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PubMed1979

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Animals↗
PubMed1990

Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state.

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.

Actins↗
PubMed1983

The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein phosphatase-2A2.

Two homogeneous protein phosphatases, termed 'smooth muscle phosphatase-I' and 'smooth muscle phosphatase-II', isolated from turkey gizzard as enzymes active against the 20-kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purified as an enzyme active against protein synthesis initiation factor eIF-2, were classified using the criteria defined by Ingebritsen and Cohen [Eur. J. Biochem. (1983) 132, 255-261]. All three enzymes were type-2 protein phosphatases based on their specificity for the alpha-subunit of phosphorylase kinase and insensitivity to inhibitor-1 and inhibitor-2. The substrate specificities of smooth muscle phosphatase-I and the eIF-2 phosphatase were similar to the catalytic subunit of protein phosphatase-2A. Smooth muscle phosphatase-I could be designated as protein phosphatase-2A1 and eIF-2 phosphatase as protein phosphatase-2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase-II allowed its assignment as protein phosphatase-2C.

Animals↗
PubMed1989

The two forms of the beta-subunit of initiation factor-2 from reticulocyte lysates arise from proteolytic degradation.

Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the beta-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed beta H (heavy, the slower-migrating species, apparent Mr = 54,300) and beta L (light, the faster-migrating species, apparent Mr = 53,100). We confirm that two forms of eIF-2 beta are present in such preparations, but present evidence that the beta L is generated from beta H during the isolation procedure. Crude reticulocyte lysates contain only the beta H species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2 beta. The beta L species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the beta H and the beta L forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2 beta, the beta L form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the beta-subunit.

Animals↗
PubMed1979

Stimulation of protein synthesis and Met-tRNAf binding by phosphorylated sugars: studies on their mechanism of action.

It has been previously reported by J. R. Lenz et al. [(1978) Biochemistry 17, 80--87] that certain phosphorylated sugars stimulate protein synthesis in extracts of mammalian cells. This effect was found to be due to a stimulation of Met-tRNAf binding to 40S ribosomal subunits, both in whole extracts and with isolated ribosomes. However, formation of a ternary complex of Met-tRNAf, initiation factor eIF-2, and GTP was not stimulated. It was also shown that the stimulation is not due solely to metabolism of the sugars. The present communication further characterizes the stimulatory effect of the sugars. They were found to prevent the inactivation of ribosomes that occurs during protein synthesis incubations. The sugars were also found to inhibit cAMP-dependent protein kinases noncompetitively. However, they stimulate Met-tRNAf binding to 40S ribosomal subunits even under conditions in which an inhibition of protein kinase has no effect. Although it has bot been possible to demonstrate a direct association of the sugars with the 40S initiation complex, the evidence suggests that their effect is mediated by an interaction with one of the components involved in the formation of this complex.

Animals↗
PubMed1979

Initiation of mammalian protein synthesis. The multiple functions of the initiation factor eIF-3.

The protein synthesis initiation factor eIF-3 (a multicomponent protein complex) was labelled with 32P by phosphorylation with a protein kinase present in a partially purified 'hemin-controlled repressor' preparation. The interaction of the labelled factor with the 40 S ribosomal subunit during the course of initiation was followed. It binds to the 40 S subunit in the absence of other initiation factors and inhibits the Mg2+-dependent reassociation of the 40 S with the 60 S ribosomal subunit. It stimulates the binding of the ternary complex (eIF-2, GTP, Met-tRNAf) to the 40 S subunit, and earlier work (Trachsel, H., Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 745-767) also showed it to be essential for the subsequent binding of mRNA. The factor is released from the 40 S initiation complex during the 60 S subunit joining reaction.

Animals↗
PubMed1983

Mechanism of interferon action: human leukocyte and immune interferons regulate the expression of different genes and induce different antiviral states in human amnion U cells.

The inhibition of virus replication and the induction of protein phosphorylation were examined in human amnion U and human fibroblast GM2767A cells treated with highly purified cloned human leukocyte and immune interferons synthesized in Escherichia coli. Both leukocyte interferon (IFN-alpha A) and immune interferon (IFN-gamma) possessed antiviral activity as measured by the single cycle yield reduction of vesicular stomatitis virus (VSV) in the human U and GM2767A cell lines. By contrast, only IFN-gamma and not IFN-alpha A inhibited the single cycle replication of reovirus in U and GM2767A cells. IFN-alpha A, but not IFN-gamma, efficiently induced the double-stranded RNA-dependent phosphorylation of the ribosome-associated protein P1 and the alpha subunit of protein synthesis initiation factor eIF-2 in U cells. However, neither IFN-alpha A nor IFN-gamma induced the phosphorylation of P1 and eIF-2 alpha in GM2767A cells. The antiviral activities of IFN-alpha A and IFN-gamma were synergistic for the inhibition of VSV but not for the inhibition of reovirus or the induction of protein phosphorylation. These results suggest that human leukocyte and immune interferons differentially regulate the expression of certain genes and induce mechanistically distinct antiviral states in human cells.

Amnion↗
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↗
PubMed1983

Partial purification and characterization of heat stable protein phosphatase inhibitor-2 from rabbit reticulocytes.

