Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

Abstract

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

Caspases shutdown nonsense-mediated mRNA decay during apoptosis

“…34 Among these proteins, eIF4G which has a central role in the pioneer round of translation through its interaction with PABPC1 and CBP80, is targeted and inactivated by caspase3. 35 The work presented here shows that UPF1 and UPF2 cleavage occurs at positions D37 and D741, respectively ( Figure 7) not only to inactivate both NMD factors per se but also to generate fragments that induce apoptosis (Figure 8a). However, this contribution to apoptosis appears to be modest and is likely due to the wide spectrum of caspase targets that influence apoptosis.…”

Conducting the initiation of protein synthesis: the role of eIF4G

“…Both groups have also used a wide spectrum of different apoptosis inducers (UV light, proteasome inhibitors and death receptor ligands) and all of them caused almost complete loss of eIF4GI. Prevention of eIF4GI cleavage by the use of general caspase inhibitors such as Z-VAD.FMK and Z-DEVD.FMK established that these enzymes were the effectors of proteolysis and further investigation identified caspase 3 as both necessary and sufficient for targeted degradation of eIF4GI in vitro and in vivo (Bushell et al, 1999;Clemens et al, 1998;Marissen and Lloyd, 1998). The proteolytic processing of eIF4GI yields a 76 kDa fragment (referred to as M-FAG for middle fragment of apoptotic cleavage of eIF4GI) that retains binding sites for eIF4E, eIF4A and eIF3 (see Fig.…”

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

“…In cells undergoing apoptosis proteolysis of eIF4GI to produce characteristic cleavage fragments is mediated by one or more caspases, 34 and caspase-3 activity is both necessary and sufficient for this process. 36,37 Protein synthesis is strongly inhibited in cells undergoing apoptosis and in some situations this inhibition, as well as the cleavage of eIF4GI, can be prevented by incubation with the cell-permeable caspase inhibitor, z-VAD.FMK. 34 ± 36 It is possible that the down-regulation of translation in apoptotic cells is due to the specific and complete cleavage of eIF4G.…”