Identification of Interprotein Interactions between the Subunits of Eukaryotic Initiation Factors eIF2 and eIF2B*

Scot R. KimballDagger , Nina K. Heinzinger§, Rick L. Horetsky, and Leonard S. Jefferson

From the Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha -subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha -subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta - and epsilon -subunits of eIF2B, and eIF2B was shown to bind only to the beta -subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta - or epsilon -subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha -subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta -subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The translation initiation phase of protein synthesis is a complex process mediated by a family of at least 12 proteins collectively referred to as eukaryotic initiation factors (reviewed in Refs. 1-3). Regulation of translation initiation plays a crucial role in maintaining protein homeostasis within cells in response to a variety of hormonal, nutritional, and environmental stimuli. One of the most tightly regulated steps in translation initiation involves two multisubunit proteins referred to as eukaryotic initiation factors eIF2 and eIF2B. During initiation, eIF2 binds GTP and initiator methionyl-tRNAi (Met-tRNAi)1, and the eIF2·GTP·Met-tRNAi ternary complex then binds to a 40 S ribosomal subunit. Upon joining of the 60 S ribosomal subunit, the GTP bound to eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosome as an eIF2·GDP binary complex. Before eIF2 can participate in another cycle of initiation, the GDP bound to the protein must be exchanged for GTP. This guanine nucleotide exchange reaction is mediated by the heteropentameric protein, eIF2B.

One of the ways through which the activity of eIF2B is regulated occurs through an unusual mechanism involving phosphorylation of one of the subunits of its substrate eIF2 (reviewed in Ref. 3). The smallest, or alpha -subunit, of eIF2 is phosphorylated in response to a variety of cellular stresses including viral infection, heat shock, heavy metals, and deprivation of amino acids or serum. Previous studies have reported that the affinity of eIF2B for eIF2(alpha P) is increased either 2-fold (4) or 150-fold (5), depending on the method used to measure the interaction. An obvious assumption from these studies is that the alpha -subunit of eIF2 would bind to one or more subunits of eIF2B. However, even though both proteins have been available in purified form for more than 15 years, the interprotein subunit interactions have remained undefined. Yet an understanding of the subunit interactions between the two proteins is vital not only to understanding the basis for the inhibition of eIF2B by phosphorylation of eIF2alpha , but also for delineating the mechanism through which eIF2B mediates guanine nucleotide exchange on eIF2.

In the present study, we have identified the interprotein interactions among the subunits of eIF2 and eIF2B. Surprisingly, eIF2B was found to bind not to the alpha -subunit of eIF2, but instead only to the beta -subunit. Furthermore, eIF2B was found to bind to a domain within approximately 70 amino acids of the C terminus of eIF2beta . Phosphorylation of eIF2alpha did not induce binding of eIF2B to the isolated alpha -subunit, but did increase the affinity of eIF2B for the eIF2 holoprotein. In addition, the delta - and epsilon -subunits of eIF2B were identified as the sites for binding of eIF2. Finally, phosphorylation of the beta -subunit of eIF2 by protein kinase A (PKA) increased the guanine nucleotide exchange activity of eIF2B.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- ECL detection reagents and horseradish peroxidase-conjugated, sheep anti-mouse Ig were purchased from Amersham Life Sciences, Inc. PVDF membrane was obtained from Bio-Rad. Casein kinase II (CK-II) was purchased from Boehringer Mannheim. PKA and protein kinase C (PKC) were from Promega.

