Regulation of Guanine Nucleotide Exchange through Phosphorylation of Eukaryotic Initiation Factor eIF2alpha
ROLE OF THE alpha - AND delta -SUBUNITS OF eIF2B*

Scot R. KimballDagger §, John R. FabianDagger , Graham D. Pavitt, Alan G. Hinnebusch, and Leonard S. JeffersonDagger

From the Dagger  Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 and the  Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(alpha P)) and exhibits little exchange activity when eIF2(alpha P) is used as substrate. In contrast, eIF2B lacking the alpha -subunit is insensitive to inhibition by eIF2(alpha P) and is able to exchange guanine nucleotide using eIF2(alpha P) 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(alpha P) and exhibits little exchange activity when eIF2(alpha P) 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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulation of translation initiation plays an important role in the control of gene expression in eukaryotic cells (reviewed in Ref. 1). By modulating different steps in the initiation pathway, regulation of the translation of mRNAs coding for specific classes of proteins as well as regulation of overall mRNA translation can be achieved. The initiation pathway is composed of a number of discrete steps involving at least 12 unique proteins referred to as eukaryotic initiation factors, or eIFs.1 Of the many steps in translation initiation, only two are thought to be important in regulating the process in vivo. The two steps include the sequential binding of first mRNA and then initiator methionyl-tRNAi (met-tRNAi) to the 40 S ribosomal subunit. In the latter step, eIF2 binds to the 40 S ribosomal subunit as a ternary complex with GTP and met-tRNAi. With formation of the 80 S initiation complex, the GTP is hydrolyzed and eIF2 is released as an eIF2·GDP binary complex. The GDP bound to eIF2 must then be exchanged for GTP, a reaction catalyzed by a guanine nucleotide exchange protein, termed eIF2B (reviewed in Ref. 2). In contrast to most guanine nucleotide exchange proteins, which are usually small and consist of a single subunit, eIF2B is composed of five dissimilar subunits present in equimolar amounts in a heteropentameric complex. Although eIF2B has been available in purified form from mammalian cells for 15 years (3), no information is available about the role of the individual subunits in catalyzing the guanine nucleotide exchange reaction. Furthermore, little is known about the role of the individual subunits in mediating regulation of the exchange activity.

The best characterized means of regulating eIF2B activity involves phosphorylation of the alpha -subunit of its substrate, eIF2, on Ser51. Phosphorylation of eIF2alpha converts eIF2 from a substrate into a competitive inhibitor (reviewed in Ref. 1). Based on genetic studies in Saccharomyces cerevisiae, it has been concluded that the eIF2B alpha -, beta -, and delta -subunits are important in mediating the effect of substrate inhibition of eIF2B (4, 5). In S. cerevisiae deprived of amino acids, eIF2alpha becomes phosphorylated, leading to increased translation of a protein termed GCN4 (reviewed in Refs. 6 and 7). Although not established experimentally, it has been assumed that eIF2B activity in yeast is inhibited in response to eIF2alpha phosphorylation and that the inhibition of eIF2B activity is responsible for the increased translation of GCN4 mRNA. This assumption is based on the finding that deletion of the alpha -subunit or point mutations identified in the alpha -, beta -, and delta -subunits of eIF2B prevent the increase in translation of GCN4 mRNA in response to amino acid deprivation (4) without having any effect on cellular growth in nonstarved cells. In addition, overexpression of the eIF2B alpha -, beta -, and delta -subunits leads to formation of a stable eIF2B subcomplex that overcomes the inhibitory effect of high level eIF2 phosphorylation, presumably through a mechanism involving sequestration of the phosphorylated eIF2 by the eIF2B subcomplex (5). The biochemical basis for the apparent insensitivity to eIF2alpha phosphorylation in cells expressing mutant forms of eIF2B subunits is unknown.

