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PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2

Madhusudan Dey,^1, Bruce Trieselmann,^1,, Emily G. Locke,¹ Jingfang Lu,¹ Chune Cao,¹ Arvin C. Dar,² Thanuja Krishnamoorthy,¹ Jinsheng Dong,¹ Frank Sicheri,² and Thomas E. Dever^1*

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland,¹ Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada²

Received 8 December 2004/ Returned for modification 10 January 2005/ Accepted 17 January 2005

	ABSTRACT

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Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2 (eIF2) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2, including those at residues flanking Ser51 and around 20 Å away in the conserved motif K₇₉GYID₈₃. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2. Thus, two structurally distinct effectors of eIF2 function, eIF2 kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2.

	INTRODUCTION

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The specificity of signaling pathways requires that protein kinases select their substrate and phosphorylate the appropriate residue from among the myriad of proteins they encounter in a cell. Crystallographic studies have revealed that protein kinase domains fold into a common structure consisting of a smaller N-terminal lobe and a larger C-terminal lobe, with the active site in a cleft between the two lobes (reviewed in references 13 and 18). It is typically thought that kinases recognize their phosphorylation sites at least in part through interactions with the immediately flanking residues, and this notion is supported by the identification of kinase consensus sequences (23, 24). However, sequences remote from the phosphorylation site also have been implicated in substrate recognition. The Jun N-terminal kinases (JNKs) bind to the N terminus of c-Jun (residues 1 to 45) and phosphorylate Ser residues at positions 63 and 73 (15), and a docking site in cyclin A facilitates substrate, and inhibitor, recruitment to the cyclin-dependent kinase cdk2 (1, 35, 39).

Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on Ser51 of its subunit is a common means to control protein synthesis. By juxtaposing different regulatory domains with a conserved eIF2 kinase domain, four eIF2 kinases transduce distinct stress signals to regulate protein synthesis (reviewed in reference 7). Binding of double-stranded RNA (dsRNA) to two dsRNA-binding domains in the N terminus of the mammalian antiviral kinase PKR promotes dimerization and activates the kinase both to autophosphorylate and to phosphorylate eIF2. Dimerization of PKR is necessary for function in vivo and for efficient phosphorylation of eIF2 on Ser51 (4, 40, 43). The kinase HRI is activated under conditions of low heme levels, whereas Perk/PEK senses endoplasmic reticulum stress. Finally, the kinase GCN2, universally conserved in all eukaryotes, responds to amino acid starvation and various other stress conditions (3, 17).

In its active GTP-bound state, eIF2 binds the initiator methionyl-tRNA (Met-tRNA_i^Met), forming the eIF2-GTP-Met-tRNA_i^Met ternary complex, and delivers the Met-tRNA_i^Met to the 40S ribosomal subunit (reviewed in reference 16). Following translation initiation, inactive eIF2-GDP is released from the ribosome. The guanine nucleotide exchange factor (GEF) eIF2B recycles eIF2-GDP to functional eIF2-GTP. Phosphorylation of eIF2 on Ser51 inhibits protein synthesis by converting eIF2 from a substrate to a competitive inhibitor of eIF2B (16). The five subunits of the eIF2B complex can be divided into two groups (reviewed in reference 16). The subunit, encoded by GCD6 in Saccharomyces cerevisiae, catalyzes nucleotide exchange and forms a complex with the structurally related subunit encoded by GCD1 (33). The , ß, and subunits, encoded by GCN3, GCD7, and GCD2, respectively, form a regulatory subcomplex in eIF2B. Removal of the subunit by deletion of GCN3 or specific mutations in any of the three subunits in the regulatory subcomplex desensitizes eIF2B to inhibition by phosphorylated eIF2 (16, 33, 34). In agreement with these in vivo findings, the regulatory subcomplex has been shown to bind directly to eIF2 in a manner dependent on the phosphorylation of Ser51 (27).

While phosphorylation of eIF2 inhibits general protein synthesis, this modification has been exploited by a few mRNAs to paradoxically increase their translation. The GCN4 protein in yeast is a transcriptional activator of a large number of genes, including most of the genes encoding amino acid biosynthetic enzymes (17). The expression of GCN4 is controlled at the translational level by the eIF2 kinase GCN2 and by regulated reinitiation at four upstream open reading frames in the GCN4 mRNA (reviewed in reference 16). When amino acids are plentiful in the medium, GCN2 is inactive, Ser51 on eIF2 is predominantly nonphosphorylated, and GCN4 is not produced. Under amino acid starvation conditions, GCN2 is activated, and it phosphorylates eIF2, resulting in inhibition of eIF2B and increased GCN4 expression (Fig. 1A). This gene-specific translational derepression of GCN4 expression is required for the survival of yeast cells under amino acid starvation conditions. Mutations of Ser51 that block phosphorylation, such as replacement by Ala (eIF2-S51A), impair translational regulation and growth of yeast under starvation conditions.