Far-Western Analysis of the Interaction of eIF2 and eIF2B-- The subunits of eIF2 and eIF2B were separated by electrophoresis on SDS-polyacrylamide gels as described previously (6). The proteins were then transferred onto PVDF membranes, and the membranes were blocked in 5% non-fat dry milk powder (ICN) in buffer A (10 mM Tris·HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) for 1 h. The membranes were incubated with either radiolabeled eIF2 (for blots containing eIF2B) or radiolabeled eIF2B (for blots containing eIF2) overnight at 4 °C. Prior to use as a probe, eIF2 was radiolabeled by phosphorylation using [32P]ATP and either heme-controlled repressor (HCR) to phosphorylate the alpha -subunit or PKA to phosphorylate the beta -subunit (7, 8). eIF2B was either radiolabeled, using phosphorylated [32P]ATP and a mixture of casein kinase I (CK-I) and glycogen synthase kinase 3 (GSK-3) to phosphorylate the epsilon -subunit (9, 10), or biotinylated, using a kit (ECL Protein Biotinylation Module) from Amersham Life Sciences, Inc. The blots were washed, incubated with radiolabeled probe, and then dried and exposed to film. Blots probed with biotinylated eIF2B were incubated with avidin coupled to horseradish peroxidase and then developed using an ECL detection kit from Amersham Life Sciences, Inc.

An assay using 96-well microtitre plates was also used to examine the interprotein subunit interactions of eIF2 and eIF2B. In these experiments, one protein was placed in multiple wells of a 96-well plate, and the plate was incubated overnight at 4 °C. The wells were then washed with buffer A, blocked with buffer A containing 5% non-fat dry milk powder, and then washed with buffer A without milk powder. The wells then received various amounts of the second protein followed by incubation at room temperature for 2 h. The wells were washed and incubated with either an anti-eIF2Bepsilon monoclonal antibody (11), when probed with eIF2B, or an anti-FLAG monoclonal antibody (IBI/Kodak), when probed with FLAG-tagged eIF2 subunits. The wells were washed, incubated with anti-mouse IgG antibody coupled to alkaline phosphatase, and then developed with Sigma 104 phosphatase substrate. The results obtained were analyzed using a Molecular Devices microtitre plate reader.

Expression of the alpha -, beta -, and gamma -Subunits of Rat eIF2 in Sf9 Insect Cells-- Recombinant baculoviruses containing cDNAs for the alpha -, beta -, and gamma -subunits of rat eIF2 were constructed using the Bac-to-Bac expression system (Life Technologies, Inc.). All constructs were made in the pFastBac1 vector. Each subunit of eIF2 was cloned with a 10-amino acid modified FLAG peptide (DYKDDDDKID) at the N terminus after the initial methionine. The cDNA for the rat eIF2alpha subunit (generously provided by Dr. J. W. B. Hershey, University of California) was cloned into the BamHI and EcoRI sites of the vector. The cDNA for the rat eIF2beta subunit was cloned into the EcoRI and KpnI sites of the vector. The cDNA for rat eIF2gamma was obtained by screening a rat brain cDNA library (Stratagene) using an expressed sequence-tagged clone (EST78998) with similarity to human eIF2gamma , which yielded a partial length cDNA. The 5' RACE system (Life Technologies, Inc.) was then used to obtain the 5'-end of the cDNA. A full-length cDNA for eIF2gamma was reconstructed and cloned into the EcoRI and KpnI sites of the vector. The baculovirus constructs were transfected into Sf9 cells according to the Bac-to-Bac protocol (Life Technologies, Inc.), and recombinant baculovirus was obtained. Viruses underwent two rounds of amplification before use for protein production. Sf9 cells were infected at a multiplicity of infection of 10 or greater, grown in shaker flasks at 27 °C, and harvested after 2-3 days. 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 in buffer B (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxychlolate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 20 µM leupeptin, 0.4 µg/ml pepstatin, and 0.2 mM benzamidine), 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 an Amicon Centricon concentrator and stored in aliquots at -70 °C.

Phosphorylation of the beta -Subunit of eIF2-- A binary complex consisting of purified rat liver eIF2 and [3H]GDP was formed as described previously (12). Approximately 3 µg of the eIF2·[3H]GDP complex was then phosphorylated at 30 °C with either CK-II (1 milliunit), PKA (300 units), or PKC (83 milliunits) in a reaction mixture containing 40 mM Tris·HCl, pH 7.0, 11 mM beta -mercaptoethanol, 8% glycerol, 80 µM phenylmethylsulfonyl fluoride, and 100 µM [32P]ATP (4000 mCi/mmol). When eIF2 was phosphorylated with PKC, reactions additionally contained 40 µM phosphatidylserine and 0.5 mM CaCl2.