In the present study, the five subunits of rat eIF2B were co-expressed in Sf9 cells using the baculovirus expression system. A functional, five-subunit eIF2B complex was purified from the cells. In addition, a four-subunit complex lacking the alpha -subunit was expressed and purified. Both the four- and five-subunit forms of eIF2B exhibited similar specific activities, indicating that the alpha -subunit of the protein is not required for optimal guanine nucleotide exchange activity. However, whereas the four-subunit form of eIF2B was not inhibited by eIF2(alpha P), the five subunit form was. Furthermore, the exchange activity using eIF2(alpha P) as substrate was greater for four- than five-subunit eIF2B. Finally, the delta -subunit containing a double point mutation corresponding to mutations identified in yeast was expressed in combination with the other four subunits of eIF2B. The sensitivity of eIF2B containing the mutant delta -subunit to inhibition by eIF2(alpha P) was similar to that observed for eIF2B lacking the alpha -subunit. However, unlike eIF2B lacking the alpha -subunit, the ability of eIF2B containing the mutant delta -subunit to catalyze GDP exchange using eIF2(alpha P) was the same as wild-type eIF2B. Overall, the results provide the first biochemical evidence of the regulatory role of the alpha - and delta -subunits in mediating inhibition of exchange activity by substrate phosphorylation. In addition, they show that the alpha -subunit of eIF2B is not required for exchange activity.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of the Five Subunits of Rat eIF2B in Sf9 Cells-- High titer stocks of baculoviruses encoding the wild-type eIF2B alpha /delta , beta /gamma , and epsilon  subunits were generated as described previously (8). For coexpression of eIF2B subunits, 2 × 108 Sf9 insect cells were infected for 1 h in a reduced volume of 15 ml containing each of the three different virus stocks at a multiplicity of infection of 2-5 for each virus. The infected Sf9 cells were then transferred to a 250-ml Erlenmeyer flask containing 85 ml of SF-900 serum-free medium (Life Technologies, Inc.) and were maintained in culture at 28 °C using an orbital shaker (100 rpm). At 72 h after infection, 1.0-ml aliquots were removed and centrifuged in Eppendorf tubes at 2000 rpm for 3 min and pellets were stored at -70 °C until lysis.

Construction of the E311K,L315Q Mutant Form of eIF2Bdelta -- Site-directed mutagenesis (Altered Sites mutagenesis kit, Promega) was utilized to introduce specific changes to the eIF2Bdelta cDNA. Oligonucleotide GDP140 (5'-AAT TGC CTG AGA TGC CTG CAC AAT CTT CTT TTG CAC ATA CCG) was used to change Glu311 and Leu315 to Lys and Gln, respectively (eIF2Bdelta -E311K,L315Q), generating pAV1057. Plasmid pAV1153 (eIF2Bdelta -E311K,L315Q) was created by subcloning the mutated eIF2Bdelta 590-base pair BamHI-NdeI fragments from pAV1057 into identically cleaved J203 (also called pAc-2Balpha FLAG/delta FLAG). Nucleotide sequencing of the subcloned 590-base pair fragment confirmed that the plasmid contained only the expected site-directed nucleotide substitutions (underlined in the above oligonucleotide sequence).

Purification of Recombinant eIF2B from Sf9 Cells-- The proteins expressed in Sf9 cells were immunoaffinity purified by chromatography on a matrix containing an immobilized anti-FLAG monoclonal antibody (Anti-FLAG M2 Affinity Gel; IBI/Kodak). Briefly, the cells were lysed as described previously (8), and the lysate was centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was mixed with 2 ml of affinity matrix for 2 h at 4 °C, and the mixture was then poured into a plastic column. The column was washed with 30 ml of buffer B, followed by 30 ml of buffer C (20 mM Tris, pH 8.0, 150 mM NaCl), and the bound protein was eluted with 200 µg/ml FLAG octapeptide in buffer C. The protein was then concentrated using a Millipore Biomax 50K centrifugal concentrator and stored in aliquots at -70 °C.