View larger version (35K): [in this window] [in a new window]   FIG. 1. Model of translational control by eIF2 phosphorylation and conservation of eIF2 structure. (A) Model of the role of eIF2 phosphorylation in GCN4 translational control (16) and yeast cell growth. Phosphorylation of eIF2 on Ser51 converts eIF2 from a substrate to an inhibitor of eIF2B, its GEF, and thus lowers the levels of eIF2-GTP-Met-tRNAiMet ternary complexes. (Left pathway) Starvation of yeast for amino acids leads to accumulation of uncharged tRNAs that bind to and activate the eIF2 kinase GCN2. The modest reduction in ternary complex levels causes derepression of GCN4 mRNA translation, and the GCN4 protein promotes transcription of a large regulon including many of the amino acid biosynthetic genes. Increased expression of the histidine biosynthesis genes in cells expressing high levels of GCN4 confers resistance to the drug 3-AT. (Right pathway) High-level expression of PKR causes significant eIF2 phosphorylation, leading to a large decrease in ternary complex levels and a severe slow-growth or lethal phenotype in yeast. Highlighted in red are the ways in which eIF2 mutations can disrupt the pathways by either blocking Ser51 phosphorylation or preventing inhibition of eIF2B by phosphorylated eIF2. (B) Sequence alignment of various eIF2 proteins and poxvirus eIF2 mimics. The amino acid sequences of the N-terminal one-third of eIF2 from yeast, humans, and flies are aligned with the full-length sequences of the vaccinia virus K3L protein and the swinepox virus C8L protein. Secondary structural elements (arrow, ß-sheet; cylinder, -helix) of the vaccinia virus K3L protein (4) and yeast eIF2 (10) are presented below the sequences. The asterisk indicates the Ser51 phosphorylation site in eIF2 (note that Ser51 is actually residue 52 in eIF2; however, the N-terminal Met is posttranslationally cleaved, at least in mammals, and by convention the Ser is numbered as residue 51). Residues conserved among all of the eIF2 proteins and the K3L and C8L proteins are shown in reverse type and included in the consensus sequence at the bottom of the alignment. Residues conserved only among all of the eIF2 proteins are shaded.

Insights into the mechanism of substrate recognition by the eIF2 kinases have come from studies of a viral pseudosubstrate inhibitor of PKR. The vaccinia virus K3L protein resembles the OB-fold domain in the N-terminal third of eIF2 (Fig. 1B) and blocks PKR activity both in vivo and in vitro (2, 5, 20). Mutational studies on the K3L protein have identified a conserved KGYID sequence motif, located 30 residues C-terminal of Ser51 in eIF2 (see Fig. 1B), as critical for kinase inhibition (4, 20). The crystal structure of the K3L protein revealed that the KGYID motif is part of a conserved patch on the K3L protein surface located 21.5 Å from the phosphorylation site (4). It is anticipated that the corresponding residues in eIF2 may also be important for kinase recognition, as suggested by in vitro binding and kinase assays using an eIF2 variant lacking a portion of this motif (36). Additional support for the notion that residues remote from Ser51 are critical determinants for kinase recognition of eIF2 comes from in vitro kinase assays using peptide substrates versus full-length substrates. A 12-residue peptide centered on Ser51 is a poor substrate for phosphorylation by PKR (K_m for the peptide, 1.08 mM; K_m for eIF2, 0.626 µM) (30), whereas the cAMP-dependent protein kinase PKA efficiently phosphorylates a peptide substrate containing its consensus sequence (see reference 21). In addition, heat denaturation of eIF2 significantly reduces its phosphorylation by HRI (26). Thus, kinase recognition of Ser51 is likely to depend on the proper tertiary structure of eIF2.

In this paper we describe a systematic mutational analysis of eIF2 to identify mutations that block translational regulation. Our data reveal that substrate recognition by the eIF2 kinases requires residues far removed from the phosphorylation site. Interestingly, eIF2 mutations that block Ser51 phosphorylation and mutations that lower the binding of phosphorylated eIF2 to eIF2B map to residues that form a contiguous molecular surface extending more than 20 Å from the Ser51 phosphorylation site. We propose that this conserved surface on eIF2 forms the recognition site for both the eIF2 kinases and eIF2B.