Measurement of eIF2B Activity-- The activity of eIF2B was measured as described previously (12) except that the eIF2·[3H]GDP binary complex was phosphorylated with CK-II, PKA, PKC, or no added kinase and nonradiolabeled ATP as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The eIF2 and eIF2B holoproteins used in these studies were purified from rat liver to greater than 95% homogeneity (Fig. 1). Far Western blot analysis was used to identify the subunits of eIF2B to which eIF2 binds and vice versa. In the first set of studies, the subunits of eIF2B were separated by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was then probed with eIF2 that had been phosphorylated using [32P]ATP and either HCR, to phosphorylate the alpha -subunit of eIF2, or PKA, to phosphorylate the beta -subunit. As shown in Fig. 2, eIF2 that was radiolabeled on either the alpha - or beta -subunit bound to both the delta - and epsilon -subunits of eIF2B, with binding to the delta -subunit being consistently stronger than to the epsilon -subunit. No binding of eIF2 to the alpha -, beta -, or gamma -subunits of eIF2B was detected. In the experiments shown in Fig. 2, the binding of eIF2 phosphorylated with HCR to eIF2Bepsilon appeared to be weaker than that observed for eIF2 phosphorylated with PKA, suggesting that phosphorylation of eIF2 on the alpha -subunit decreased the affinity of eIF2 for eIF2Bepsilon . However, this apparent difference was not consistent from experiment to experiment.


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Fig. 1.   Purification of eIF2 and eIF2B from rat liver. eIF2 and eIF2B were purified from rat liver as described previously (6, 11). Approximately 3 µg of eIF2 and 5 µg of eIF2B were resolved on a 12.5% polyacrylamide gel and then stained with Coomassie R-250. The subunits of eIF2 and eIF2B are indicated on the left of both panels. Molecular mass standards are indicated in kilodaltons on the right of each panel.


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Fig. 2.   Far-Western analysis of the interprotein subunit interactions between eIF2 and eIF2B. The interactions among the subunits of eIF2 and eIF2B were examined by far-Western blot analysis as described under "Experimental Procedures." In panels A and B, approximately 7.5 µg of eIF2B was resolved on a 12.5% polyacrylamide gel and transferred to a PVDF membrane. The membranes were probed with eIF2 that had been phosphorylated with [32P]ATP and either HCR (panel A) or PKA (panel B). In panels C and D, 7.5 µg of unphosphorylated eIF2 (lanes 1) or eIF2 that had been phosphorylated with nonradiolabeled ATP and HCR (lanes 2) was resolved on a 12.5% polyacrylamide gel and then transferred to PVDF. The membrane was probed with either biotinylated eIF2B (panel C) or eIF2B that had been phosphorylated with [32P]ATP and a mixture of GSK-3 and CK-I (panel D). The analyses were repeated at least three times for each condition with similar results. The results of typical blots are shown.

In similar experiments, the binding of eIF2B to the subunits of eIF2 was examined by far-Western blot analysis. For these studies, an aliquot of eIF2 was phosphorylated with HCR with nonradiolabeled ATP, and phosphorylated and unphosphorylated eIF2 were electrophoresed in parallel lanes on a polyacrylamide gel. The proteins were transferred to a PVDF membrane, and the membrane was probed with eIF2B that had either been biotinylated or phosphorylated using [32P]ATP and a mixture of GSK-3 and CK-I. As shown in Fig. 2, eIF2B bound to the beta -subunit of eIF2; no detectable binding of eIF2B to either the alpha - or gamma -subunits of eIF2 was observed. Furthermore, both biotinylated and phosphorylated eIF2B bound exclusively to eIF2beta , indicating that the method used to label eIF2B did not affect the interaction of the two proteins. It is noteworthy that phosphorylation of eIF2 by HCR did not promote binding of eIF2B to eIF2alpha .

