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Biomedical subjects

C G Proud

Publications and source records attributed to C G Proud.

At least 91 records · Page 5Linked to original sources
PubMed1994

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B.

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.

Animals↗
PubMed1994

Guanine nucleotide exchange factor for eukaryotic initiation factor-2. Cloning of cDNA for the delta-subunit of rabbit translation initiation factor-2B.

Peptide sequence data for rabbit eIF-2B delta were obtained and used to design redundant oligonucleotides for PCR. RNA was isolated from rabbit liver and used to direct the synthesis of total cDNA. A rabbit eIF-2B delta transcript was then amplified by PCR and sequenced. The PCR product was used to isolate a clone from a rabbit liver cDNA library. RACE (rapid amplification of cDNA ends) was used to obtain further 5' sequence. Subsequently, a full length cDNA was obtained from a rabbit reticulocyte library. PCR was used to confirm that the sequence is the same for the liver factor. The sequence obtained shows strong homology to that of yeast eIF-2B delta, the GCD2 gene product.

Amino Acid Sequence↗
PubMed1994

Peptide-chain elongation in eukaryotes.

The elongation phase of translation leads to the decoding of the mRNA and the synthesis of the corresponding polypeptide chain. In most eukaryotes, two distinct protein elongation factors (eEF-1 and eEF-2) are required for elongation. Each is active as a complex with GTP. eEF-1 is a multimer and mediates the binding of the cognate aminoacyl-tRNA to the ribosome, while eEF-2, a monomer, catalyses the movement of the ribosome relative to the mRNA. Recent work showing that bacterial ribosomes possess three sites for tRNA binding and that during elongation tRNAs may occupy 'hybrid' sites is incorporated into a model of eukaryotic elongation. In fungi, elongation also requires a third factor, eEF-3. A number of mechanisms exist to promote the accuracy or 'fidelity' of elongation: eEF-3 may play a role here. cDNAs for this and the other elongation factors have been cloned and sequenced, and the structural and functional properties of the elongation factors are discussed. eEF-1 and eEF-2 can be regulated by phosphorylation, and this may serve to control rates of elongation in vivo.

Animals↗
PubMed1993

Cloning of cDNA for the beta-subunit of rabbit translation initiation factor-2 using PCR.

RNA was isolated from rabbit liver and used to direct the synthesis of total cDNA. Rabbit eIF-2 beta transcripts were then specifically amplified by PCR and sequenced. RACE (rapid amplification of cDNA ends) was used to obtain 3' and 5' sequences. Comparison of the deduced amino acid sequence with that of human eIF-2 beta reveals a very high degree of sequence identity.

Amino Acid Sequence↗
PubMed1993

Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.

Adenoviridae↗
PubMed1993

Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B.

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of elongation factor-2 from the lepidopteran insect, Spodoptera frugiperda.

In mammalian cells, protein synthesis can be regulated at the level of elongation by the phosphorylation of elongation factor 2 (eEF-2) by a highly specific Ca2+/calmodulin-dependent kinase. In this report, we show that eEF-2 from a cell line derived from the insect, Spodoptera frugiperda, is a substrate for mammalian eEF-2 kinase and that phosphorylation is Ca(2+)-dependent. Furthermore, two-dimensional peptide mapping shows that the kinase phosphorylates the same sites in Spodoptera eEF-2 as those phosphorylated in the rabbit protein. However, we were unable to detect an eEF-2 kinase in Spodoptera cells.

Amino Acid Sequence↗
PubMed1993

The role of the beta-subunit of initiation factor eIF-2 in initiation complex formation.

The functional properties of preparations of protein synthesis initiation factor eIF-2 which lack the beta-subunit (as confirmed immunologically) were compared with those of the heterotrimeric factor. The former can bind guanine nucleotides but not initiator tRNA, and also exhibits a substantially reduced rate of initiation factor eIF-2B-mediated GDP/GTP-exchange.

Animals↗
PubMed1993

Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation factor-2 kinase and induces calcium-independent activity.

The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) phosphorylated purified calcium/calmodulin-dependent eukaryotic elongation factor-2 (eEF-2) kinase, isolated from rabbit reticulocyte lysates. It maximally incorporated about 1 mol of phosphate/mol of eEF-2 kinase. The Km of eEF-2 kinase for PKA was calculated to be 7 microM. Phosphorylation of eEF-2 kinase by PKA induced calcium-independent activity which amounted to 40-50% of the total activity measured in the presence of calcium. Furthermore, the level of calcium-independent activity induced by phosphorylation by PKA was similar to that induced by the calcium-stimulated autophosphorylation of eEF-2 kinase. Phosphopeptide mapping of eEF-2 kinase labelled by autophosphorylation and by PKA revealed a number of common phosphopeptides. This suggests that PKA may phosphorylate the same site(s) which are phosphorylated autocatalytically and which are responsible for the induction of calcium-independent activity. The possible implications these findings have for the control of translation are discussed.

Animals↗
PubMed1993

Regulation of elongation factor-2 by multisite phosphorylation.