	MATERIALS AND METHODS

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Yeast strains and plasmids. Strain H1643 (MATa ura3-52 leu2-3 leu2-112 trp1-63 sui2 p[SUI2, URA3] GCN4-lacZ, TRP1@TRP1), used for screening eIF2 mutants, has been described previously (9), as has the isogenic gcn2 strain H1925 (41). Yeast strain H2767 (MAT leu2 trp1 ura3-52 prb1-1122 pep4-3 gal2 gcd6 gcd7 p1871[GCD7, GCD2, GCN3, URA3] pTK1.11[GCD1-2xFlag-His, GCD6, LEU2]), overexpressing eIF2B subunits and used for binding assays, has been described previously (27).

The LEU2 low-copy-number plasmids p1097 (9) and pC171, carrying the yeast SUI2 gene encoding eIF2, were used to screen for mutants. Plasmid pC171 was constructed in three steps. First, the BglII site encoding residues 291 to 292 in yeast eIF2 was destroyed by a silent mutation. Second, a BglII site was inserted at the codons for residues Arg56 and Ser57. Third, the modified SUI2 gene was inserted at the BamHI site of a version of the pRS315 vector (37) in which the polylinker was modified to remove all of the sites from SalI to EcoRV. Thus, in pC171 the AvrII site in the SUI2 promoter, the BglII site at Arg56, the SalI site at Val75, and the HindIII site at Glu142 are all unique. Plasmid pC1655 was constructed by first removing the XbaI site from the multiple cloning site in pC171 and then inserting an XbaI site by a single point mutation 12 bases 5' of the eIF2 AUG start site.

The URA3 high-copy-number plasmids p1420 and p1246, encoding human PKR and HRI, respectively (8), pTK4, encoding FLAG- and His-tagged PKR (27), and pDH103, encoding FLAG- and His-tagged GCN2 (11), all under the control of a yeast GAL-CYC1 hybrid promoter, have been described previously. The glutathione S-transferase (GST)-eIF2 expression vector p2565, described previously (27), was constructed by inserting the full yeast eIF2 coding region into the pGEX-2T vector at the BamHI site. Point mutations S51A, RRR_52-54VKA, K79A, and D83A were introduced into p2565 by subcloning an internal HpaI-HindIII fragment encoding eIF2 residues 22 to 143. DNA fragments encoding full-length and C-terminally truncated forms of eIF2 were generated by PCR and subcloned between the BamHI and XbaI or XhoI sites of vectors pEGKT (31) and pGEX-6P (Amersham Biosciences) for expression in yeast and bacteria, respectively. By using the same strategy, PCR products were generated and subcloned into pGEX-6P to express GST-eIF2 mutants with the L35A, I45N, L50S, K79A, Y81S, or D83E mutation in bacteria.

Plasmids pC2252 and pC2256, encoding eIF2 residues 1 to 200 with a polyhistidine tag [His₆-eIF2(1-200)] and His₆-eIF2(1-200)-D83E, respectively, were generated by subcloning PCR products between the NdeI and BamHI sites of vector pET-15b (Novagen).

Mutagenesis and screening. The eIF2 codons for the residues flanking Ser51 and those in the KGYID motif were randomly mutated by PCR. For the residues around Ser51, the 5' oligonucleotide primer included the AvrII site and the 3' primer included the new BglII site at residues 56 and 57. The 3' primer was designed to randomly incorporate all four nucleotides at the three positions specifying a particular codon. To mutate the residues in the KGYID motif, the 5' primer included the SalI site and the mutated codon, and the 3' primer included the HindIII site. Products of the PCRs were digested with the appropriate restriction enzymes and subcloned into pC171. Separate DNA libraries were generated for each randomly mutated residue and were independently transformed into yeast strain H1643. Approximately 250 independent yeast transformants (4 x 64, the number of possible mutants) were selected and replica printed to 5-fluoroorotic acid (5-FOA) medium in order to select for cells that had lost the URA3 plasmid encoding wild-type (WT) eIF2. The 5-FOA plate was incubated for 2 days at 30°C and then replica printed to a 3-aminotriazole (3-AT) plate. From the screen of 250 yeast transformants, the number of 3-AT-sensitive (3-AT^s) mutants isolated at each residue was as follows: 128 at Glu49, 27 at Leu50, 81 at Lys79, 6 at Gly80, 93 at Tyr81, 110 at Ile82, 215 at Asp83, and 74 at Arg88. Plasmids were isolated from the 3-AT^s colonies and retested, and then as many as 40 plasmids from each mutated residue were sequenced. Thus, the screening and sequencing may not have identified all of the 3-AT^s mutations at each residue. The sequence of the entire PCR product was determined to confirm that no additional mutations had been inserted during the PCR procedures.

