Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2.
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In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2. (+info)
Regulation of protein synthesis after acute resistance exercise in diabetic rats.
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These studies determined whether insulin-like growth factor-I (IGF-I) involvement in exercise-stimulated anabolic processes becomes more evident during hypoinsulinemia. Male Sprague-Dawley rats (n = 6-12/group) were made diabetic (blood glucose congruent with 300 mg/dl) by partial pancreatectomy (PPX) or remained nondiabetic (glucose congruent with 144 mg/dl). Rats performed acute resistance exercise by repetitive standing on the hindlimbs with weighted backpacks (ex), or they remained sedentary (sed). Resistance exercise caused increases in rates of protein synthesis (nmol Phe incorporated. g muscle-1. h-1, measured for gastrocnemius muscle in vivo 16 h after exercise) for both nondiabetic [sed = 154 +/- 6 (SE) vs. ex = 189 +/- 7] and diabetic rats (PPXsed = 152 +/- 11 vs. PPXex = 202 +/- 14, P < 0.05). Arterial plasma insulin concentrations in diabetic rats, congruent with180 pM, were less than one-half those found in nondiabetic rats, congruent with444 pM, (P < 0.05). The activity of eukaryotic initiation factor 2B (eIF2B; pmol GDP exchanged/min) was higher (P < 0.05) in ex rats (sed = 0.028 +/- 0.006 vs. ex = 0.053 +/- 0.015; PPXsed = 0.033 +/- 0.013 vs. PPXex = 0.047 +/- 0.009) regardless of diabetic status. Plasma IGF-I concentrations were higher in ex compared with sed diabetic rats (P < 0.05). In contrast, plasma IGF-I was not different in nondiabetic ex or sed rats. Muscle IGF-I (ng/g wet wt) was similar in ex and sed nondiabetic rats, but in diabetic rats was 2- to 3-fold higher in ex (P < 0.05) than in sed rats. In conclusion, moderate hypoinsulinemia that is sufficient to alter glucose homeostasis does not inhibit an increase in rates of protein synthesis after acute moderate-intensity resistance exercise. This preserved response may be due to a compensatory increase in muscle IGF-I content and a maintained ability to activate eIF2B. (+info)
Phosphorylated seryl and threonyl, but not tyrosyl, residues are efficient specificity determinants for GSK-3beta and Shaggy.
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Glycogen synthase kinase-3 is involved in diverse functions including insulin signalling and development. In a number of substrates, phosphorylation by glycogen synthase kinase-3 is known to require prior phosphorylation at a Ser in the +4 position relative to its own phosphorylation site. Here we have used synthetic peptides derived from a putative glycogen synthase kinase-3 site in the Drosophila translation initiation factor eIF2B epsilon to investigate the efficacy of residues other than Ser(P) as priming residues for glycogen synthase kinase-3beta and its Drosophila homologue Shaggy. Glycogen synthase kinase-3beta phosphorylated peptides with Ser(P) and Thr(P) in the priming position, but peptides with Tyr(P), Thr, Glu or Asp were not phosphorylated. The Vmax for the Thr(P) peptide was three times higher than that of the Ser(P) peptide. These data suggest that glycogen synthase kinase-3 is unique among phosphate-directed kinases. The priming site specificity of Shaggy is similar to that of mammalian glycogen synthase kinase-3beta. This unpredicted efficacy of Thr(P) in the priming position suggests that there may be other unidentified substrates for these kinases. (+info)
Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E.
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The present study examined potential mechanisms for the inhibition of protein synthesis in skeletal muscle after chronic alcohol consumption. Rats were maintained on an alcohol-containing diet for 14 wk; control animals were pair fed. Alcohol-induced myopathy was confirmed by a reduction in lean body mass as well as a decrease in the weight of the gastrocnemius and psoas muscles normalized for tibial length. No alcohol-induced decrease in total RNA content (an estimate of ribosomal RNA) was detected in any muscle examined, suggesting that alcohol reduced translational efficiency but not the capacity for protein synthesis. To identify mechanisms responsible for regulating translational efficiency, we analyzed several eukaryotic initiation factors (eIF). There was no difference in the muscle content of either total eIF2alpha or the amount of eIF2alpha in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bepsilon in muscle was also not different. In contrast, alcohol decreased eIF2B activity in psoas (fast-twitch) but not in soleus or heart (slow-twitch) muscles. Alcohol feeding also dramatically influenced the distribution of eIF4E in the gastrocnemius (fast-twitch) muscle. Compared with control values, muscle from alcohol-fed rats demonstrated 1) an increased binding of the translational repressor 4E-binding protein 1 (4E-BP1) with eIF4E, 2) a decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a decrease in eIF4G associated with eIF4E. In summary, these data suggest that chronic alcohol consumption impairs translation initiation in muscle by altering multiple regulatory sites, including eIF2B activity and eIF4E availability. (+info)
Nutrients differentially regulate multiple translation factors and their control by insulin.
