Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines

Abstract

We have investigated the e ect of inducing apoptosis in BJAB and Jurkat cells on the cellular content of several polypeptide chain initiation factors. Serum deprivation results in inhibition of protein synthesis and induction of apoptosis in BJAB cells; at early times, there is selective degradation of polypeptide initiation factor eIF4G but no major losses of other key initiation factors. The disappearance of full length eIF4G is accompanied by the appearance of smaller forms of the protein, including a major product of approximately 76 kDa. Apoptosis induced by cycloheximide results in similar e ects. Both total cytoplasmic eIF4G and eIF4G associated with eIF4E are degraded with a half-life of 2 ± 4 h under these conditions. Treatment of serum-starved or cycloheximide-treated cells with Z-VAD.FMK or Z-DEVD.FMK, which inhibit caspases required for apoptosis, protects eIF4G from degradation and blocks the appearance of the ca. 76 kDa product. Exposure of BJAB cells to rapamycin rapidly inhibits protein synthesis but does not lead to acute degradation of eIF4G. In both BJAB and Jurkat cells induction of apoptosis with anti-Fas antibody or etoposide also results in the selective loss of eIF4G, which is inhibitable by Z-VAD.FMK. These data suggest that eIF4G is selectively targeted for cleavage as cells undergo apoptosis and is a substrate for proteases activated during this process.

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

“…38 We also assessed the levels of several other initiation factors in the BJAB cells under the same conditions. Figure 1B shows that, in agreement with our previous results, 34 the levels and integrity of poly(A)-binding protein (PABP), eIF4A and total eIF4E were unaffected in apoptotic cells. However, use of antiserum which was specific for the phosphorylated form of eIF4E 39 indicated that the phosphorylation status of eIF4E decreased at later times following the induction of apoptosis ( Figure 1B, lanes 4 ± 6).…”

“…We have reported previously that all of these treatments also result in the complete cleavage of eIF4GI into a series of characteristic fragments. 34 Figure 1B illustrates the time-course of eIF4GI cleavage in response to induction of apoptosis with cycloheximide, as visualised with a number of domain and sequence-specific antisera (W, RL, E; see Materials and Methods and Figure 3). In agreement with our previous data, 34,35 antiserum generated against a large Cterminal fragment of eIF4G (W) showed that cycloheximide induced the cleavage of total cytoplasmic eIF4GI to yield the novel ca.…”

“…34 ± 36 This effect is selective since several other initiation factors, including eIF4E and eIF4A, are relatively stable under these conditions. 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.…”

DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling

“…Inhibition of eIF4E and p70 S6 kinase is therefore clearly not a consequence of apoptosis. It also precedes the cleavage of 4E-BP1, which we observed in cells treated with any of the DNAdamaging agents used at later times when caspases are active (Figures 1b, 4c, 5a,b), and the cleavage of eIF4G 1 (Figure 2f), which occurs as a consequence of caspase-3 activation in cells which are actively undergoing apoptosis (Clemens et al, 1998;Marissen and Lloyd, 1998;Bushell et al, 1999). The broad-speci®city caspase inhibitor Z-VAD.FMK did not prevent the inhibition of protein synthesis (Figure 6), the inactivation of p70 S6 kinase (Figure 2a), the dephosphorylation of 4E-BP1 (Figure 2c) or the dissociation of eIF4E:eIF4F complexes (data not shown), demonstrating that all these e ects are independent of caspase activity.…”

“…This event is thus also independent of caspase activation and the onset of apoptosis. eIF4G 1 has previously been shown to be degraded in cells undergoing apoptosis (Marissen and Lloyd, 1998;Clemens et al, 1998) and this requires caspase activity/ activation (Marissen and Lloyd, 1998;Bushell et al, 1999). We therefore analysed the total level of eIF4G 1 in ) were used in the in vitro p70 S6 kinase activity assay as described in Materials and methods.…”