To randomly mutate eIF2 codons 1 to 48, the appropriate region of the SUI2 gene was amplified by error-prone PCR (Stratagene) under low-stringency conditions (1 to 2 mutations per cycle) and the products were subcloned between the XbaI and BglII sites of plasmid pC1655. Similarly, the region of the SUI2 gene encoding amino acids 89 to 288 was amplified by error-prone PCR and subcloned between the SalI and BglII sites of plasmid p1097. Sequencing of randomly selected clones from these two mutagenesis procedures revealed that 40 to 50% of the plasmids contained a mutation. The eIF2 mutant libraries were screened as described above by transformation into yeast strain H1643. Approximately 3,000 independent yeast transformants were picked, replica printed to 5-FOA medium, and subsequently printed to 3-AT medium as described above. Twenty-four plasmids from the residue-1-to-48 screen and six plasmids from the residue-89-to-288 screen conferred a 3-AT^s phenotype that was confirmed following isolation and retesting of the plasmids. Sequencing of the plasmids identified the mutations G30R, A31T, L35S, M44K, I45N, and E49G from the residue-1-to-48 screen. Because the sequences of the six plasmids from the residue-89-to-288 screen revealed multiple mutations and Western blot analyses using phosphospecific antibodies against Ser51 indicated that the mutations did not interfere with eIF2 phosphorylation, these mutants were not examined further.

eIF2B binding assays. GST-eIF2 fusion proteins were expressed in Escherichia coli BL21(DE3) plus cells (Novagen), and the recombinant proteins were purified as previously described (27). Whole-cell extracts (WCEs) were prepared from a saturated culture of yeast strain H2767 that overexpresses all five eIF2B subunits. Cells were suspended in breaking buffer [40 mM Tris-HCl (pH 7.5), 2 µM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, 15 mM disodium EDTA, 45 mM NaF, 25 mM ß-glycerophosphate, 120 mM (NH₄)₂SO₄, 10 mM 2-aminopurine] and broken by vortexing with glass beads.

GST-eIF2 fusions were phosphorylated by PKR in vitro with 1 mM ATP in 50 µl of kinase buffer (20 mM Tris-HCl [pH 8.0], 50 mM KCl, 25 mM MgCl₂, 1 µM PMSF) at room temperature for 30 min. Phosphorylated eIF2 proteins were immobilized on glutathione-Sepharose beads (Amersham Biosciences; 1 µg of eIF2 per 50 µl of beads [bed volume]) in 950 µl of binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM KCl, 2.5 mM MgCl₂, 0.1 mM disodium EDTA, 1 mM dithiothreitol, 0.1% Triton X-100, 5 mM NaF, 0.1 mM ATP, and 1 protease inhibitor tablet [Roche] per 50 ml of buffer) at 4°C for 1 h. The Sepharose beads were washed, resuspended in 900 µl of the same buffer, mixed with 100 µl (1 mg) of yeast WCE from strain H2767, and incubated for 2 h at 4°C with continuous slow shaking. The beads were collected and washed repeatedly with binding buffer; then bound proteins were eluted with glutathione and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and probed with appropriate antisera.

In vitro kinase assay and kinetics. His₆-eIF2 fusion proteins were purified by nickel-silica resin (QIAGEN) using the manufacturer's protocol, and FLAG-tagged PKR was overexpressed and purified from yeast as described previously (27). Various amounts of His-tagged eIF2 proteins (25 nM to 5 µM) were mixed with FLAG-tagged PKR (2.5 nM) in kinase buffer (20 mM Tris-HCl [pH 8.0], 50 mM KCl, 25 mM MgCl₂, 1 µM PMSF) with 10 µCi of [-³³P]ATP. Reactions were stopped after 20 min by addition of 2x SDS dye (Invitrogen), and products were separated by SDS-PAGE. The relative incorporation of radioactive phosphate into eIF2 was determined by PhosphorImager analysis, and the data were analyzed by using KaleidaGraph.

