in a variety of cell types leads to inhibition of pr otein synthesis. Recently, the cleavage of eukaryotic translation initiatio n factor 4G (eIF4G) by caspase 3 was described as a possible event contribu ting to translation inhibition, Here, we report the cleavage of another ini tiation factor in apoptotic cells, eIF2 alpha, that could contribute to reg ulation of translation during apoptosis, This cleavage event could be compl etely inhibited by pretreatment of HeLa cells with Z-VAD-fmk, In vitro anal ysis using purified eIF2 and purified caspases showed cleavage of eIF2 alph a by caspase 3, 6, 8, and 10 but not 9, Caspase 3 most efficiently cleaved eIF2 alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro, Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2 alp ha revealed a modest preference of the caspases for the nonphosphorylated f orm, When eIF2.2B complex was used as substrate, only caspase 3 was capable of eIF2 alpha cleavage, which was not affected by phosphorylation of the c u subunit, The eIF2 GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the c leavage 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 eIF2 alpha results in functional al teration of the eIF2 complex, which no longer stimulated upstream AUG selec tion on a mRNA containing a viral internal ribosome entry site and was no l onger capable of stimulating overall translation. In conclusion, we describ e here the cleavage of a translation initiation factor, eIF2 alpha that cou ld contribute to inhibition or alteration of protein synthesis during the l ate stages of apoptosis.