Measurement of eIF2B Activity-- The guanine nucleotide exchange activity of eIF2B was measured as described previously (9) using eIF2 purified from rat liver as substrate. In some assays, eIF2B (0.5 µg) was preincubated for 2 min at 37 °C with 1 µg of either unphosphorylated eIF2 or eIF2 phosphorylated on Ser51 of the alpha -subunit using the eIF2alpha kinase, HCR (10). The guanine nucleotide exchange activity of eIF2B was then measured as the exchange of [3H]GDP bound to eIF2 for nonradiolabeled GDP with time.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The five rat eIF2B subunits were coexpressed in Sf9 cells using the baculovirus expression system as described previously (8). Each of the recombinant proteins was expressed with an amino-terminal octapeptide extension referred to as FLAG to aid in purification. As shown in the left panel of Fig. 1, a single immunoaffinity purification step utilizing an anti-FLAG monoclonal antibody coupled to a solid matrix resulted in isolation of an approximately equimolar mixture of the five eIF2B subunits at a purity of greater than 90%. Likewise, expression of just the eIF2B beta -, gamma -, delta -, and epsilon -subunits yielded a complex lacking the alpha -subunit (Fig. 1, right panel). The four-subunit complex lacking the alpha -subunit will be hereafter referred to as eIF2B(-alpha ).


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Fig. 1.   Purification of rat eIF2B expressed in Sf9 cells. FLAG epitope-tagged rat eIF2B subunits were co-expressed in Sf9 cells as described previously (8). The expressed proteins were immunoaffinity purified by chromatography on a matrix containing an immobilized anti-FLAG monoclonal antibody as described under "Materials and Methods." Approximately 5 µg of either the five-subunit eIF2B holoprotein (left panel) or a four-subunit eIF2B complex lacking the alpha -subunit (right panel) were electrophoretically resolved on a 12.5% polyacrylamide gel and stained with Coomassie R-250. The positions of the subunits are indicated in the center of the figure, and molecular mass standards are indicated in kilodaltons on the right.

The specific activities of eIF2B and eIF2B(-alpha ) were compared in a guanine nucleotide exchange assay using eIF2·[3H]GDP as substrate. As shown in Fig. 2, exchange activities were the same when equal amounts of eIF2B and eIF2B(-alpha ) were added to the assay. The results show that the alpha -subunit of eIF2B is not required for exchange activity.


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Fig. 2.   Specific activities of 4- and 5-subunit forms of eIF2B. A binary complex consisting of purified rat liver eIF2 and [3H]GDP was formed as described previously (9). Thirty-five µl (approximately 1.4 µg of eIF2) were then added to a reaction mixture containing 0.5 µg of either four-subunit (square ) or five-subunit (bullet ) eIF2B. The activity of eIF2B was measured as the exchange of [3H]GDP bound to eIF2 for nonradiolabeled GDP with time as described under "Materials and Methods." The results represent the mean ± S.E. of three experiments. Within each experiment, two independent assays were performed for each condition.

In a previous study, incubation of Sf9 cell extracts expressing rat eIF2B with eIF2 phosphorylated with the eIF2alpha kinase, HCR, resulted in a decrease in exchange activity compared with extracts incubated with unphosphorylated eIF2 (8). In contrast, extracts of cells expressing eIF2B(-alpha ) showed no decrease in exchange activity when incubated with eIF2 phosphorylated on the alpha -subunit. In the present study, purified eIF2B and eIF2B(-alpha ) were incubated with either unphosphorylated eIF2 or eIF2(alpha P) prior to assay. As shown in the inset to Fig. 3 (lane 1), there was no detectable unphosphorylated eIF2alpha in the phosphorylated eIF2 preparation. Similarly, no phosphorylated eIF2alpha was detected in the unphosphorylated preparation (Fig. 3, lane 2). In confirmation of the results obtained previously with crude cell extracts, incubation of purified eIF2B with eIF2(alpha P) (Fig. 3, open symbols) prior to assay resulted in a substantial decrease in exchange activity compared with incubation with unphosphorylated eIF2 (Fig. 3, closed symbols). In contrast, the activity of eIF2B(-alpha ) was nearly unaffected by phosphorylated eIF2.