	RESULTS

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The N-terminal 180 residues of eIF2 are required for kinase recognition. Structural studies revealed that residues 1 to 175 in yeast eIF2 and residues 3 to 182 in human eIF2 (10, 32) form a stable protein consisting of two domains, with an N-terminal OB-fold domain (residues 1 to 89) that resembles the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR (4). Previous in vitro studies demonstrated that the two-domain form of eIF2 (residues 1 to 197) was efficiently phosphorylated by PKR (27). To define the minimal kinase recognition domain in yeast eIF2 in vivo, GST-eIF2 fusion proteins truncated at their N or C termini were expressed in a yeast strain in which the chromosomal eIF2 gene (SUI2) was replaced with the phosphorylation site mutant eIF2-S51A. Cells were treated with 3-AT, an inhibitor of the HIS3 gene product that causes histidine starvation, leading to activation of GCN2. WCEs were subjected to Western blotting using antibodies that recognize eIF2 phosphorylated on Ser51, followed by antibodies that recognize GST (Fig. 2A). C-terminally truncated GST-eIF2(1-200) was expressed and phosphorylated to a similar extent as a full-length fusion protein, GST-eIF2(1-304) (Fig. 2A, lanes 1, 4, 6, and 9); however, the larger C-terminal truncations in GST-eIF2(1-150) and GST-eIF2(1-160) rendered these proteins unstable in yeast (Fig. 2A, lane 10, and 2B, lanes 1 to 4). GST-eIF2 fusions consisting of residues 1 to 190 or 1 to 180 were readily expressed and phosphorylated in a GCN2-dependent manner in yeast (Fig. 2B, lanes 9 to 16). In contrast, while expression of GST-eIF2(1-170) was only slightly impaired, the protein was poorly phosphorylated by GCN2 in yeast (Fig. 2B, lanes 5 to 8). Truncation of 10 residues from the N terminus of eIF2 in GST-eIF2(10-304) reduced Ser51 phosphorylation by GCN2 in vivo (data not shown). However, deletion of the first 10 residues of native (untagged) eIF2 had no effect on yeast cell growth or Ser51 phosphorylation. Truncation of 15 residues from the N terminus of eIF2 was lethal in yeast (data not shown). Taking these findings together, we conclude that both the OB-fold and -helical domains of eIF2, residues 10 to 180, are required for protein stability and efficient Ser51 phosphorylation in vivo. Confirming these in vivo results, in vitro kinase assays using PKR revealed efficient phosphorylation of recombinant GST-eIF2(1-180) but not GST-eIF2(1-170) or GST-eIF2(1-160) (Fig. 2C). Finally, replacement of Ser51 by Ala in GST-eIF2(1-180) blocked phosphorylation by PKR in these in vitro assays, confirming the specificity of the in vitro kinase assays (Fig. 2D). These results support our in vivo finding that GCN2 requires the N-terminal 180 residues of eIF2 for Ser51 phosphorylation.

View larger version (46K): [in this window] [in a new window]   FIG. 2. eIF2 residues 1 to 180 are necessary and sufficient for efficient phosphorylation of Ser51 both in vivo and in vitro. (A) The C-terminal half of eIF2 is not required for Ser51 phosphorylation in vivo. GST-eIF2 fusion proteins with the indicated C-terminal residue were expressed under the control of a galactose-inducible promoter in the yeast strain H2653, in which the chromosomal SUI2 gene encoding eIF2 contains the nonphosphorylatable mutation S51A. WCEs were prepared from cells treated with 3-AT to activate GCN2, and proteins were separated by SDS-PAGE and analyzed by Western blotting using antibodies specific for the Ser51-phosphorylated form of eIF2 (left) (Quality Controlled Biochemicals, Inc.), as described previously (43). The membrane was stripped and probed with anti-GST antisera to analyze the expression of the GST-eIF2 fusion proteins (right). (B) Fine mapping of eIF2 residues required for Ser51 phosphorylation in vivo. The indicated GST-eIF2 fusion proteins were expressed under the control of a galactose-inducible promoter in SUI2-S51A yeast strains either containing or lacking GCN2 as indicated. WCEs, in 1x and 10x amounts, were separated by SDS-PAGE and analyzed by Western blotting for Ser51 phosphorylation and total GST-eIF2 expression as described for panel A. (C) eIF2 residues 1 to 180 are required for efficient phosphorylation of Ser51 in vitro. The indicated GST-eIF2 fusion proteins were purified from bacteria and incubated with recombinant human PKR purified from yeast and [-33P]ATP for the indicated times. Reaction mixtures were resolved by SDS-PAGE, proteins were detected by Coomassie staining (lower panels), and eIF2 phosphorylation was analyzed by autoradiography (upper panels). (D) PKR phosphorylation of eIF2 in vitro is dependent on Ser51. GST-eIF2(1-180) and GST-eIF2-S51A(1-180) fusion proteins were prepared and utilized for in vitro kinase assays with human PKR as described for panel C.

Identification of mutations in eIF2 that impair translational control by GCN2.

As detailed in Materials and Methods, the codons for residues 48 to 50 and 52 to 54 in a plasmid-borne SUI2 allele, encoding yeast eIF2, were independently subjected to random mutagenesis. Pools of the mutated plasmids were introduced into the sui2 yeast strain H1643 as the sole source of eIF2 by plasmid shuffling, and the cells were tested for defects in GCN4 expression. Translational derepression of GCN4 is essential for growth of yeast under amino acid starvation conditions. Yeast strains lacking the GCN2 kinase or expressing the nonphosphorylatable eIF2-S51A mutant are unable to grow on a medium containing 3-AT (Fig. 1A) (9). Thus, the inability to grow on a medium containing 3-AT is a simple screen for identification of eIF2 mutants that impair translational regulation by GCN2.