Previous studies indicated that the species of type 1 and type 2 protein phosphatases (PP-1, PP-2) in rabbit reticulocytes are similar to those of rabbit skeletal muscle and rabbit liver. Reticulocyte PP-1 was found to be selectively inhibited by the heat stable protein phosphatase inhibitor-2 (I-2) from rabbit skeletal muscle. Of interest was the observation that muscle I-2 appeared to regulate protein synthesis in reticulocyte lysates by inhibiting an eIF-2 alpha phosphatase with type 1 properties. In this study we have characterized reticulocyte inhibitor-2 (I-2) and find that its properties are similar to those of skeletal muscle I-2. (i) Both I-2 species are stable to boiling and to acid treatment, and have similar chromatographic profiles on DEAE-cellulose and on Blue Sepharose CL-6B. (ii) The two I-2 species migrate electrophoretically as 26-28,000 dalton polypeptides in SDS-acrylamide gels. (iii) Both skeletal muscle I-2 and reticulocyte I-2 selectively inhibit isolated reticulocyte PP-1 and endogenous PP-1 in the lysate. (iv) Reticulocyte I-2 co-chromatographs with PP-1 on DEAE-cellulose, and over 90% of lysate I-2 can be isolated from this partially purified PP-1. (v) Both inhibitor-2 species are active in the unphosphorylated state, but upon addition to lysates, both are phosphorylated by endogenous cAMP-independent protein kinase(s). In addition a preliminary analysis using a polyclonal antibody against muscle inhibitor-1 confirmed biochemical analyses which indicate that lysates are deficient in inhibitor-1.

Animals↗
PubMed1984

Protein synthesis in rabbit reticulocytes: requirements for Met-tRNAf . 40S preinitiation complex formation with AUG-codon and physiological mRNAs.

Under standard conditions, in the presence of GTP, highly purified eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNAf binding to 40S ribosomes. Replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP, in the above system, gave significant stimulation of Met-tRNAf binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNAf binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5'-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. This is the first report on the formation of a stable Met-tRNAf . 40S initiation complex dependent on physiological mRNAs and the factor requirements for such complex formation.

Animals↗
PubMed1988

Purification of a novel heat-stable translational inhibitor from rabbit reticulocyte lysates.

We have purified to apparent homogeneity a novel heat-stable (HS) factor from postribosomal supernatants of rabbit reticulocyte lysates by heating for 10 min at 80 degrees C, fractionation on Sephadex, anion-exchange chromatography on QMA Accell, and gel filtration HPLC. The apparent molecular mass of HS is 500-1000 Da on the basis of its behaviour on gel filtration. Like a factor from bovine heart [(1982) Proc. Natl. Acad. Sci. USA 79, 3134-3137], the reticulocyte HS inhibits translation in hemin-supplemented lysates with biphasic kinetics similar to hemin deficiency and promotes phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF-2. It is active at nanomolar concentrations. Reticulocyte HS appears to be neither a peptide nor an oligonucleotide since HS activity was insensitive to proteolytic or nucleolytic digestion.

Animals↗
PubMed1985

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2.

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.

Adenosine Triphosphate↗
PubMed1989

Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2.

We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.

Amino Acids↗
PubMed1992

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.

Animals↗
PubMed1978

Phosphorylation of eukaryotic protein synthesis initiation factors.

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.

Animals↗
PubMed1985

Adenovirus VAI RNA prevents phosphorylation of the eukaryotic initiation factor 2 alpha subunit subsequent to infection.

The virus-associated VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of mRNAs late after infection. Deletion mutant dl331 fails to produce this RNA and, as a result, grows poorly. Three lines of evidence suggest that VAI RNA facilitates translation by preventing inactivation of the function of eukaryotic initiation factor 2 (eIF-2). First, the mutant's translational defect can be relieved by addition of eIF-2 or eIF-2B (GTP recycling factor). Second, extracts of mutant-infected cells exhibit enhanced protein P1/eIF-2 alpha subunit kinase activity. Third, dl331 can grow with nearly normal kinetics in cells that do not express the kinase.

Adenoviridae Infections↗
PubMed1989

Mutations in the structural genes for eukaryotic initiation factors 2 alpha and 2 beta of Saccharomyces cerevisiae disrupt translational control of GCN4 mRNA.

The SUI2 and SUI3 genes of Saccharomyces cerevisiae encode the alpha and beta subunits, respectively, of translation initiation factor eIF-2 (eukaryotic initiation factor 2). Previously isolated mutations in these genes restore expression from his4 mutant alleles lacking an ATG initiation codon. The SUI mutations also lead to increased levels of HIS4 mRNA. We show that the latter phenotype exists because the SUI mutations elevate expression of GCN4, an activator of HIS4 transcription. Increased GCN4 expression in the SUI mutants occurs independently of the GCN2 and GCN3 gene products that are normally required to stimulate translation of GCN4 mRNA under conditions of amino acid starvation. Derepression of GCN4 expression in the SUI mutants requires the multiple AUG codons in the leader of the GCN4 transcript that normally mediate its translational control by amino acid availability. In these respects, the SUI mutations resemble mutations in GCD genes whose products function as translational repressors of GCN4. Thus, in addition to its general role in AUG start codon selection, eIF-2 appears to be an important factor in GCN4 translational control. We also show that deletion of GCN3 in sui2-1 strains is lethal, suggesting that GCN3 contributes to eIF-2 alpha function in addition to its role as a translational activator of GCN4.

Chromosome Deletion↗
PubMed1991

Complex formation by positive and negative translational regulators of GCN4.

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.

Amino Acids↗