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Fig. 3.   The five-subunit eIF2B holoprotein, but not eIF2B lacking the alpha -subunit, is inhibited by phosphorylated eIF2. eIF2 was phosphorylated using the eIF2alpha kinase, HCR, and then separated from ATP and HCR by chromatography on a phosphocellulose column (24). The phosphorylated and unphosphorylated forms of eIF2alpha were resolved by slab gel isoelectric focusing and then electrophoretically transferred to a polyvinylidene difluoride membrane. The proteins were then visualized by protein immunoblot analysis using a monoclonal anti-eIF2alpha antibody (inset). Lane 1, eIF2 incubated in the absence of HCR; lane 2, eIF2 phosphorylated with HCR; lane 3, an extract from a rat liver perfused with histidinol showing resolution of a mixture of phosphorylated and unphosphorylated eIF2alpha . Approximately 0.5 µg of the five-subunit (bullet , open circle ) or four-subunit (black-square, square ) forms of eIF2B were incubated for 2 min at 37 °C with 1 µg of either unphosphorylated eIF2 (bullet , black-square) or eIF2 phosphorylated on the alpha -subunit (open circle , square ). The guanine nucleotide exchange activity of eIF2B was then measured as described in the legend to Fig. 2 using a binary complex of unphosphorylated eIF2 and [3H]GDP. The results represent the mean ± S.E. of three experiments. Within each experiment, two independent assays were performed for each condition.

The sensitivity of the exchange activity of eIF2B and eIF2B(-alpha ) to eIF2 phosphorylation was further examined using an eIF2(alpha P)·[3H]GDP complex as substrate. As shown in the top panel of Fig. 4, both eIF2B and eIF2B(-alpha ) catalyzed GDP exchange using phosphorylated eIF2, although the exchange activity using eIF2(alpha P) as substrate (open symbols) was significantly slower than that observed using an equimolar amount of unphosphorylated eIF2·[3H]GDP (closed symbols). In addition, eIF2B(-alpha ) catalyzed GDP exchange using eIF2(alpha P) as substrate at a significantly faster rate than did five subunit eIF2B. The exchange activity observed was not influenced by substrate dephosphorylation since the amount of eIF2(alpha P) was the same at the beginning and end of the assay (Fig. 4, bottom panel). Likewise, the difference in exchange activity between the four- and five-subunit forms of eIF2B was not due to substrate dephosphorylation.


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Fig. 4.   eIF2B is able to exchange GDP using phosphorylated eIF2·[3H]GDP as substrate. Binary complexes consisting of [3H]GDP and either phosphorylated (open circle , square , triangle ) or unphosphorylated (bullet , black-square, black-triangle) eIF2 were formed as described in the legend to Fig. 2. The binary complexes (1.4 µg) were then used as substrate for either four-subunit (black-square, square ) or five-subunit (bullet , open circle ) forms of eIF2B (top panel). In addition, the exchange of [3H]GDP bound to eIF2 for free nonradiolabeled GDP was measured in the absence of eIF2B (black-triangle, triangle ). The results represent the mean ± S.E. of three experiments. Within each experiment, two independent assays were performed for each condition. Bottom panel, an aliquot of the eIF2B reaction mixture was analyzed for eIF2alpha phosphorylation state prior to the start of the assay (t = 0) and at the end of the assay (t = 6 min). The reaction mixture was resolved by SDS-polyacrylamide gel electrophoresis, followed by protein immunoblot analysis using an antibody specific for eIF2alpha phosphorylated on Ser51 (25).

