Abstract
Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed. Cell Death and Differentiation (2000) 7, 628–636
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Abbreviations
- Ct:
-
C-terminal fragment of eIF4G
- eIFs:
-
eukaryotic initiation factors
- FAG:
-
fragment of the apoptotic cleavage of eIF4G
- FMDV:
-
foot-and-mouth-disease virus
- m7GTP:
-
7-methylguanosine triphosphate
- PABP:
-
poly(A) binding protein
- PARP:
-
poly(ADP-ribose) polymerase
- z-VAD.FMK:
-
Z-Val-Ala-Asp.fluo-romethylketone
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Acknowledgements
We thank Dr Anne Gatignol (ICGM, Paris) for the gift of the Hela cell cDNA library, P Vende (Jouy-en-Josas, France) for the cloning and sequencing of eIF4GI constructs, Dr R Rhoads (Shreveport, Louisiana, USA) for the gift of pHFC1, A Gillman-Smith for help with mass spectrometry and N Castagné for skilful technical assistance. We also thank Dr Jenny Pain, Dr Linda McKendrick, Dr Ruth Simon, Ms Wendy Wood and Ms Gill Carpenter for helpful discussions. This research was supported by grants from The Royal Society, The Leukaemia Research Fund, The Cancer Prevention Research Trust and The Wellcome Trust (grant numbers 045619 and 056778). During the performance of this work M Bushell was funded by a BBSRC Industrial CASE Studentship in SJ Morley's laboratory, in collaboration with Roche Discovery (Welwyn Garden City). SJ Morley is a Senior Research Fellow of the Wellcome Trust.
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Bushell, M., Poncet, D., Marissen, W. et al. Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage. Cell Death Differ 7, 628–636 (2000). https://doi.org/10.1038/sj.cdd.4400699
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DOI: https://doi.org/10.1038/sj.cdd.4400699
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