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S J Morley

Publications and source records attributed to S J Morley.

At least 37 records · Page 2Linked to original sources
PubMed2000

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

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.

Apoptosis↗
PubMed2000

Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis.

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Cellular stress in xenopus kidney cells enhances the phosphorylation of eukaryotic translation initiation factor (eIF)4E and the association of eIF4F with poly(A)-binding protein.

Eukaryotic initiation factor (eIF) 4E binds to the 5'-cap structure of eukaryotic mRNA and has a central role in the control of cell proliferation. We have shown previously that the stimulation of cultured Xenopus kidney cells with serum resulted in the activation of protein synthesis, enhanced phosphorylation of eIF4E and increased binding of the adapter protein, eIF4G, and poly(A)-binding protein (PABP) to eIF4E to form the functional initiation factor complex, eIF4F/PABP. We now show that cellular stresses such as arsenite, anisomycin and heat shock also result in increased phosphorylation of eIF4E, eIF4F complex formation and the association of PABP with eIF4G, in conditions under which the rate of protein synthesis is severely inhibited. In contrast with reported effects on mammalian cells, the stress-induced increase in eIF4F complex formation occurs in the absence of changes in the association of eIF4E with its binding proteins 4E-BP1 or 4E-BP2. The stress-induced changes in eIF4E phosphorylation were totally abrogated by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and were partly inhibited by the phosphoinositide 3-kinase inhibitor LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. However, eIF4E phosphorylation was unaffected by extracellular signal-regulated protein kinase (MAP kinase) inhibitor PD98059. These results indicate that cellular stresses activate multiple signalling pathways that converge at the level of eIF4F complex formation to influence the interactions between eIF4E, eIF4G and PABP.

Animals↗
PubMed1999

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.

Apoptosis↗
PubMed1999

The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells.

Serum stimulation of cultured Xenopus kidney cells results in enhanced phosphorylation of the translational initiation factor (eIF) 4E and promotes a 2.8-fold increase in the binding of the adapter protein eIF4G to eIF4E, to form the functional initiation factor complex eIF4F. Here we demonstrate the serum-stimulated co-isolation of the poly(A)-binding protein (PABP) with the eIF4F complex. This apparent interaction of PABP with eIF4F suggests that a mechanism shown to be important in the control of translation in the yeast Saccharomyces cerevisiae also operates in vertebrate cells. We also present evidence that the signaling pathways modulating eIF4E phosphorylation and function in Xenopus kidney cells differ from those in several mammalian cell types studied previously. Experiments with the immunosuppressant rapamycin suggest that the mTOR signaling pathway is involved in serum-promoted eIF4E phosphorylation and association with eIF4G. Moreover, we could find little evidence for regulation of eIF4E function via interaction with the specific binding proteins 4E-BP1 or 4E-BP2 in these cells. Although rapamycin abrogated serum-enhanced rates of protein synthesis and the interaction of eIF4G with eIF4E, it did not prevent the increase in association of eIF4G with PABP. This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E.

Amino Acid Sequence↗
PubMed1999

Translation initiation factor 4E.

Translation initiation factor 4E (eIF4E) binds the 7-methylguanosine cap structure of mRNA and mediates recruitment of mRNA to ribosomes, with the potential of regulating the overall rate of translation and discriminating between different RNAs. Increased translation is required for progress through the cell cycle, and it is therefore not surprising that eIF4E has oncogenic properties when overexpressed. The function of this review is to summarise what is known about eIF4E gene and protein structure, biological function and medical relevance.

Amino Acid Sequence↗
PubMed1998

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

We have investigated the effect 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 effects. 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.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Cleavage of translation initiation factor 4G (eIF4G) during anti-Fas IgM-induced apoptosis does not require signalling through the p38 mitogen-activated protein (MAP) kinase.

Initiation factor (eIF) 4G plays a key role in the regulation of translation, acting as a bridge between eIF4E and eIF3, to allow an mRNA molecule to associate with the 40S ribosomal subunit. In this study, we show that activation of the Fas/CD95 receptor complex in Jurkat cells induces the degradation of eIF4G, the inhibition of total protein synthesis and cell death. These responses were prevented by the caspase inhibitors, zVAD.FMK and zDEVD.FMK. We also show that, in contrast to Saccharomyces cerevisiae, although rapamycin caused a modest inhibition of protein synthesis it did not induce apoptosis or the cleavage of eIF4G. Studies with the specific inhibitor, SB203580, have shown that signalling through the p38 MAP kinase pathway is not required for either the Fas/CD95-induced cleavage of eIF4G or cell death. These data suggest that the cleavage of eIF4G and the inhibition of translation play an integral role in Fas/CD95-induced cell death in Jurkat cells.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability.

The immunosuppressant rapamycin has been shown previously to inhibit the G1/S transition in several cell types by prolonging the G1 phase of the cell cycle. This process appears to be controlled, in part, by the rapamycin-sensitive FK506-binding protein-rapamycin-associated protein-p70 S6 kinase (p70(S6k)) pathway and the cyclin-dependent kinases (Cdk). We now show that in serum-stimulated NIH 3T3 cells, rapamycin treatment delays the accumulation of cyclin D1 mRNA during progression through G1. Rapamycin also appears to affect stability of the transcript. The combined transcriptional and post-transcriptional effects of the drug ultimately result in decreased levels of cyclin D1 protein. Moreover, degradation of newly synthesized cyclin D1 protein is accelerated by rapamycin, a process prevented by inclusion of the proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal. The overall effect of rapamycin on cyclin D1 leads, in turn, to impaired formation of active complexes with Cdk4, a process which triggers retargeting of the p27(Kip1) inhibitor to cyclin E/Cdk2. In view of this novel experimental evidence, we discuss a possible mechanism for the rapamycin-induced cell cycle arrest at the G1/S transition.