No 3-AT^s mutants were isolated among the SUI2 alleles mutated at residues Ser48, Arg52, Arg53, and Arg54. To test the possibility that the three Arg residues play redundant roles in specifying phosphorylation of Ser51 by GCN2, an eIF2 triple mutant with R52V, R53K, and R54A (RRR_52-54VKA) was constructed. Each single-site mutant maintained the WT 3-AT-resistant (3-AT^r) phenotype, as was also found for the triple mutant (Fig. 3A). Mutations that lower eIF2 activity confer 3-AT^r phenotypes independently of GCN2 and eIF2 phosphorylation (14, 42). In cells lacking GCN2, the RRR_52-54VKA mutant conferred a weak 3-AT^r phenotype; however, growth on 3-AT medium was significantly better in cells containing GCN2 (Fig. 3A). These results, and complementary findings from GCN4-lacZ reporter assays (data not shown), are consistent with the notion that the RRR_52-54VKA mutation lowers eIF2 activity but the mutant protein remains a substrate for phosphorylation by GCN2.

View larger version (67K): [in this window] [in a new window]   FIG. 3. eIF2 mutations near and remote from Ser51 impair GCN4 translational control. Effects of eIF2 mutations either in residues flanking Ser51 (A), in residues conserved among all eIF2 and K3L protein homologs (B), or isolated in random screens (C) on amino acid analog sensitivity as a measure of GCN4 translational regulation. Yeast cells expressing the indicated eIF2 alleles were grown to confluence on a synthetic dextrose (SD) plate and replica plated to a 3-AT plate (30 mM) and an SD plate, as indicated. Plates were incubated for 3 days at 30°C. Where indicated, the yeast strain lacked the GCN2 kinase (gcn2).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Summary of screen to identify eIF2 mutants with defects in GCN4 translational control, eIF2 phosphorylation, or eIF2B binding. The indicated eIF2 residues were subjected to site-directed or random mutagenesis, and the mutated alleles were screened in yeast to identify mutations that blocked growth on 3-AT medium and thus eliminated GCN4 regulation (second column). Yeast strains expressing the eIF2 alleles that blocked growth on 3-AT medium were also tested for phosphorylation of Ser51 (see Fig. 5), and mutations that eliminated or reduced Ser51 phosphorylation in vivo are listed in the third column. Mutations that conferred a 3-ATs phenotype but did not impair Ser51 phosphorylation were tested for eIF2B binding (see Fig. 7), and mutations that eliminated or reduced eIF2B binding are listed in the last column. Mutations previously reported (27) to impair the binding of eIF2B to phosphorylated eIF2 are also listed (italicized and underlined) in the last column.

In addition to these site-specific mutagenesis protocols, two regions of the SUI2 gene, encoding eIF2 residues 1 to 48 and 89 to 288, were independently mutated by error-prone PCR as described in Materials and Methods. Pools of the mutated plasmids were introduced into the sui2 yeast strain H1643 by plasmid shuffling, and the resulting strains were tested for defects in GCN4 expression. Five eIF2 mutations, G30R, A31T, L35S, M44K, and I45N, were found to confer a 3-AT^s phenotype (Fig. 3C and 4).

A subset of the eIF2 mutations impairs GCN2 phosphorylation of Ser51. Mutations in eIF2 that disrupt translational control of GCN4 could affect either the ability of GCN2 to phosphorylate Ser51 or the ability of phosphorylated eIF2 to inhibit eIF2B (Fig. 1A). Thus, all of the eIF2 mutants with 3-AT^s phenotypes identified in Fig. 4 were screened for their effects on Ser51 phosphorylation. Western blot analyses of WCEs were performed by using phosphospecific antibodies against eIF2 phosphorylated on Ser51 as well as polyclonal eIF2 antibodies. As shown in Fig. 5, Ser51 phosphorylation in cells expressing WT eIF2 increased when cells were treated with 3-AT (Fig. 5A, lane 2 versus lane 1) and was fully dependent on GCN2 (Fig. 5A, lane 4 versus lane 2) and Ser51 (Fig. 5B, lane 2). The eIF2 mutations G30R, A31T, M44K, Y81V, and D83A nearly abolished Ser51 phosphorylation (Fig. 4 and 5B, lanes 3, 4, 6, 11, and 12). Consistent with the 3-AT^r phenotype in cells expressing the RRR_52-54VKA triple mutant, Ser51 phosphorylation was stimulated in a GCN2-dependent manner in cells treated with 3-AT (Fig. 5A, lanes 7 to 10). We observed that mutations at residues Glu49 and Leu50 completely eliminated detection of eIF2 by the Ser51 phosphospecific antibodies, including the nonspecific detection of unphosphorylated eIF2 in gcn2 strains (data not shown), indicating that the primary epitope for the antibodies includes the residues N-terminal to the phosphorylation site. Thus, a second assay, isoelectric focusing (IEF) gel electrophoresis (9), was used to monitor the effects of the Glu49 and Leu50 mutations on Ser51 phosphorylation. As shown in Fig. 5C, the abundance of the Ser51-phosphorylated isoform of eIF2 increased in cells treated with 3-AT (compare lanes 1 and 2). The E49R and L50P mutations greatly reduced and almost completely eliminated phosphorylation of Ser51, respectively (Fig. 5C, lanes 4 and 10). In contrast, the E49V and L50S mutations did not impair Ser51 phosphorylation (Fig. 5C, lanes 6 and 8). It is noteworthy that all of the 3-AT^s mutants in eIF2 had a recessive phenotype (data not shown), indicating that the eIF2 mutants did not function as dominant inhibitors of GCN2.