Pavitt et al. (4) recently described nine eIF2Bdelta mutations that yielded a phenotype similar to that observed in cells lacking eIF2Balpha , i.e. increased eIF2alpha phosphorylation was not accompanied by reduced cell growth rates and increased GCN4 translation. The results suggested that, like eIF2B lacking the alpha -subunit, eIF2B with any one of these point mutations in the delta -subunit should be resistant to inhibition by eIF2(alpha P). Four of the amino acids that were found to be mutated in yeast eIF2Bdelta are conserved in the amino acid sequence of the rat protein (4). Of the conserved residues, substitutions of Glu377 and Leu381 with Lys and Gln, respectively, yielded a phenotype exhibiting the least apparent sensitivity to substrate phosphorylation. When a double mutation was made in yeast eIF2Bdelta combining these two substitutions, the phenotype observed was identical to the phenotype of the L381Q single mutation. Therefore, in the present study, the same double mutation was made in rat eIF2Bdelta and the mutant protein was coexpressed with the other four wild-type subunits. Recombinant eIF2B containing the mutant delta -subunit (referred to hereafter as eIF2B(delta *)) was purified from Sf9 cells as described above for wild-type eIF2B. Unexpectedly, over 50% of the mutant eIF2Bdelta was degraded during the purification procedure, even in the presence of a mixture of eight different protease inhibitors. Because of the difficulty in obtaining purified eIF2B(delta *) with equimolar amounts of all five subunits, the exchange activity of eIF2B(delta *) was assessed in cell extracts rather than with the purified protein. As shown in the inset to Fig. 5, no degradation of eIF2Bdelta occurred during preparation of extracts from cells expressing either the wild-type or mutant protein. It can also be seen that the amount of expressed protein was essentially the same for each of the five. As observed using purified eIF2B, incubation of extracts of Sf9 cells expressing all five wild-type eIF2B subunits with eIF2(alpha P) prior to assay (Fig. 5, open symbols) significantly reduced exchange activity compared with extracts incubated with unphosphorylated substrate (Fig. 5, closed symbols). In addition, preincubation of extracts of cells expressing eIF2B(-alpha ) with either eIF2(alpha P) or eIF2 resulted in little difference in exchange activity. Similar to the results observed for eIF2B(-alpha ), the exchange activity of eIF2B(delta *) was only minimally inhibited by preincubation with eIF2(alpha P). The results show directly for the first time that both the alpha - and delta -subunits of eIF2B are important in mediating the inhibition of exchange activity by eIF2(alpha P).


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Fig. 5.   eIF2B(delta *) is not inhibited by phosphorylated eIF2. Rat eIF2B subunits were coexpressed in Sf9 cells, and cell extracts were prepared for assay of guanine nucleotide exchange activity as described under "Materials and Methods." An aliquot of the extracts from cells expressing the five wild-type eIF2B subunits (lane 1), four wild-type subunits plus the E311K,L315Q mutant delta -subunit (lane 2), and cells expressing four wild-type subunits other than the alpha -subunit (lane 3) were analyzed by protein immunoblot analysis using an anti-FLAG antibody (inset). In addition, eIF2 was phosphorylated using HCR as described in the legend to Fig. 2. Extracts of Sf9 cells expressing the five wild-type rat eIF2B subunits (bullet , open circle ), the four eIF2B subunits other than the alpha -subunit (black-square, square ), or four wild-type eIF2B subunits plus the E311K,L315Q mutant delta -subunit (black-triangle, triangle ) were prepared as described previously for the wild-type protein (8). Aliquots (5 µl) of cell extracts were incubated for 2 min at 37 °C with approximately 1 µg of either unphosphorylated eIF2 (bullet , black-square, black-triangle) or eIF2 phosphorylated on the alpha -subunit (open circle , square , triangle ). Guanine nucleotide exchange activity was then measured as described in the legend to Fig. 2, using a preformed binary complex of unphosphorylated eIF2 and [3H]GDP. The results represent the mean ± S.E. of three experiments. Within each experiment, two independent assays were performed for each condition.

Finally, the exchange activity of eIF2B(delta *) was examined using phosphorylated eIF2 as substrate. As seen in Fig. 6, both wild-type eIF2B and eIF2B(delta *) catalyzed GDP exchange using eIF2(alpha P) as substrate (open symbols), although the activity was substantially lower using phosphorylated compared with unphosphorylated eIF2 (closed symbols). However, unlike eIF2B lacking the alpha -subunit, the exchange activity using phosphorylated eIF2 as substrate was nearly the same for eIF2B(delta *) as for wild-type eIF2B. Thus, although eIF2B(delta *) was less sensitive to the inhibitory effect of eIF2(alpha P) on nucleotide exchange using unphosphorylated eIF2 as substrate (Fig. 5), it remained largely incapable of using phosphorylated eIF2 as substrate.


