Phosphorylation of initiation factor elF-2 and the control of reticulocyte protein synthesis

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.     

Depression of Neuronal Protein Synthesis Initiation by Protein Tyrosine Kinase Inhibitors

Abstract— Growth factors stimulate cellular protein synthesis, but the intracellular signaling mechanisms that regulate initiation of mRNA translation in neurons have not been clarified. A rate-limiting step in the initiation of protein synthesis is the formation of the ternary complex among GTP, eukaryotic initiation factor 2 (elF-2), and the initiator tRNA. Here we report that genistein, a specific tyrosine kinase inhibitor, decreases tyrosine kinase activity and the content of phosphotyrosine proteins in cultured primary cortical neurons. Genistein inhibits protein synthesis by >80% in a dose-dependent manner (10–80 μg/ml) and concurrently decreases ternary complex formation by 60%. At the doses investigated, genistein depresses tyrosine kinase activity and concomitantly stimulates PKC activity. We propose that a protein tyrosine kinase participates in the initiation of protein synthesis in neurons, by affecting the activity of elF-2 directly or through a protein kinase cascade.     

Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF-2α) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of elF-2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of elF-2 that delivers charged initiator tRNA_i^Met to the ribosome. When the levels of elF-2·GTP·Met-tRNA_i^Met ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF-2 by the protein kinase GCN2 decreases the concentration of elF-2·GTP·Met-tRNA_i^Met complexes by inhibiting the guanine nucleotide exchange factor for elF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF-2.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Association of HSP90 with the heme-regulated eukaryotic initiation factor 2α kinase—A collaboration for regulating protein synthesis

Among the various heat shock proteins (HSPs), members of the HSP70 and HSP90 families have drawn particular attention due to their heat shock-unrelated functions. HSP90, an ubiquitous and abundant member of the HSP90 family has been shown to be associated with a large array of protein factors. These proteins reside in the nucleus as well as in the cytoplasm and are involved in various physiological processes, such as, regulation of chromatin structure, cell cycle, cytoskelelal architecture, protein trafficking and protein synthesis. In this article, we focus our interest on the role of HSP90 in protein synthesis. Recent data obtained from a few laboratories strongly suggest that HSP90 interacts with the heme-regulated eukaryotic initiation factor 2α (elF-2α) kinase, also called the heme-regulated inhibitor, and causes its activation which leads to inhibition of protein synthesis. On the basis of data reported from various laboratories, including our own, we propose a possible model on the mechanism of HSP90-mediated activation of heme-regulated inhibitor and regulation of protein synthesis.     

The alpha subunit of eucaryotic initiation factor 2 is phosphorylated in mengovirus-infected mouse L cells.

Infection of mouse L cells with mengovirus resulted in the activation of a protein kinase (PK) that selectively phosphorylated the small, 38,000-molecular-weight alpha subunit of eucaryotic initiation factor 2 (eIF-2) in vitro. The mengovirus-activated kinase was detected in vitro approximately 3 h after virus adsorption. The ratio of phosphorylated to unphosphorylated eIF-2 also increased in vivo between 3 and 7 h after adsorption. The virus-activated kinase fractionated with the ribosomal pellet and had a high affinity for DEAE-cellulose and Mono Q ion-exchange columns. Gel electrophoresis of the kinase activity eluting from the Mono Q column and silver staining of the gel revealed only one protein band with a molecular mass of 70 kilodaltons. The optimal assay conditions for the mengovirus-activated kinase paralleled those of the double-stranded RNA-activated PK (dsRNA-PK). Lysates from infected cells contained elements capable of activating partially purified dsRNA-PK. These elements were identified as double-stranded RNA by their sensitivity to double-stranded RNase. The phosphorylation of the alpha subunit of eIF-2 coincided with the synthesis of dsRNA in infected cells, suggesting that the mengovirus-activated kinase is the dsRNA-PK. The phosphorylation of the alpha subunit of eIF-2 correlated with the global inhibition of protein synthesis that occurs at late times after infection.     

Purification of Dictyostelium myosin II heavy chain kinase a based on the increase in negative charge accompanying hyperphosphorylation

The initial step in the purification of Dictyostelium myosin 11 heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg²⁺ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 m KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M_r greater than 700,000).     

