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S J Morley

Publications and source records attributed to S J Morley.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1995

Translational regulation during activation of porcine peripheral blood lymphocytes: association and phosphorylation of the alpha and gamma subunits of the initiation factor complex eIF-4F.

Mature peripheral blood lymphocytes exist in a resting state both in vivo and when maintained in culture, exhibiting low translation rates consistent with their non-proliferative state. Previously we have shown that activation of these quiescent cells with either phorbol ester or concanavalin A leads to a rapid increase in the rate of protein synthesis and phosphate-labelling of initiation factor eIF-4 alpha [Morley, Rau, Kay and Pain (1993) Eur. J. Biochem. 218, 39-48]. We now show that neither the early enhanced translation rate nor the early increased phosphate-labelling of eIF-4 alpha requires the activity of the 70 kDa form of ribosomal protein S6 kinase. In addition, we demonstrate that eIF-4 gamma is phosphorylated in response to cell activation, an event which is correlated with phosphorylation of eIF-4 alpha and enhanced eIF-4F complex formation. In these studies, isoelectric focusing and immunoblot analysis of eIF-4 alpha indicate that phosphate-labelling of eIF-4 alpha following cell activation reflects a modest increase in steady-state phosphorylation, mediated by the enhanced activity of eIF-4 alpha kinase(s) and inhibition of eIF-4 alpha phosphatase activity. In the resting cell, eIF-4 alpha is associated with heat- and acid-stable insulin-responsive protein (PHAS-I; 4E-BP1); following acute stimulation with phorbol ester, there is a 40% decrease in the amount of PHAS-I associated with eIF-4 alpha. Incubation of anti-PHAS-I immunoprecipitates with extracts containing activated or immunprecipitated mitogen-activated protein kinase resulted in a small increase in phosphorylation of recovered PHAS-I and a modest release of eIF-4 alpha from the PHAS-I-eIF-4 alpha complex. These data suggest a possible role for PHAS-I in the regulation of eIF-4F complex formation and the rate of translation in primary cells.

Amino Acid Sequence↗
PubMed1995

The phosphodiesterase inhibitor SQ 20006 selectively blocks mitogen activation of p70S6k and transition to S phase of the cell division cycle without affecting the steady state phosphorylation of eIF-4E.

In quiescent cells high levels of protein synthesis are required in order to re-enter the cell cycle upon stimulation. Initiation of polypeptide synthesis is the step most often subject to regulation, controlled in part by phosphorylation of 40 S ribosomal protein S6 and a number of initiation factors. The kinase responsible for S6 phosphorylation is p70S6k. We now show that the p70S6k pathway can be selectively blocked by the aminopurine analogue, SQ 20006. This agent is known to raise cAMP levels, resulting in activation of protein kinase A. We present evidence that the increase in cAMP is not responsible for the inhibitory effect observed. We also show that SQ 20006 can prevent the activation of p70S6k in a rapid and reversible manner. The compound does not exert its inhibitory activity on p70S6k but can inhibit in vitro two protein kinase C isozymes (alpha and gamma). In a B lymphoblastoid cell line, treatment with SQ 20006 results in inhibition of protein synthesis at the initiation stage. In contrast, when tested directly upon the translational machinery in the reticulocyte lysate, inhibition is manifest at both the level of initiation and elongation. The role of protein kinase A in the modulation of p70S6k and the rate of translation is discussed.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Inhibition of protein synthesis by the heme-controlled eIF-2 alpha kinase leads to the appearance of mRNA-containing 48S complexes that contain eIF-4E but lack methionyl-tRNA(f).

Phosphorylation of the initiation factor eIF-2 by the heme-regulated eIF-2 alpha kinase (HCR) results in pronounced inhibition of protein synthesis and of binding of Met-tRNA(f) to 40S subunits in reticulocyte lysates. This inhibition is associated with the appearance of a more rapidly sedimenting 48S complex; this contains mRNA detectable by poly(U) hybridization, but not Met-tRNA(f). In contrast, 48S complexes that accumulate in the presence of the initiation inhibitor edeine contain both Met-tRNA(f) and mRNA. We have compared the composition of the particles that accumulate in the presence of HCR with those seen in the presence of edeine and find that both particles contain the cap binding protein, eIF-4E. Moreover, both particles exhibit a buoyant density of 1.40 g/ml in CsCl equilibrium density gradients. This is consistent with the presence of 500-700 kDa of protein additional to ribosomal structural protein, and suggests the presence of eIF-3 on both types of 48S complex. Lysates pre-treated with HCR and then treated with edeine show the ability to accumulate 48S complexes containing Met-tRNA(f), though at a slower rate than control lysates. These observations are discussed in the light of mechanisms previously suggested for the appearance of 48S particles following HCR treatment. In addition, we have observed association of eIF-4E with polysomes and 80S monosomes in reticulocyte lysates, suggesting that this factor may not be released immediately following the binding of the 40S ribosomal subunit to the 5' end of the mRNA.

Animals↗