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Fig. 6.   Guanine nucleotide exchange activity using phosphorylated eIF2 as substrate. Binary complexes consisting of [3H]GDP and either phosphorylated (open circle , square ) or unphosphorylated (bullet , black-square) eIF2 were formed as described in the legend to Fig. 2. The binary complexes were then used as substrate for measuring eIF2B activity in extracts of Sf9 cells expressing either the five wild-type rat eIF2B subunits (bullet , open circle ) or four wild-type eIF2B subunits plus the E311K,L315Q mutant delta -subunit (black-square, square ) as described in the legend to Fig. 4. The results represent the mean ± S.E. for three different cell preparations for each condition.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulation of translation initiation through phosphorylation of the alpha -subunit of eIF2 occurs in response to a variety of stimuli including deprivation of amino acids (10-12), glucose (12), purines (13), or serum (12). Phosphorylation of eIF2alpha does not directly inhibit formation of either the eIF2·GTP·Met-tRNAi ternary complex or the 43 S preinitiation complex (i.e. a 40 S ribosomal subunit associated with eIF-1A, eIF-3, and the eIF2 ternary complex), as these reactions proceed efficiently in vitro with the phosphorylated factor (14). Instead, phosphorylation of the alpha -subunit of eIF2 is thought to impede eIF2B activity by sequestering eIF2B into an inactive complex. Three lines of evidence support this assumption. (a) eIF2B reportedly does not catalyze GDP exchange on eIF2(alpha P) (15); (b) eIF2(alpha P) displaces unphosphorylated eIF2 bound to eIF2B but unphosphorylated eIF2 does not (16); and (c) eIF2(alpha P) inhibits the activity of eIF2B in the presence of low concentrations of substrate (i.e. <10-fold molar excess of eIF2·GDP to eIF2(alpha P)) but at higher substrate concentrations the inhibition caused by eIF2(alpha P) is negligible (15), suggesting that eIF2(alpha P) is acting as a competitive inhibitor of eIF2B.

Phosphorylation of eIF2alpha plays a critical role in the general control response in S. cerevisiae. Starvation of S. cerevisiae for any one of at least 10 amino acids leads to phosphorylation of eIF2alpha and increased translation of the mRNA coding for the transcription factor GCN4 (reviewed in Refs. 6 and 7). The latter effect is dependent upon the presence of eIF2B (reviewed in Refs. 7 and 17). In yeast, it has been suggested that three of the five subunits of eIF2B are involved in recognition of the phosphorylation status of eIF2alpha . In particular, deletion of eIF2Balpha has no effect on cellular growth under nonstarvation conditions (18). However, eIF2Balpha is required for induction of GCN4 translation under amino acid starvation conditions (19) and the induction of GCN4 is dependent upon phosphorylation of eIF2alpha (20). Moreover, deletion of eIF2Balpha reduces the growth-inhibitory effect of high level eIF2 phosphorylation catalyzed by overexpression of the human double-stranded RNA-activated eIF2alpha protein kinase, PKR (21). A more recent study has identified point mutations in the alpha -subunit of eIF2B that are even more effective than deletion of the subunit in reversing the effects of eIF2 phosphorylation on translation and growth (4). These results suggest that the primary function of the alpha -subunit of eIF2B is to mediate the inhibitory effects of eIF2alpha phosphorylation on exchange activity.