Because the finding that eIF2B did not bind to the alpha -subunit of eIF2 was so unexpected, we used a second method to examine the binding of the individual subunits of eIF2 to eIF2B. In these experiments, the subunits of eIF2 were individually expressed in Sf9 cells using the baculovirus system. Each of the subunits was expressed with an 8-amino acid extension, referred to as FLAG, at the N terminus of the protein to aid in purification and identification. As shown in the top panel of Fig. 3, immunoaffinity purification of the FLAG-tagged eIF2 subunits resulted in isolation of proteins that were greater than 90% homogeneous. The purified subunits were used to probe wells of a microtitre plate in which the eIF2B holoprotein had been immobilized. As shown in the bottom panel of Fig. 3, only the beta -subunit of eIF2 exhibited significant binding to eIF2B. Similar to what was observed in the far-Western blot analysis, phosphorylation of eIF2alpha by HCR did not enhance binding of eIF2alpha to immobilized eIF2B.


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Fig. 3.   Purification of the individual subunits of eIF2 expressed in Sf9 cells and protein interaction analysis of the subunits of eIF2 and eIF2B. The subunits of eIF2 were expressed in and purified from Sf9 cells as described under "Experimental Procedures." In the top panel, approximately 2 µg of each subunit was resolved on a 12.5% polyacrylamide gel and stained with Coomassie R-250. The positions of the subunits are indicated on the left, and molecular mass standards are indicated in kilodaltons on the right. In the bottom panel, 0.25 µg of eIF2B was bound to the wells of a microtitre plate and then probed with the individual subunits of eIF2. The amounts of eIF2 subunits bound to eIF2B were quantitated using an anti-FLAG monoclonal antibody as described under "Experimental Procedures." The values represent the mean ± S.E. of three experiments. Within each experiment, each condition was analyzed in triplicate.

To identify the region of eIF2beta to which eIF2B binds, two deletion mutants of the eIF2beta cDNA were constructed where the C-terminal 65 or 133 amino acids (termed 268 and 200, respectively) were removed from the coding sequence. As shown in Fig. 4, removal of the C-terminal 65 residues reduced the binding of eIF2beta to eIF2B by greater than 80%. No significant binding of the 200 mutant to eIF2B was detected. In an attempt to confirm the specificity of the interaction between eIF2B and the beta -subunit of eIF2, the interactions between the wild-type and deletion mutants of eIF2beta and the beta -, delta -, and epsilon -subunits of eIF2B were examined. In these experiments, the beta -, delta -, and epsilon -subunits of eIF2B were expressed in and purified from Sf9 cells using the baculovirus system (13). In confirmation of the results obtained by far-Western blot analysis, the isolated delta - and epsilon -subunits of eIF2B bound to wild-type eIF2beta , whereas the beta -subunit did not (Fig. 5). Furthermore, removal of the C-terminal 65 residues of eIF2beta reduced the binding of both eIF2Bdelta and epsilon  by greater than 75%. These results confirm both the specificity of the interaction between the beta -subunit of eIF2 and the delta - and epsilon -subunits of eIF2B as well as the importance of the C terminus of eIF2beta in the interaction between eIF2 and eIF2B.


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Fig. 4.   Deletion analysis of the eIF2B binding site on eIF2beta . As described under "Experimental Procedures," two deletion mutants of the eIF2beta cDNA were constructed such that the expressed protein was truncated at amino acids 200 (mutant 200) or 268 (mutant 268), respectively, as illustrated on the left. Each of the proteins was expressed with the modified FLAG epitope at the N terminus after the initial methionine, and the amino acid sequence is numbered starting with the first amino acid of the eIF2beta coding region. The binding of eIF2B to full-length eIF2beta and to the eIF2beta mutants was assessed by the protein interaction assay described under "Experimental Procedures." In this analysis, 0.2 µg of eIF2B was bound to wells of a microtitre plate and probed with either full-length eIF2beta (wt), eIF2beta truncated at amino acid 268 (268), or eIF2beta truncated at amino acid 200 (200). Quantitation of the binding of eIF2beta to eIF2B was assessed using an anti-FLAG monoclonal antibody. The results represent the mean ± S.E. of three experiments. Within each experiment, each condition was analyzed in triplicate. A typical SDS-polyacrylamide gel of the mutant 200 (200), mutant 268 (268), and full-length (wt) eIF2beta proteins purified from Sf9 cells is shown in the insert in the right panel. Molecular mass standards are indicated in kilodaltons on the left of the insert.