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos

The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation. The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II. Among the exogenous proteins initiation factor eIF2 is the best substrate and is 7.8 times more efficiently phosphorylated than casein. Endogenous mRNP protein substrates have a Mr of 125000, 65000, 38000, 26000 and 23500. The main phosphate acceptor is the Mr38000 poly(A)-binding protein. Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property. The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed.Abbreviations PMSF phenylmethanesulfonyl fluoride - mRNP messenger ribonucleoprotein - iRNA small inhibitor RNA - eIF2 eukaryotic initiation factor 2     

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and “ribosome adaptor,” eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.     

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.     

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr¹⁷² in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca²⁺/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a–Rheb–AMPK signaling axis.     

Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells

The association of mRNA and ribosomes with the cytoskeleton of eucaryotic cells may be important for protein synthesis and its regulation. HeLa cells were gently lysed with detergent, and soluble and cytoskeletal framework subfractions were prepared by centrifugation. We analyzed these fractions for ribosomes and confirmed earlier findings that polysomes are preferentially associated with the cytoskeletal fraction. The levels of initiation factors elF-2, elF-3, elF-4A, and elF-4B were quantitated by immunoblotting; all are enriched in the cytoskeletal fraction relative to the soluble fraction. Heat shock, fluoride, pactamycin, and cytochalasin caused the release of both ribosomes and initiation factors into the soluble fraction. However, treatment of the cytoskeletal fraction with EDTA or low levels of ribonuclease resulted in polysome degradation but no release. Therefore initiation factor association with the cytoskeletal framework correlates with the presence of ribosomes, whereas ribosome association does not require intact mRNA.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity.

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.     

Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.     

Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.     

Phosphorylation in vitro of eukaryotic initiation factors IF-E2 and IF-E3 by protein kinases.

Purified protein synthesis initiation factors IF-E2 and IF-E3 from rabbit reticulocytes were phosphorylated in vitro with protein kinases isolated from the same source. The highest levels of phosphorylation resulted from incubation of the factors with a cyclic nucleotide-independent protein kinase previously shown to have specificity for acidic proteins. The extent of phosphorylation of initiation factor IF-E2 was between 0.3 and 0.4 mol of phosphate per mol of factor complex, with either ATP or GTP as phosphoryl donor. Initiation factor IF-E2 is composed of three nonidentical polypeptides; only the polypeptide with a molecular weight of 52,000 was phosphorylated. The extent of phosphorylation of initiation factor IF-E3 was between 0.7 and 1.0 mol of phosphate per mol of factor complex with GTP as phosphoryl donor; with ATP, less phosphorylation of the factor was obtained. Initiation factor IF-E3 is composed of 9 to 11 nonidentical polypeptides; only 2 of these, with molecular weights of 120,000 and 70,000, were phosphorylated. A lower level of phosphorylation of initiation factor IF-E3 was found with the cyclic AMP-dependent protein kinase; the polypeptide of molecular weight 140,000 was the major site of phosphorylation.     

UIS2: A Unique Phosphatase Required for the Development of Plasmodium Liver Stages

Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.     

Maskin is a CPEB-associated factor that transiently interacts with elF-4E

In Xenopus, the CPE is a bifunctional 3' UTR sequence that maintains maternal mRNA in a dormant state in oocytes and activates polyadenylation-induced translation during oocyte maturation. Here, we report that CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor we term maskin. Maskin contains a peptide sequence that is conserved among elF-4E-binding proteins. Affinity chromatography demonstrates that CPEB, maskin, and elF-4E reside in a complex in oocytes, and yeast two-hybrid analyses indicate that CPEB and maskin bind directly, as do maskin and elF-4E. While CPEB and maskin remain together during oocyte maturation, the maskin-elF-4E interaction is substantially reduced. The dissolution of this complex may result in the binding of elF-4E to elF-4G and the translational activation of CPE-containing mRNAs.     

Substrate specificity of liver calmodulin-dependent glycogen synthase kinase

A number of proteins were tested as potential substrates for purified rabbit liver calmodulin-dependent glycogen synthase kinase. It was found that liver phenylalanine hydroxylase and several brain proteins including tyrosine hydroxylase, microtubule-associated protein 2, and synapsin I were readily phosphorylated. Brain tubulin was very poorly phosphorylated. These results suggest that calmodulin-dependent glycogen synthase kinase may be a more general protein kinase involved in the regulation of several cellular Ca²⁺-dependent functions.