The yeast eIF2B beta - and delta -subunits exhibit regions of amino acid sequence similarity to eIF2Balpha (22), suggesting that these other two subunits might also be involved in regulating the activity of eIF2B in response to eIF2alpha phosphorylation. In support of this suggestion, overexpression in yeast of the eIF2B alpha -, beta -, and delta -subunits together results in formation of a stable subcomplex in vivo whose presence neutralizes the effects of eIF2 phosphorylation on translation (5). It was proposed that this subcomplex can sequester eIF2(alpha P) and permit native five-subunit eIF2B to catalyze guanine nucleotide exchange on unphosphorylated eIF2. In addition, point mutations have been identified in both the beta - and delta -subunits of yeast eIF2B that result in the expression of the same phenotype as is observed in cells in which eIF2Balpha has been deleted, i.e. phosphorylation of eIF2alpha does not result in increased translation of GCN4 (4, 23). However, eIF2B activity has not been measured in extracts of S. cerevisiae deprived of amino acids and the mechanism involved in the putative change is still speculative.

The mutations identified in yeast eIF2B alpha -, beta -, and delta -subunits could lead to the observed phenotype through several distinct mechanisms. For example, the mutations could lead to an overall increase in the specific activity of eIF2B, be permissive for guanine nucleotide exchange using phosphorylated eIF2 as substrate, or decrease the affinity of eIF2B for eIF2alpha (P) such that it is no longer a competitive inhibitor of eIF2B. In the present study, rat eIF2B lacking the alpha -subunit had the same specific activity as the wild-type protein. Although the specific activity of eIF2B(delta *) could not be directly measured, the rate of guanine nucleotide exchange was similar in extracts of Sf9 cells expressing the five wild-type eIF2B subunits and cells expressing a mutant form of the delta -subunit in combination with the remaining four wild-type subunits. Assuming that the catalytic properties of yeast eIF2B are similar to the rat protein, the results suggest that the phenotype observed in yeast lacking eIF2Balpha or expressing mutant forms of eIF2Bdelta in response to increased eIF2alpha phosphorylation is not a result of an overall increase in the specific activity of eIF2B. In addition, the results of the present study suggest that, although wild-type eIF2B can catalyze GDP exchange using eIF2(alpha P) as substrate and eIF2B(-alpha ) catalyzes GDP exchange using eIF2(alpha P) as substrate at a faster rate than wild-type eIF2B, the difference in rate observed between wild-type eIF2B and eIF2B(-alpha ) using eIF2(alpha P) as substrate may not be sufficient to account for the phenotype observed in yeast in response to phosphorylation of eIF2alpha . In contrast, both eIF2B(-alpha ) and eIF2B(delta *) appear to be completely resistant to inhibition by eIF2(alpha P) when the proteins are incubated with eIF2(alpha P) prior to assay using unphosphorylated eIF2 as substrate. This result suggests that the affinity for eIF2(alpha P) is significantly less for the mutant forms of eIF2B than for the wild-type protein.

In summary, the present study provides the first biochemical demonstration that both the alpha - and delta -subunits of eIF2B play important roles in regulating the guanine nucleotide exchange activity of the protein in response to phosphorylation of eIF2alpha . In particular, eIF2B lacking the alpha -subunit or containing a mutant form of the delta -subunit is completely resistant to inhibition by eIF2(alpha P). Finally, lack of the alpha -subunit allows for faster GDP exchange using eIF2(alpha P)·[3H]GDP as substrate.

    ACKNOWLEDGEMENT

We acknowledge the excellent technical assistance of Lynne Hugendubler.

    FOOTNOTES

* This work was supported in part by Grants DK13499 and DK15658 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, P. O. Box 850, Hershey, PA 17033. Tel.: 717-531-8970; Fax: 717-531-7667; E-mail: skimball{at}psu.edu.