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Fig. 5.   Binding of the individual subunits of eIF2B to deletion mutants of eIF2beta . Two deletion mutants of the beta -subunit of eIF2 were expressed in and purified from Sf9 cells as described in the legend to Fig. 4. In addition, the beta -, delta -, and epsilon -subunits of eIF2B were expressed in Sf9 cells with the FLAG sequence at the N terminus as described previously (13) and purified as described for the subunits of eIF2 under "Experimental Procedures." The binding of the individual subunits of eIF2B to full-length eIF2beta and the eIF2beta mutants was assessed by the protein interaction assay described under "Experimental Procedures." In this analysis, approximately 3.3 µg of eIF2Bbeta , delta , or epsilon  was bound to wells of a microtitre plate and probed with either full-length eIF2beta (wt), eIF2beta truncated at amino acid 268 (268), or eIF2beta truncated at amino acid 200 (200). Quantitation of the binding of eIF2beta to eIF2B was assessed using an anti-FLAG monoclonal antibody. The results represent the mean ± S.E. of three determinations. n.d., none detected

In an attempt to gain greater insight into the interaction between the beta -subunit of eIF2 and eIF2B, the amino acid sequence of eIF2beta was submitted to the Protein Sequence Analysis server at the BioMolecular Engineering Research Center at Boston University (Boston, MA) for type 2 analysis for multimeric proteins. The secondary structure prediction indicates a high probability that the region of eIF2beta , including amino acids 260-285, will form an alpha -helix. Most of the predicted alpha -helical structure is removed in the eIF2beta deletion mutant truncated at amino acid 268, and the probability that the remainder of the domain will form an alpha -helix is greatly reduced. This result is noteworthy because of the finding in the present study that binding of the eIF2beta 268 mutant to either wild-type eIF2B, eIF2Bdelta , or eIF2Bepsilon is reduced by greater than 75% compared with the full-length protein, suggesting that this putative alpha -helical domain is important in the interaction of the two proteins.

A caveat to the above experiments is that they do not directly demonstrate an interaction between eIF2B and the C terminus of the beta -subunit of eIF2. An alternative explanation for the results is that deletion of the C-terminal portion of the protein interferes with the correct folding of the remainder. If the correct folding of the remainder of the protein is prevented, but necessary for binding of eIF2B, then deletion of the C terminus could indirectly prevent binding of eIF2B to the beta -subunit of eIF2.

To demonstrate that the methods used above could detect a difference in affinity of eIF2B for eIF2 following phosphorylation of eIF2alpha , unphosphorylated eIF2 holoprotein and eIF2 holoprotein phosphorylated with HCR were bound to wells in parallel rows in a microtitre plate. The wells were then probed with various amounts of eIF2B, and the binding of eIF2B to eIF2 was quantitated using a monoclonal antibody to eIF2Bepsilon . As shown in Fig. 6, the titration curve for the binding of eIF2B to eIF2 phosphorylated on the alpha -subunit was shifted to the left of the curve for binding to unphosphorylated eIF2. The amount of eIF2B required to obtain half-maximal binding to unphosphorylated eIF2 was approximately 3-fold greater than for the phosphorylated protein, suggesting that phosphorylation of eIF2 by HCR increases the affinity of eIF2B for eIF2 by a factor of ~3.


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Fig. 6.   Effect of phosphorylation of the alpha -subunit of eIF2 on the affinity of eIF2B for eIF2. The affinity of eIF2B for phosphorylated and unphosphorylated eIF2 was examined using a modification of the ELISA assay as described under "Experimental Procedures." Pure eIF2 was phosphorylated with HCR, and 0.5 µg of phosphorylated (black-square) or unphosphorylated (open circle ) protein was bound to the wells of a 96-well microtitre plate. The wells were then probed with various amounts of eIF2B as noted in the figure. The amount of eIF2B bound to eIF2 was quantitated using an anti-eIF2Bepsilon monoclonal antibody as described under "Experimental Procedures." The analysis was repeated three times with similar results.

Finally, the effect of phosphorylation of the beta -subunit of eIF2 on the activity of eIF2B was examined. The beta -subunit of eIF2 is a substrate for at least three different protein kinases. CK-II phosphorylates Ser2 and Ser67, PKC phosphorylates Ser13, and PKA phosphorylates Ser218 (8). In these studies, the eIF2·[3H]GDP complex was formed and then phosphorylated with nonradiolabeled ATP using either CK-II, PKA, or PKC prior to use as a substrate for eIF2B. As shown in the top panel of Fig. 7, all three kinases incorporated similar amounts of radioactivity into the beta -subunit of eIF2; no incorporation of 32P into eIF2beta was observed in the absence of added kinase. Furthermore, no incorporation of 32P into eIF2alpha was observed (data not shown). Phosphorylation by either CK-II or PKC had no effect on the activity of eIF2B (Fig. 7, bottom panel). In contrast, phosphorylation of eIF2beta by PKA stimulated the guanine nucleotide exchange activity of eIF2B by 44% (p < 0.01). Thus, phosphorylation of eIF2beta in the C-terminal part of the protein, but not in the N-terminal region, resulted in stimulation of eIF2B activity.


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Fig. 7.   Effect of phosphorylation of the beta -subunit of eIF2 on the activity of eIF2B. A binary complex consisting of purified rat liver eIF2 and [3H]GDP was formed as described previously (12). Approximately 3 µg of the eIF2·[3H]GDP complex was then phosphorylated using [32P]ATP and either CK-II, PKA, or PKC as described under "Experimental Procedures." At the times indicated, 15 µl of the reaction mixture (0.9 µg of eIF2) were removed from the tube and subjected to SDS-polyacrylamide gel electrophoresis. The gel was then stained, dried, and exposed to film. An autoradiograph is shown (top panel). Alternatively, 2.6 µg of the eIF2·[3H]GDP complex was phosphorylated using nonradiolabeled ATP and either CK-II, PKA, or PKC as described under "Experimental Procedures." Thirty-five µl of the reaction mixture (approximately 1 µg of eIF2) was then added to a reaction mixture containing 0.4 µg of purified rat liver eIF2B (11). The activity of eIF2B was measured as the exchange of [[3H]GDP bound to eIF2 for nonradiolabeled GDP with time (12) (bottom panel).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study we demonstrate for the first time the identification of the subunits of eIF2B to which eIF2 binds and the subunit of eIF2 to which eIF2B binds. We were surprised to find that eIF2B exhibits little or no binding to the alpha -subunit of eIF2 regardless of whether or not the alpha -subunit of eIF2 has been phosphorylated by the eIF2alpha kinase, HCR. Previous studies have shown that eIF2B has a greater affinity for eIF2 phosphorylated by HCR than for the unphosphorylated protein, suggesting that eIF2B might bind to eIF2alpha (4, 5, 14). In two separate studies (5, 14), the affinity of eIF2B for the eIF2·GDP binary complex was determined by kinetic analysis of the guanine nucleotide exchange reaction. In the reported studies, a comparison of the Km value obtained for eIF2·GDP with the KI value for eIF2(alpha P) suggested that the affinity of eIF2B for eIF2(alpha P) was 150-fold higher than that for unphosphorylated eIF2 (5). A study using fluorescence anisotropy to determine the dissociation constants (KD) of the eIF2B·eIF2 and eIF2B·eIF2(alpha P) complexes also found that eIF2B has a higher affinity for eIF2(alpha P) than for unphosphorylated eIF2 although the difference in KD between phosphorylated and unphosphorylated eIF2 was only 2-fold in the presence of GDP (4) rather than the 150-fold difference noted above (5). Furthermore, it was reported that the affinity of eIF2B for eIF2 varied 5-fold as GDP concentration was altered (4). In the present study, it was found that eIF2B had a 3-fold greater affinity for eIF2(alpha P) than for unphosphorylated eIF2, a value closer to that observed using fluorescence anisotropy (4) than was seen using kinetic measurements (5). Although GDP was not included in the buffers in the present study, we have found that as much as 70% of the eIF2 purified from rat liver has GDP bound to it (unpublished observation), which may explain why the value obtained herein is closer to the lower of the two values reported by Goss et al. (4). The simplest explanation for the above observations is that phosphorylation of eIF2alpha results in a conformational change in the eIF2 holoprotein that alters the affinity of eIF2B for eIF2beta . In support of this model, we have found that, like the eIF2B holoprotein, the alpha -subunit of eIF2 binds to eIF2beta ,2 which could provide a basis for a conformational change in eIF2beta in response to phosphorylation of eIF2alpha . A second explanation for the observations is that, in addition to binding to the beta -subunit of eIF2, eIF2B might bind to an area on eIF2 that is shared between the individual subunits and would only be detected when the subunits were present in the holoprotein. Such an interaction would not have been detected by far-Western analysis.