View larger version (32K): [in this window] [in a new window]   FIG. 5. A subset of the eIF2 mutations impairs Ser51 phosphorylation. (A and B) Immunoblot analysis using eIF2 Ser51 phosphospecific antibodies. WCEs were prepared from strains expressing the indicated eIF2 alleles. The strains were grown in SD minimal medium or in SD medium supplemented with 10 mM 3-AT (lanes marked with plus signs in panel A; all samples in panel B), where the GCN2 kinase is activated. Total proteins were subjected to immunoblot analysis with antibodies against Ser51-phosphorylated eIF2 (upper panels) as described for Fig. 2A. The blot was stripped and reprobed with a polyclonal antiserum against total yeast eIF2 (lower panels). This polyclonal antiserum was raised against the C-terminal half of yeast eIF2, far from the Ser51 phosphorylation site, and thus should detect unphosphorylated and Ser51-phosphorylated forms of yeast eIF2 equally well. (C) IEF analysis of eIF2 phosphorylation. WCEs were prepared from yeast expressing the indicated eIF2 alleles and were grown as described for panel A. Total proteins were separated by IEF on a vertical slab gel, and eIF2 was detected by immunoblot analysis using the polyclonal anti-yeast eIF2 antiserum, as described previously (9). As indicated, the eIF2 isoform phosphorylated on Ser51 focuses higher in the gel than the basal form. (D) eIF2 mutations impair Ser51 phosphorylation by GCN2 in vitro. GCN2 bearing N-terminal FLAG and polyhistidine tags was overexpressed and purified from yeast as described previously (11). The GCN2 (0.25 µg) was mixed with [-33P]ATP and 0.5 µg of the indicated recombinant GST-eIF2 fusion protein purified from bacteria. The kinasebuffer and reaction conditions were identical to those described previously (11). Reaction mixtures were resolved by SDS-PAGE, proteins were detected by Coomassie staining (lower panel), and eIF2 phosphorylation was analyzed by autoradiography (upper panel).

The results of the comprehensive analysis of Ser51 phosphorylation for all of the eIF2 mutants are summarized in Fig. 4 (third column). Interestingly, only 5 of the 19 3-AT^s mutations at Glu49 and only 1 of 10 mutations at Leu50 impaired Ser51 phosphorylation. Similarly, only a subset of the mutations at Lys79, Tyr81, Ile82, and Arg88 reduced Ser51 phosphorylation (Fig. 4). Asp83, 32 residues from the Ser51 phosphorylation site, appeared to be the most critical determinant for phosphorylation. Nearly all mutations at Asp83 impaired GCN4 expression and Ser51 phosphorylation. Only the conservative substitution of Glu for Asp83 did not block phosphorylation, suggesting that the eIF2 kinases require an acidic charge at this position for efficient substrate recognition.

Translational regulation and phosphorylation of Ser51 by the four eIF2 kinases are dependent on the same determinants in eIF2. To determine whether the other three eIF2 kinases utilized the same eIF2 determinants as GCN2 for substrate recognition, we expressed human PKR or HRI or Caenorhabditis elegans Perk/PEK under the control of a galactose-inducible promoter in a gcn2 yeast strain expressing various forms of eIF2. High-level expression of PKR or Perk/PEK on galactose medium was lethal, while high-level expression of HRI conferred a 3-AT^r phenotype in cells expressing WT eIF2 (Fig. 6A). The PKR and Perk/PEK toxicity, and the HRI phenotype, were suppressed in cells expressing nonphosphorylatable eIF2-S51A, eIF2-D83A, or eIF2-D76A,Y81Q (Fig. 6A). In contrast, the growth of yeast expressing the eIF2-RRR_52-54VKA mutant was severely inhibited by PKR or Perk/PEK (Fig. 6A), consistent with the ability of GCN2 to phosphorylate this mutant. The correlation between the impact of these eIF2 mutations on GCN2 phosphorylation and PKR, Perk/PEK, and HRI suppression suggests that all four kinases recognize the same determinants on eIF2.