1 The abbreviations used are: eIF, eukaryotic initiation factor; HCR, heme-controlled repressor.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771[Abstract]
  2. Price, N., and Proud, C. G. (1994) Biochimie 76, 748-760[CrossRef][Medline] [Order article via Infotrieve]
  3. Siekierka, J., Mauser, L., and Ochoa, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2537-2540[Abstract]
  4. Pavitt, G. D., Yang, W., and Hinnebusch, A. G. (1997) Mol. Cell. Biol. 17, 1298-1313[Abstract]
  5. Yang, W., and Hinnebusch, A. G. (1996) Mol. Cell. Biol. 16, 6603-6616[Abstract]
  6. Hinnebusch, A. G. (1994) Trends Biochem. Sci. 19, 409-414[CrossRef][Medline] [Order article via Infotrieve]
  7. Hinnebusch, A. G. (1997) J. Biol. Chem. 272, 21661-21664[Free Full Text]
  8. Fabian, J. R., Kimball, S. R., Heinzinger, N. D., and Jefferson, L. S. (1997) J. Biol. Chem. 272, 12359-12365[Abstract/Free Full Text]
  9. Kimball, S. R., Everson, W. V., Flaim, K. E., and Jefferson, L. S. (1989) Am. J. Physiol. 256, C28-C34[Abstract/Free Full Text]
  10. Kimball, S. R., Antonelli, D. A., Brawley, R. M., and Jefferson, L. S. (1991) J. Biol. Chem. 266, 1969-1976[Abstract/Free Full Text]
  11. Marton, M. J., Crouch, D., and Hinnebusch, A. G. (1993) Mol. Cell. Biol. 13, 3541-3556[Abstract]
  12. Scorsone, K. A., Panniers, R., Rowlands, A. G., and Henshaw, E. C. (1987) J. Biol. Chem. 262, 14538-14543[Abstract/Free Full Text]
  13. Rolfes, R. J., and Hinnebusch, A. G. (1993) Mol. Cell. Biol. 13, 5099-5111[Abstract]
  14. Trachsel, H., and Staehelin, T. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 204-208[Abstract]
  15. Rowlands, A. G., Panniers, R., and Henshaw, E. C. (1988) J. Biol. Chem. 263, 5526-5533[Abstract/Free Full Text]
  16. Safer, B., Jagus, R., Konieczny, A., and Crouch, D. (1982) in Interactions of Translational and Transcriptional Controls in the Regulation of Gene Expression (Grunberg-Manago, M., and Safer, B., eds), pp. 311-325, Elsevier Science Publishing Co., Inc., Amsterdam
  17. Hinnebusch, A. G. (1993) Mol. Microbiol. 10, 215-223[Medline] [Order article via Infotrieve]
  18. Cigan, A. M., Foiani, M., Hannig, E., and Hinnebusch, A. G. (1991) Mol. Cell. Biol. 11, 3217-3228[Medline] [Order article via Infotrieve]
  19. Hinnebusch, A. G., and Klausner, R. D. (1991) in Translation in Eukaryotes (Trachsel, H., ed), pp. 243-272, CRC Press, Inc., Boca Raton, FL
  20. Dever, T. E., Feng, L., Wek, R. C., Cigan, A. M., Donahue, T. F., and Hinnebusch, A. G. (1992) Cell 68, 585-596[Medline] [Order article via Infotrieve]
  21. Dever, T. E., Chen, J.-J., Barber, G. N., Cigan, A. M., Feng, L., Donahue, T. F., London, I. M., Katze, M. G., and Hinnebusch, A. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4616-4620[Abstract]
  22. Bushman, J. L., Asuru, A. I., Matts, R. L., and Hinnebusch, A. G. (1993) Mol. Cell. Biol. 13, 1920-1932[Abstract]
  23. Vazquez de Aldana, C. R., and Hinnebusch, A. G. (1994) Mol. Cell. Biol. 14, 3208-3222[Abstract]
  24. Kimball, S. R., Everson, W. V., Myers, L. M., and Jefferson, L. S. (1987) J. Biol. Chem. 262, 2220-2227[Abstract/Free Full Text]
  25. DeGracia, D. J., Sullivan, J. M., Neuman, R. W., Alousi, S. S., Hikade, K. R., Pittman, J. E., Rafols, J. A., and Krause, G. S. (1997) J. Cereb. Blood Flow Metab. 17, 1291-1302[Medline] [Order article via Infotrieve]


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