In the present study we also obtained the novel finding that eIF2 binds to the delta - and epsilon -subunits of eIF2B. In related studies in Saccharomyces cerevisiae, Hinnebusch and co-workers (15) have identified point mutations in the alpha -, beta -, and delta -subunits of eIF2B that result in a phenotype characterized by an apparent insensitivity to phosphorylation of eIF2alpha . Based on these results, it was proposed that the alpha -, beta -, and delta -subunits of S. cerevisiae eIF2B perform a regulatory role in recognizing that the alpha -subunit of eIF2 is phosphorylated and that one or more of this group of subunits binds to eIF2. However, of the three subunits, it is unlikely that the primary interaction between eIF2 and eIF2B occurs through binding of eIF2 to the alpha -subunit of eIF2B because cells deleted for eIF2Balpha grow at the same rate as wild-type cells, whereas deletion of any of the other four subunits of eIF2B is lethal (reviewed in Ref. 16). In the model proposed by Hinnebusch (16), the remaining two subunits, i.e. the gamma - and epsilon -subunits, would presumably play a role in catalyzing the guanine nucleotide exchange reaction. The results of the present study confirm the postulate that eIF2 binds to the delta -subunit of eIF2B but do not support a model where eIF2 binds to either the alpha - or beta -subunits of eIF2B.

In summary, the present study reports the identification of the subunits of eIF2 and eIF2B that interact with each other and suggest that eIF2B binds to the C terminus of eIF2beta . The results presented herein both cast doubt on the long held belief that eIF2B binds to the alpha -subunit of eIF2 and require the construction of new models where the predominant interactions between eIF2B and eIF2 occur through the C terminus of the beta -subunit of eIF2 and the delta - and epsilon -subunits of eIF2B.

    ACKNOWLEDGEMENT

We acknowledge the excellent technical assistance of Joan McGwire.

    FOOTNOTES

* This work was supported in part by Grants DK13499 and DK15658 from the National Institutes of Health (to L. S. J.) and Research Grant 195058 from the Juvenile Diabetes Foundation International (to S. R. K.).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.

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

§ Recipient of a Postdoctoral Fellowship from the Juvenile Diabetes Foundation International and supported in part by Training Grant DK07684 from the National Institutes of Health.

1 The abbreviations used are: Met-tRNAi, methionyl-tRNAi; PKA, protein kinase A; PKC, protein kinase C; PVDF, polyvinylidene difluoride; CK-II, casein kinase II; HCR, heme-controlled repressor; GSK-3, glycogen synthase kinase 3.

2 S. R. Kimball, N. K. Heinzinger, R. L. Horetsky, and L. S. Jefferson, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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