Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.     

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.     

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.     

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.     

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry

Being quite experienced in the field of gynaecologic surgery and hysterectomies especially, being familiar with recent innovations in laparoscopic surgery and also having some own experience in laparoscopic surgery, the authors discuss the advantages and disadvantages of all surgical methods of hysterectomy. Comparing the techniques, the duration, bearing in mind the the risks, overall costs and all other surgical details, the authors concluded that laparoscopically assisted vaginal hysterectomy is the best choice because it is the least invasive, less risky, no scars are left, the postoperative recovery is quick, there are numerous indications for it, the preparation obtained as a whole can be used for further clinical examination. The only disadvantage is it is too costly and sometimes the operation itself lasts too long, so it should not be applied in some cases.     

GTP-binding protein gamma12 subunit phosphorylation by protein kinase C--identification of the phosphorylation site and factors involved in cultured cells and rat tissues in vivo

We have demonstrated previously that the GTP-binding protein gamma12 subunit is a selective substrate for phosphorylation by protein kinase C among various gamma subunits in vitro, and that a serine residue in the N-terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N-terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated gamma12 and Swiss 3T3 cells rich in this protein, gamma12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12-myristate 13-acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of gamma12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of G(i/o). Phosphorylation of gamma12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet-derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP-binding proteins and protein kinase C in combination. Phosphorylated gamma12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of G(i/o) in vivo.     

The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli. In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA to E. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2-N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the N-formylmethionyl-tRNA-ribosome-mRNA ternary complex.     

Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons

The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.     

The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.     

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.     

Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.     

PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A

Our previous study has shown that the phases of circadian rhythms of ocular melatonin and dopamine are always opposite and intraocular melatonin injection suppresses dopamine release. Therefore, it is possible that dopamine rhythms result from inhibitory action of melatonin. We have examined this possibility in the following experiments. In the first experiment effects of continuous light on melatonin and dopamine release were examined. The data indicated that continuous light exposure resulted in loss of circadian rhythmicity of melatonin and dopamine by suppressing melatonin and enhancing dopamine levels throughout the day. To further examine the effects of light in the second experiment, 2 h light pulse was applied during the night, then temporal changes of melatonin and dopamine release were studied. The light pulse rapidly suppressed melatonin release, whereas it rapidly increased dopamine release. These changes occurred within 30 min in both melatonin and dopamine. However, the recovery after the cessation of the light stimulus was slower in melatonin than dopamine. In the third experiment it was tested if dopamine release was increased by lowering melatonin release with an intraocular injection of the D2 agonist, quinpirol. Although quinpirol strongly inhibited melatonin release independently of the time of injection, dopamine did not always increase by the inhibition of melatonin. These results indicate that ocular dopamine rhythms are not simply produced by melatonin inhibitory action.     

ATP-regulated K+ channel and cytosolic Ca2+ mobilization in cultured rat spinal neurons

ATP activated the K+ channel responsible for outwardly rectifying currents via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein in cultured rat spinal neurons. The evoked currents were inhibited by a selective protein kinase C inhibitor, GF109203X, whereas a phospholipase C inhibitor, neomycin had no effect. These indicate that the currents are regulated by phospholipase C-independent protein kinase C activation. In addition, ATP enhanced intracellular free Ca2+ concentration. The increase in intracellular free Ca2+ concentration was inhibited by a broad G-protein inhibitor, GDP beta S, but not affected by neomycin or an inositol 1,4,5-triphosphate receptor antagonist, heparin, suggesting that the cytosolic Ca2+ mobilization is regulated by a mechanism independent of a phospholipase C-mediated phosphatidylinositol signaling. These results, thus, demonstrate that ATP has dual actions on the coupled K+ channel and cytosolic Ca2+ release.     

Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel alpha subunit

Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI5-24. cBIMPS had similar effects on type IIA brain Na+ channel alpha subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na+ channel alpha subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (LI-II) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in LI-II of the Na+ channel alpha subunit, causing a reduction in Na+ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.     

Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2alpha. Role of the alpha- and delta-subunits of eiF2b

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.     

A difference in the rate of ribosomal elongation balances the synthesis of eukaryotic translation initiation factor (eIF)-2 alpha and eIF-2 beta

Eukaryotic translation initiation factor 2 (eIF-2) is a heterotrimer composed of three subunits designated alpha, beta, and gamma. These proteins exist in equimolar amounts in the cell and have not been detected as isolated subunits. Our research examines the basis of their balanced synthesis. Northern analysis of K562 cell mRNA revealed that eIF-2 beta was five times more abundant than eIF-2 alpha. However, immunoprecipitation of pulse-labeled K562 cells showed an equimolar rate of synthesis of eIF-2 alpha and -beta despite the 5-fold difference in the size of their mRNA pools. Addition of equal amounts of synthetic capped mRNA for eIF-2 alpha and eIF-2 beta to an in vitro translation reaction produced five times more eIF-2 alpha protein than eIF-2 beta. Determination of the polysome profile for alpha and beta mRNA in K562 cells indicated eIF-2 alpha was translated more efficiently than eIF-2 beta. Substitution of either the initiation codon context or the leader of the beta mRNA for that of alpha had only a minor effect on the translational efficiency of beta. Comparison of the rate of ribosomal elongation for the two mRNAs indicated that ribosomes associated with the beta mRNA elongate at a rate 4-fold less than that of eIF-2 alpha. Thus, the balanced translation of alpha and beta mRNA is primarily the result of a 4-fold difference in the rate of ribosomal elongation.     

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein alpha subunit Galpha13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits were characterized in transfection systems. Galphaq, Galpha12, and Galpha13, but not Galphai, activate SRF through RhoA. When Galphaq, alpha12, or alpha13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13, but not Galphaq or Galpha12, showed synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Galpha13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Galpha13- and Galpha12-induced, but not GEF115 itself- or Galphaq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF.