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Cleavage of Polypeptide Chain Initiation Factor Eif4gi

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Cleavage of Polypeptide Chain Initiation Factor Eif4gi Cell Death and Differentiation (2000) 7, 628 ± 636 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage M Bushell1, D Poncet2, WE Marissen3, H Flotow4, RE Lloyd3, Abbreviations: Ct, C-terminal fragment of eIF4G; eIFs, eukar- MJ Clemens*,1 and SJ Morley5 yotic initiation factors; FAG, fragment of the apoptotic cleavage of eIF4G; FMDV, foot-and-mouth-disease virus; m7GTP, 7-methyl- 1 Department of Biochemistry and Immunology, Cellular and Molecular guanosine triphosphate; PABP, poly(A) binding protein; PARP, Sciences Group, St George's Hospital Medical School, Cranmer Terrace, poly(ADP-ribose) polymerase; z-VAD.FMK, Z-Val-Ala-Asp.¯uo- London SW17 ORE, UK romethylketone 2 Laboratoire de Virologie et Immunologie Moleculaires, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas, Cedex, France 3 Department of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Introduction 4 Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AY, UK In eukaryotic cells the polypeptide chain initiation factor eIF4G 5 Biochemistry Group, School of Biological Sciences, University of Sussex, plays an essential role in the mechanism of translation by Brighton BN1 9QG, UK acting as a molecular bridge between other components of * Corresponding author: MJ Clemens, Department of Biochemistry and the ribosomal initiation complex (reviewed in references1±3). Immunology, Cellular and Molecular Sciences Group, St George's Hospital Two forms of eIF4G, which are products of different genes, Medical School, Cranmer Terrace, London SW17 ORE, UK, 4 Tel: +44 20 8725 5770; Fax: +44 20 8725 2992; have been identified. eIF4GI is a single polypeptide chain of 5 E-mail: [email protected] at least 154 kDa; recently extensions to the original N- terminal sequence of this protein have been described,6±8 Received 22.12.99; revised 22.3.00; accepted 28.3.00 and it is possible that the true N-terminus has not yet been Edited by RA Knight identified. In vivo eIF4G exists partly in the form of a complex with the mRNA cap-binding protein eIF4E and the ATP- dependent RNA helicase eIF4A, constituting the initiation Abstract factor eIF4F.9 Within the sequence of eIF4GI9 there are 10 10,11 11 Polypeptide chain initiation factor eIF4GI undergoes caspase- domains that interact with eIF4E, eIF4A, , eIF3, the poly(A) binding protein (PABP)7,12±14 and the eIF4E kinase mediated degradation during apoptosis to give characteristic 15 fragments. The most prominent of these has an estimated Mnk1. Interaction of PABP with eIF4GI has been suggested to facilitate the functional association of the 3' end of an mass of approximately 76 kDa (Middle-Fragment of Apoptotic mRNA with the 5' end.14 The association of eIF4G with eIF4E cleavage of eIF4G; M-FAG). Subcellular fractionation of the markedly enhances the binding of the latter to the mRNA BJAB lymphoma cell line after induction of apoptosis cap16 and is necessary to permit cap-dependent translation. It indicates that M-FAG occurs in both ribosome-bound and is also likely that the phosphorylation of eIF4E, which has soluble forms. Affinity chromatography on m7GTP-Sephar- been correlated with enhanced translational activity in cells ose shows that M-FAG retains the ability of eIF4GI to associate treated with mitogens and growth factors,17 ± 20 is facilitated by with both the mRNA cap-binding protein eIF4E and initiation the binding of the eIF4E kinase Mnk1 to the C-terminal part of factor eIF4A and that the ribosome-bound form of M-FAG is eIF4GI.15 Over-expression of either eIF4G or eIF4E has been 21,22 also present as a complex with eIF4E and eIF4A. These data shown to transform cells to a tumorigenic phenotype, and abnormally high levels of both factors have been observed in suggest that the binding sites for eIF4E, eIF4A and eIF3 on 23 ± 25 eIF4GI areretained in the caspase-generated fragment. M-FAG several types of human tumours. There is a well-characterised loss of integrity of both is also a substrate for cleavage by the Foot-and-Mouth- eIF4GI (reviewed in reference2)andeIF4GII4,26 in Disease Virus-encoded L protease. These properties,together mammalian cells after infection with picornaviruses such with the pattern of recognition by a panel of antibodies, define as Polio or Foot-and-Mouth-Disease viruses (FMDV). This the origin of the apoptotic cleavage fragment. N-terminal is a result of cleavage at specific internal sites by the sequencing of the products of caspase-3-mediated eIF4GI virally-encoded 2A and L proteases, respectively. Since cleavage hasidentified the major cleavagesites.Thepatternof these cleavages occur in a region between the eIF4E and eIF4GI degradation and the possible roles of the individual eIF3 binding sites27 they destroy the bridging function of cleavage products in cells undergoing apoptosis are eIF4G and thus lead to the inhibition of cap-dependent 