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Results for “eIF-2 Kinase”

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PubMed1979

Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA.

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.

Animals↗

PubMed1985

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1.

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.

Animals↗

PubMed1979

Protein synthesis in rabbit reticulocytes: characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (Met-tRNAfMet.eIF-2.GTP) formation by heme-regulated inhibitor.

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex (Met-tRNAfMet.eIF-2.GTP) and AUG-dependent transfer of Met-tRNAfMet to 40S ribosomal subunits. A factor (sRF) that reverses protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleimide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF.

Animals↗

PubMed1991

Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity.

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.

Animals↗

PubMed1979

The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.

Animals↗

PubMed1979

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.

Animals↗

PubMed1978

Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor.

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.

Adenosine Triphosphate↗

PubMed1978

Mode of action of the hemin-controlled inhibitor of protein synthesis: studies with factors from rabbit reticulocytes.

Previously [de Haro, C., Datta, A & Ochoa, S. (1978) Proc. Natl. Acad. Sci. USA 75, 243--247] it was shown with initiation factors from Artemia salina embryos that the activity of the initiator methionyl-tRNA binding factor eIF-2 is stimulated by another factor (ESP, for eIF-2 stimulating protein) present, like eIF-2, in ribosomal salt washes. Incubation of eIF-2 with translational inhibitor from rabit reticulocytes, in the presence of ATP, abolished the ESP effect. At physiological concentrations eIF-2 was virtually inactive without ESP. These observations indicated that the translational inhibitor acts by converting eIF-2 to a form that is not stimulated by ESP. The same observations have now been made with eIF-2 and ESP from rabbit reticulocytes but, in this case, the dependence of eIF-2 activity on ESP is much more pronounced than with the A. salina factors. eIF-2 from reticulocytes interacts with ESP from A. salina and conversely.

Animals↗

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↗

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↗

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↗

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↗

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↗