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S J Morley

Publications and source records attributed to S J Morley.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1995

Proteolytic cleavage of initiation factor eIF-4 gamma in the reticulocyte lysate inhibits translation of capped mRNAs but enhances that of uncapped mRNAs.

Infection of cells with the foot-and-mouth-disease virus, a member of the picornavirus family, results in the shut-off of host protein synthesis. A major contributory mechanism is the proteolytic destruction of the gamma subunit of the complex eIF-4, which functions in translation to promote the binding of the 43S ribosomal preinitiation complex to the 5' end of the cellular mRNA molecules bearing a 5' terminal cap structure. Picornavirus RNA molecules, which are uncapped, use a distinct mechanism for translational initiation, which can operate in the absence, or at low levels, of eIF-4. The proteolysis of eIF-4 gamma in cells infected by foot-and-mouth-disease virus results from expression of a virus-encoded cysteine proteinase known as Leader (or L) protease. We have used a transcription plasmid encoding this protease as a tool to deplete in vitro translation systems of eIF-4 gamma in order to elucidate in more detail the role of this polypeptide in the control of translation. Using in vitro transcribed mRNAs we have observed a marked contrast between capped and uncapped transcripts in the response of their translation to the proteolysis of eIF-4 gamma. Translation of capped mRNAs is, as expected, severely impaired, and is restored by addition of eIF-4 complex containing the intact gamma-subunit. On the other hand, translation of uncapped transcripts, normally inefficient, is substantially enhanced. The data suggest that the translation of uncapped mRNAs may be stimulated in this system by one or more of the proteolytic degradation products of eIF-4 gamma.

3C Viral Proteases↗
PubMed1995

Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation.

Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At laser times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4 alpha and eIF-4 gamma and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4 alpha may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4 alpha is shown to reflect activation of eIF-4 alpha kinase(s), which coincides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.

Animals↗
PubMed1994

Signal transduction mechanisms in the regulation of protein synthesis.

Control of polypeptide synthesis plays an important role in cell proliferation and translation rates generally reflect the growth state of the cultured eukaryotic cell. Physiological regulation of protein synthesis is almost always exerted at the level of polypeptide chain initiation, with the binding of mRNA to the small ribosomal subunit a rate-limiting step in many cell systems. Studies have indicated key roles in the regulation of protein synthesis for the structural features of mRNA molecules and phosphorylation of initiation factors which catalyse this process. This review focuses on translational regulation at the level of mRNA binding to the ribosome and the role of phosphorylation of initiation factors in mediating both quantitative and qualitative control. The identity of putative kinases which may mediate these processes is addressed and a possible model for the role of a transient activation of initiation factors in cell growth or differentiation is presented.

Animals↗
PubMed1993

Increased phosphorylation of eukaryotic initiation factor 4 alpha during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation.

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non-proliferative state. When cultured in the presence of the appropriate mitogen, there is a 2-4-fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahern, T. and Atkins, M. (1971) Biochim. Biophys. Acta 247, 322-334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519-521]. In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester phorbol 12-myristate 13-acetate or concanavalin A leads to a rapid 2-4-fold increase in the rate of protein synthesis within 1 h or 4 h, respectively, which is insensitive to the transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2-4-fold increase in labelling of the eukaryotic initiation factor eIF-4 alpha with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF-4 alpha. Similarly, with the human leukaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT-3, or treatment with phorbol ester induces a 2-3-fold increase in eIF-4 alpha phosphorylation within 30 min. Analysis of phosphorylation by two-dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF-4 alpha kinase activity rather than phosphate turnover on eIF-4 alpha. Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF-4 alpha recovered in high-molecular-mass complexes. These data suggest that, in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF-4 alpha and eIF-4 gamma into high-molecular-mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.

Amino Acid Sequence↗
PubMed1993

Stimulation of translation in 3T3-L1 cells in response to insulin and phorbol ester is directly correlated with increased phosphate labelling of initiation factor (eIF-) 4F and ribosomal protein S6.

One of the earliest responses to growth-promoting compounds in quiescent or serum-starved cells is stimulation of phosphorylation of ribosomal protein S6. Exposure of 3T3-L1 cells to insulin or phorbol ester (phorbol 12-myristate 13-acetate, PMA) also promotes phosphorylation of specific initiation factors. In this study, stimulation of phosphate labelling of S6, eIF-4F and eIF-4B in response to insulin and PMA has been examined in 3T3-L1 cells and compared with changes in protein synthesis. The rate of phosphate incorporation into eIF-4F and S6 is rapid and parallels the transient stimulation of protein synthesis observed with insulin. Whilst a similar correlation exists with PMA, the response is not as great as with insulin, but is more sustained. A role for the co-ordinate phosphorylation of initiation factors and ribosomal protein S6 in the stimulation of protein synthesis is discussed.

3T3 Cells↗
PubMed1992

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

3T3 Cells↗
PubMed1992

Identification and early activation of a Xenopus laevis p70s6k following progesterone-induced meiotic maturation.

Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.

3T3 Cells↗
PubMed1991

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation.

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.

Animals↗
PubMed1991

Intracellular messengers and the control of protein synthesis.

The molecular events responsible for controlling cell growth and development, as well as their coordinate interaction is only beginning to be revealed. At the basis of these controlling events are hormones, growth factors and mitogens which, through transmembrane signalling trigger an array of cellular responses, initiated by receptor-associated tyrosine kinases, which in turn either directly or indirectly mediate their effects through serine/threonine protein kinases. Utilizing the obligatory response of activation of protein synthesis in cell growth and development, we describe efforts to work backwards along the regulatory pathway to the receptor, identifying those molecular components involved in modulating the rate of translation. We begin by describing the components and steps of protein synthesis and then discuss in detail the regulatory pathways involved in the mitogenic response of eukaryotic cells and during meiotic maturation of oocytes. Finally we discuss possible future work which will further our understanding of these systems.

Animals↗
PubMed1990

Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters.

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.

Animals↗