28 ± 32 discussed. Cell Death and Differentiation (2000) 7, 628 ± 636. initiation. The remaining C-terminal fragment of eIF4G can nevertheless still support initiation that is cap- independent28 or that involves ribosome binding to internal Keywords: apoptosis; BJAB cells; caspases; eIF4G; initiation 33 factors; protein sequencing sites on mRNAs. Caspase cleavage fragments of eIF4GI MBushellet al 629 We have shown that eIF4GI is rapidly cleaved during the novel ca. 76 kDa fragment (M-FAG; Figure 1B, lanes 4 ± 6). process of apoptosis (cell death) that can be induced by a Cleavage of eIF4GI correlated temporally with that of the variety of treatments in several cell lines.34 ± 36 This effect is classical apoptotic substrate, poly(ADP-ribose) polymerase selective since several other initiation factors, including (PARP; lanes 4 ± 6). M-FAG is actually a doublet, the levels of eIF4E and eIF4A, are relatively stable under these both components of which remain constant within cells for conditions. In cells undergoing apoptosis proteolysis of several hours after induction of apoptosis. In addition, with this eIF4GI to produce characteristic cleavage fragments is antiserum we have observed the transient generation of mediated by one or more caspases,34 and caspase-3 smaller amounts of eIF4GI fragments of approximately activity is both necessary and sufficient for this pro- 150 kDa (p150, see Figure 2A) and 120 kDa (p120, faintly cess.36,37 Protein synthesis is strongly inhibited in cells visible in Figure 1B and 2A), which appear within 2 h of undergoing apoptosis and in some situations this inhibition, cycloheximide treatment. The N-terminal sequence-specific as well as the cleavage of eIF4GI, can be prevented by antiserum (RL) visualised a group of previously described incubation with the cell-permeable caspase inhibitor, z- cleavage products (eIF4GcpN),36 which we designate N- VAD.FMK.34±36 It is possible that the down-regulation of FAG, and the C-terminal sequence-specific antiserum (E) translation in apoptotic cells is due to the specific and recognised a novel fragment of approximately 50 kDa (C- complete cleavage of eIF4G. However the remaining FAG) (Figure 1B). The same pattern of cleavages was protein synthetic activity (which amounts to some 35% of observed after treatment with other inducers of apoptosis the control rate35) may be maintained by the continued (serum deprivation, anti-Fas antiserum and etoposide ± data presence of one or more cleavage fragments that retain the not shown). Similar results were also seen using antisera ability to interact with other key initiation factors. If this is specific to eIF4GII, although in this case a different pattern of the case it is also likely that the properties of the cleavage cleavages occurred.38 fragments that appear during apoptosis are distinct from We also assessed the levels of several other initiation those of the fragments of eIF4G produced in cells during factors in the BJAB cells under the same conditions. Figure picornavirus infection, which do not support any cap- 1B shows that, in agreement with our previous results,34 dependent initiation.28±32 the levels and integrity of poly(A)-binding protein (PABP), To begin to test these hypotheses we have identified the eIF4A and total eIF4E were unaffected in apoptotic cells. sites of the caspase-mediated cleavages of eIF4GI that However, use of antiserum which was specific for the occur rapidly during cellular apoptosis and have charac- phosphorylated form of eIF4E39 indicated that the terised the properties of the major cleavage products (N- phosphorylation status of eIF4E decreased at later times FAG, M-FAG and C-FAG) that persist in cells for several following the induction of apoptosis (Figure 1B, lanes 4 ± 6). hours after fragmentation of the full-length factor. Our results indicate that the cleavages that occur and the nature of the fragments that result, are quite distinct from Association of the cleavage fragments of eIF4G those seen in picornavirus-infected cells. The properties of with eIF4E and ribosomes the M-FAG fragment observed in apoptotic cells are Initiation factor eIF4E and associated proteins were isolated compatible with the possibility that this protein can still by m7GTP-Sepharose chromatography (Figure 1C). Immuno- support cap-dependent initiation but with a substantially blotting of the m7GTP-Sepharose-purified proteins revealed impaired efficiency. that M-FAG (lanes 4 ± 6), p120 (lanes 4, 5) and eIF4A (lanes 4 ± 6) remained associated with eIF4E in cells in which essentially all of the eIF4GI had been cleaved. However, Results when protein recovered by m7GTP-Sepharose was visualised with antisera specific for N-FAG or C-FAG, these character- Characterisation of the cleavage fragments of istic fragments were absent, indicating that neither of these eIF4G in apoptotic cells products remains associated with eIF4E (Figure 1C, lanes 4 ± Treatment of the EBV-negative Burkitt's lymphoma BJAB cell 6).
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