Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.     

Stage-specific initiation factors for protein synthesis during insect development

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor.     

Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.     

Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) is a major mechanism regulating protein synthesis in rabbit reticulocytes. To determine whether phosphorylation of eIF-2 alpha is a likely regulatory mechanism in the Ehrlich cell, we have measured the percent of cellular eIF-2 alpha which is phosphorylated in cells exposed to heat shock, 2-deoxyglucose, or amino acid deprivation, conditions which rapidly decrease the concentration of 40 S initiation complexes and inhibit protein synthesis. eIF-2 alpha and eIf-2 alpha (P) were separated by isoelectric focusing and were detected by immunoblotting with a monoclonal antibody we developed for this purpose. Under the above three inhibitory conditions, phosphorylation of eIF-2 alpha increased rapidly, and this increase correlated in time with the rapid inhibition of protein synthesis. In heat-shocked cells which were returned to 37 degrees C, both phosphorylation and protein synthesis remained unchanged for 10 min and then returned toward control values slowly and in parallel. The close temporal correspondence between changes in protein synthesis and phosphorylation supports an important regulatory role for phosphorylation in protein synthesis. An increase of 25-35 percentage points, to 50-60% phosphorylation from control levels of 20-30% phosphorylation, correlated with an 80-100% inhibition of protein synthesis. This steep curve of inhibition is consistent with a mechanism in which eIF-2 alpha (P) saturates and inhibits the guanine-nucleotide exchange factor.     

Protamine kinase phosphorylates eukaryotic protein synthesis initiation factor 4E.

Up to 1 mol of phosphoryl groups was incorporated per mol of eukaryotic protein synthesis initiation factor (eIF) 4E following incubation of purified preparations of this factor with purified preparations of a protamine kinase from bovine kidney cytosol. By contrast, purified preparations of two forms of mitogen-activated protein kinase, casein kinase II and two forms of a distinct autophosphorylation-activated protein kinase exhibited little activity, if any, with eIF-4E. Together with previous observations, the results indicate that the protamine kinase could contribute to the insulin-stimulated phosphorylation of eIF-4E.     

Control of initiation of eukaryotic protein synthesis by guanine nucleotides and methionyl-tRNAi.

The influence of changing concentrations of GDP, methionyl-tRNAi, eIF-2 and eIF-2B on possible rates of initiation of protein synthesis have been explored in calculations based on previously derived rate constants for interaction of the components involved in formation of ternary or quaternary complexes of eIF-2B, eIF-2, GTP and Met-tRNAi. When allowance is made for the limitation of diffusional coefficients imposed on macromolecules by the intracellular milieu it is apparent that recent estimates by Rowlands et al. (Eur. J. Biochem. 175, 93:1988) of higher concentrations of eIF-2 and eIF-2B in cells than hitherto proposed become necessary to support known rates of initiation. Under these conditions changing concentrations of met-tRNAi as proposed by Cooper and Braverman (J. Biol. Chem. 256, 7461:1981) are likely to have an important regulating influence.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.     

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.     

The essential vertebrate ABCE1 protein interacts with eukaryotic initiation factors

The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2alpha and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.     

How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.     

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.     

Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.     

Sequences for two cDNAs encoding Arabidopsis thaliana eukaryotic protein synthesis initiation factor 4A.

Two distinct cDNAs encoding protein synthesis initiation factor 4A (eIF-4A) were isolated from an Arabidopsis thaliana cDNA library and sequenced. The deduced amino acid sequences from the two cDNAs were compared to eIF-4A from tobacco, mouse and Saccharomyces cerevisiae. The putative ATP-binding sites and RNA helicase motifs were identified.