3T3 Cells↗
PubMed1997

Signalling through either the p38 or ERK mitogen-activated protein (MAP) kinase pathway is obligatory for phorbol ester and T cell receptor complex (TCR-CD3)-stimulated phosphorylation of initiation factor (eIF) 4E in Jurkat T cells.

Initiation factor (elF) 4E plays a key role in the regulation of translation. Its activity is modulated both by phosphorylation and by its association with an inhibitory protein, 4E-BP1, which precludes its interaction with eIF4G. Although increased eIF4E phosphorylation has been correlated with the activation of protein synthesis in T cells, the kinase(s) and/or phosphatase(s) involved have not been characterised. There is evidence for phosphorylation of eIF4E mediated by both protein kinase C-dependent and -independent signalling pathways. In these studies, I show that activation of protein kinase C with phorbol ester, stimulation via the T cell receptor complex with the monoclonal antibody OKT3 and cellular stresses increase the phosphorylation of eIF4E in Jurkat T cells. In contrast to published data, inhibition of either the ERK MAP kinase or p38 MAP kinase signalling pathways does not affect the PMA- or OKT3-stimulated increase in eIF4E phosphorylation. However, simultaneous inhibition of both of these pathways with selective inhibitors is required to completely abrogate the enhanced phosphorylation of eIF4E. These data show that in Jurkat cells, protein kinase C modulates the phosphorylation status of eIF4E indirectly via the ERK and/or p38 MAP kinase signalling pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Involvement of stress-activated protein kinase and p38/RK mitogen-activated protein kinase signaling pathways in the enhanced phosphorylation of initiation factor 4E in NIH 3T3 cells.

The initiation factor (eIF) 4E is regulated by modulating both the phosphorylation and the availability of the protein to participate in the initiation process. Here we show that either serum treatment or activation of the stress-activated protein kinase (JNK/SAPK) led to enhanced phosphorylation of eIF4E in quiescent NIH 3T3 cells. Although the immunosuppressant, rapamycin, was found to stabilize the association of eIF4E with its negative regulator, 4E-BP1, this drug did not prevent the early effects of serum stimulation on the overall rate of translation, polysome formation, the phosphorylation status of eIF4E, or the recruitment of eIF4E into the eIF4F complex. However, the rapid enhancement of eIF4E phosphorylation in response to serum was largely prevented by the inhibitor of mitogen-activated protein (MAP) kinase activation, PD98059. Activation of the JNK/SAPK signaling pathway with anisomycin resulted in enhanced phosphorylation of eIF4E, which was prevented by either rapamycin or the highly specific p38 MAP kinase inhibitor, SB203580. These data illustrate that multiple signaling pathways, including those of distinct members of the MAP kinase family, mediate the phosphorylation of eIF4E and that the association of eIF4E with 4E-BP1 does not necessarily prevent phosphorylation of eIF4E in vivo.

3T3 Cells↗
PubMed1997

The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is prevented by eIF4E binding protein (PHAS-I; 4E-BP1) in the reticulocyte lysate.

A common feature of viral infection is the subversion of the host cell machinery towards the preferential translation of viral products. In some instances, this is partly mediated by the expression of virally encoded proteases which lead to the cleavage of initiation factor eIF4G. The foot-and-mouth disease virus encodes two forms of a cysteine proteinase (L protease) which bisects the eIF4G polypeptide into an N-terminal fragment containing the eIF4E binding site, and a C-terminal fragment which contains binding sites for eIF4A and eIF3 and which associates with the 40S ribosomal subunit. Previously, we have demonstrated that the cleavage of eIF4G by L protease stimulates the translation of uncapped transcripts encoding cellular proteins and supports internal initiation driven by picornavirus internal ribosome entry segment (IRES) elements. Use of reticulocyte lysates manipulated to deplete them of eIF4E and the N-terminal fragment suggests that the C-terminal fragment of eIF4G is responsible for these effects, and we have now confirmed this by purifying the C-terminal fragment and analysing its effects directly in the absence of L protease. Interestingly, we find that pre-incubation of reticulocyte lysates or ribosomal salt wash fractions with the specific eIF4E binding protein, PHAS-I (eIF4E-BP1), blocks the proteolytic cleavage of eIF4G by L protease. This effect can be reversed by addition of recombinant eIF4E. These data are consistent with a model whereby the L protease cleavage site in eIF4G is inaccessible until a change in conformation is induced by the binding of eIF4E. This may have implications for a role for eIF4E binding in triggering changes that expose other domains in the eIF4G molecule during initiation of translation.

Animals↗
PubMed1996

A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate.

The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (approximately 75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported. The association of a small proportion of eIF4E with the ribosome fraction appears to be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate.

Amino Acid Sequence↗
PubMed1996

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.

Animals↗