We have studied the phosphorylation of protein synthesis elongation factor eEF-2, the effects of phosphorylation on its activity and the dephosphorylation of phosphorylated eEF-2 by protein phosphatases-2A and -2C. Extensive analysis of phosphopeptides generated from eEF-2 phosphorylated in vitro by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates: in this system monophosphorylated eEF-2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono-phosphorylated eEF-2 detected are phosphorylated at Thr56. The eEF-2 kinase could phosphorylate a synthetic peptide based on residues 49-60 of eEF-2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high Km, compared to eEF-2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53. In the light of the existence of two phosphorylation sites in eEF-2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)-directed synthesis of polyphenylalanine, where both the bis- and mono-phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non-phosphorylated eEF-2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF-2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained. Protein phosphatases-2A and -2C (PP-2A and PP-2C) can each efficiently dephosphorylate phosphorylated eEF-2. While bis-phosphorylated eEF-2 was a better substrate for PP-2A than monophosphorylated factor (phosphorylated at Thr56), the converse was true for PP-2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP-2C by the presence of phosphate on Thr58. Nevertheless, PP-2C exhibited a preference for dephosphorylation of Thr56 in bis-phosphorylated eEF-2, while PP-2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.

Amino Acid Sequence↗
PubMed1993

Purification and phosphorylation of elongation factor-2 kinase from rabbit reticulocytes.

Eukaryotic elongation-factor-2 kinase has been purified to homogeneity from rabbit reticulocytes through a seven-step procedure and has been identified as a protein with a molecular mass of approximately 103 kDa as judged by SDS/PAGE. A degradation product of about 95 kDa was also evident in some preparations. The activity of the purified kinase was completely dependent on calcium and calmodulin. The kinase rapidly underwent extensive autophosphorylation, incorporating 1 mol phosphate/mol within 1 min; 5 mol phosphate/mol were incorporated within 1 h. The autophosphorylation was Ca2+/calmodulin-dependent; phosphopeptide mapping revealed multiple phosphopeptides even after just 0.5 min of autophosphorylation, suggesting that a number of sites became rapidly phosphorylated. Autophosphorylation occurred on serine and threonine residues. Preincubation in the presence of Ca2+, Mg2+ and ATP produced a rapid 2-3-fold activation of the kinase and also induced partial Ca(2+)-independent activity. Preincubation in the absence of the ligands showed that all three were required for full activation and induction of Ca(2+)-independent activity.

Adenosine Triphosphate↗
PubMed1993

Evidence for a role for protein kinase C in the stimulation of protein synthesis by insulin in swiss 3T3 fibroblasts.

We have examined the role of protein kinase C (PKC) in the stimulation of protein synthesis by insulin using two complementary approaches. In the first, fibroblasts were pretreated with phorbol esters to down-regulate PKC. In these cells, the effects of insulin and of phorbol esters on protein synthesis were completely abolished, although serum still elicited an effect approaching that seen in control cells. Secondly, we used newly developed inhibitors of PKC which, again, blocked the effects of insulin and phorbol esters without greatly reducing the response to serum. Thus PKC apparently plays an important role in the stimulation of translation by insulin.

3T3 Cells↗
PubMed1993

Purification and characterisation of an initiation-factor-2 kinase from uninduced mouse erythroleukaemia cells.

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MKu) which phosphorylates the alpha subunit of protein-synthesis-initiation factor 2 (eIF-2 alpha). In this paper, we show that this kinase phosphorylates both eIF-2 alpha and a synthetic peptide based on the phosphorylation site in eIF-2 alpha at Ser51, the target residue for other eIF-2 alpha kinases. Consistent with this, prior treatment of eIF-2 with MKu impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF-2B. Using a modified cell-free translation system, we have shown that MKu inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF-2B-mediated guanine-nucleotide exchange. MKu has been purified and its properties have been compared with those of the haem-controlled repressor eIF-2 alpha kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MKu contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg(2+)-ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF-2 alpha are inhibited by the same, low, (10 microM) concentrations of haemin. Phosphorylation of the 98-kDa components present in the MEL-cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.

Amino Acid Sequence↗
PubMed1992

Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates.

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.

Animals↗
PubMed1992

Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid activation of the guanine-nucleotide-exchange factor by insulin and growth factors.

Insulin, whole serum, phorbol esters and epidermal growth factor each rapidly stimulate protein synthesis in serum-depleted Swiss 3T3 fibroblasts. The activation of protein synthesis by each of these agents is associated with stimulation of the activity of the guanine-nucleotide-exchange factor (GEF). This protein recycles the initiation factor eIF-2 by promoting exchange of GDP bound to eIF-2 for GTP. Activation of GEF is rapid, becoming maximal within 15 min. The degree of activation of GEF by these stimuli (to greater than 170% of control for insulin, serum or epidermal growth factor; 120% for phorbol dibutyrate) is more than enough to account for their effects on the overall rate of translation. Stimulation of protein synthesis and GEF activity occurs at low nanomolar insulin concentrations, indicating they are mediated through the insulin receptor. The best-characterized mechanism for regulating GEF activity is through changes in the phosphorylation of the smallest subunit of eIF-2 (eIF-2 alpha); however, none of the stimuli studied altered the level of phosphorylation of eIF-2 alpha in Swiss fibroblasts. It seems that direct regulation of GEF activity may be occurring here, and possible mechanisms for this are discussed.

3T3 Cells↗
PubMed1991

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.

Eukaryotic Initiation Factor-2↗