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Eukaryotic initiation factor eIF2B and eukaryotic elongation factor eEF2 each mediate regulatory steps important for the overall regulation of mRNA translation in mammalian cells and are activated by insulin. Here, we demonstrate that their activation by insulin requires the presence, in the medium in which the cells are maintained, of both amino acids and glucose: insulin only induced activation of eIF2B and the dephosphorylation of eEF2 when cells were exposed to both types of nutrient. Other translational regulators, e.g. the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the eIF4E binding protein 1, 4E-BP1, are also regulated by insulin but their control does not require glucose, only amino acids. The effects of nutrients on the activation of eIF2B do not reflect changes in the phosphorylation of eIF2 (and, by inference, operation of a kinase analogous to yeast Gcn2p), or a requirement for nutrients for inactivation of glycogen synthase kinase-3 or dephosphorylation of eIF2B. Nutrients did not affect the ability of insulin to activate protein kinase B. These data show that activation by insulin of p70 S6 kinase, which modulates the translation of specific mRNAs, depends on the availability of amino acids whereas regulation of factors involved in overall activation of translation (eIF2B, eEF2) requires both amino acids and glucose. These results add substantially to the emerging evidence that nutrients themselves modulate functions of mammalian cells and indicate that (i) nutrients modulate the activation of eIF2B and eEF2 through as-yet unidentified mechanisms and (ii) regulation of p70 S6 kinase and 4E-BP1 by insulin requires other inputs in addition to protein kinase B. (+info)
Leucine, glutamine, and tyrosine reciprocally modulate the translation initiation factors eIF4F and eIF2B in perfused rat liver.
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Leucine, glutamine, and tyrosine, three amino acids playing key modulatory roles in hepatic proteolysis, were evaluated for activation of signaling pathways involved in regulation of liver protein synthesis. Furthermore, because leucine signals to effectors that lie distal to the mammalian target of rapamycin, these downstream factors were selected for study as candidate mediators of amino acid signaling. Using the perfused rat liver as a model system, we observed a 25% stimulation of protein synthesis in response to balanced hyperaminoacidemia, whereas amino acid imbalance due to elevated concentrations of leucine, glutamine, and tyrosine resulted in a protein synthetic depression of roughly 50% compared with normoaminoacidemic controls. The reduction in protein synthesis accompanying amino acid imbalance became manifest at high physiologic concentrations and was dictated by the guanine nucleotide exchange activity of translation initiation factor eIF2B. Paradoxically, this phenomenon occurred concomitantly with assembly of the mRNA cap recognition complex, eIF4F as well as activation of the 70-kDa ribosomal S6 kinase, p70(S6k). Dual and reciprocal modulation of eIF4F and eIF2B was leucine-specific because isoleucine, a structural analog, was ineffective in these regards. Thus, we conclude that amino acid imbalance, heralded by leucine, initiates a liver-specific translational fail-safe mechanism that deters protein synthesis under unfavorable circumstances despite promotion of the eIF4F complex. (+info)
Amino acid-induced stimulation of translation initiation in rat skeletal muscle.
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Amino acids stimulate protein synthesis in skeletal muscle by accelerating translation initiation. In the two studies described herein, we examined mechanisms by which amino acids regulate translation initiation in perfused skeletal muscle hindlimb preparation of rats. In the first study, the effects of supraphysiological amino acid concentrations on eukaryotic initiation factors (eIF) 2B and 4E were compared with physiological concentrations of amino acids. Amino acid supplementation stimulated protein synthesis twofold. No changes were observed in eIF2B activity, in the amount of eIF4E associated with the eIF4E-binding protein (4E-BP1), or in the phosphorylation of 4E-BP1. The abundance of eIF4E bound to eIF4G and the extent of phosphorylation of eIF4E were increased by 800 and 20%, respectively. In the second study, we examined the effect of removing leucine on translation initiation when all other amino acids were maintained at supraphysiological concentrations. Removal of leucine from the perfusate decreased the rate of protein synthesis by 40%. The inhibition of protein synthesis was associated with a 40% decrease in eIF2B activity and an 80% fall in the abundance of eIF4E. eIF4G complex. The fall in eIF4G binding to eIF4E was associated with increased 4E-BP1 bound to eIF4E and a reduced phosphorylation of 4E-BP1. In contrast, the extent of phosphorylation of eIF4E was unaffected. We conclude that formation of the active eIF4E. eIF4G complex controls protein synthesis in skeletal muscle when the amino acid concentration is above the physiological range, whereas removal of leucine reduces protein synthesis through changes in both eIF2B and eIF4E. (+info)
Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells.
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In PC12 phaeochromocytoma cells, protein synthesis is activated by epidermal and nerve growth factors (EGF and NGF). EGF and NGF also regulate a number of components of the translational machinery in these cells. Here we show that the ability of EGF and NGF to induce the phosphorylation of the 70 kDa ribosomal protein, S6 kinase, and the eukaryotic initiation factor (eIF), 4E-binding protein 1, is dependent upon the presence of amino acids (but not glucose) in the medium. This resembles the regulation of these proteins by insulin, which also requires amino acids. Glucose, but not amino acids, is required for the activation of eIF2B by EGF and NGF. In contrast, EGF and NGF can still activate protein synthesis in the absence of nutrients, suggesting that other regulatory events are important in this. In nutrient-deprived cells, an increase in the phosphorylation of eIF4E, and the assembly of the eIF4F complex by EGF and NGF, coincided with the activation of protein synthesis. In serum-starved cells, activation of protein synthesis, phosphorylation of eIF4E, and formation of the eIF4F complex, were blocked by inhibition of MEK, a component of the extracellular regulated kinase (ERK) signalling pathway. Thus the ERK pathway plays a key role in the regulation of protein synthesis in PC12 cells. (+info)