View larger version (28K): [in this window] [in a new window]   FIG. 6. The kinases PKR, Perk/PEK, HRI, and GCN2 rely on the same eIF2 determinants for substrate recognition. (A) The eIF2 D83A mutation suppresses PKR, Perk/PEK, HRI, and GCN2 phenotypes in yeast. Plasmid p722, bearing GCN2, and plasmids p1420, p1246, and p551 (38), expressing human PKR, human HRI, and C. elegans PEK/Perk (residues 26 to 1077), respectively, under the control of a yeast GAL-CYC1 hybrid promoter, were introduced into derivatives of the gcn2 yeast strain H1925 expressing the indicated eIF2 alleles. Transformants were grown to saturation in minimal SD (2% glucose) medium, and 4 µl of serial dilutions (optical density at 600 nm, 1.0 and 0.1) was spotted onto minimal SD or SGal (10% galactose) medium or the same medium containing 10 mM 3-AT, as indicated. Plates were incubated at 30°C for 6 days. (B) Immunoblot analysis of eIF2 phosphorylation in yeast expressing PKR. The yeast strain H1925 expressing the indicated eIF2 alleles was transformed with the PKR expression vector p1420, and two independent transformants of each strain were grown to exponential phase in SDmedium and then shifted to SGR medium (containing 10% galactose plus 2% raffinose) for 18 h to induce PKR expression. WCEs were prepared, and total proteins were subjected to immunoblot analysis with antibodies against Ser51-phosphorylated eIF2 (upper panel) as described for Fig. 2A. The blot was stripped and reprobed with a polyclonal antiserum against total yeast eIF2 (lower panel). (C) In vitro kinase assay of PKR phosphorylation of eIF2. Purified recombinant PKR was mixed with [-33P]ATP and the indicated recombinant GST-eIF2 fusion proteins purified from bacteria. Kinase reactions were resolved by SDS-PAGE, and gels were stained with Coomassie blue to detect GST-eIF2 (bottom panel), followed by autoradiography to visualize phosphorylated eIF2 (upper panel). (D) Kinetic analysis of PKR phosphorylation of eIF2 and eIF2 D83E. His6-eIF2(1-200) and His6-eIF2-D83E fusion proteins were purified from bacteria and examined for phosphorylation by purified PKR as described in Materials and Methods. Reaction products were resolved by SDS-PAGE, and the relative incorporation of phosphate into eIF2 was determined by using a PhosphorImager. The data are expressed in arbitrary units. Results are representative of at least three independent experiments.

A subset of eIF2 regulatory mutations impairs binding of eIF2B to phosphorylated eIF2. As shown in Fig. 5 and summarized in Fig. 4, several eIF2 mutations eliminated GCN4 translational regulation but retained Ser51 phosphorylation. We hypothesized that these mutations prevented phosphorylated eIF2 inhibition of eIF2B (Fig. 1A). The gcn3^c-R104K mutation in the (regulatory) subunit of yeast eIF2B inhibits eIF2B function in the absence of eIF2 phosphorylation and is thought to mimic the impact of phosphorylated eIF2 on eIF2B activity. Previously identified eIF2 mutations (e.g., L84F) that impaired translational regulation but not Ser51 phosphorylation were found to suppress the toxic effects of the gcn3^c-R104K mutation on yeast cell growth (41). The eIF2 mutations L35A, I45N, K79A, Y81S, and D83E, identified in this study as blocking GCN4 translational control but not Ser51 phosphorylation, were found to suppress the slow-growth phenotype associated with the gcn3^c-R104K mutation (Fig. 7A), consistent with the idea that these mutations alter the inhibition of eIF2B by phosphorylated eIF2. In contrast, the eIF2-S51A mutation, which prevents Ser51 phosphorylation, was unable to suppress the gcn3^c-R104K mutation (Fig. 7A). Notably, the E49V and L50S mutations, immediately adjacent to the phosphorylation site, failed to suppress the gcn3^c-R104K mutation, suggesting that the residues adjacent to Ser51 may not interact directly with eIF2B (GCN3). To directly examine the binding of eIF2 to eIF2B, we introduced the L35A, I45N, L50S, K79A, Y81A, and D83E mutations into a GST-eIF2(1-304) construct. Previously, it was shown that GST-eIF2 bound native or overexpressed eIF2B in yeast WCEs in a manner dependent on Ser51 phosphorylation and sensitive to mutations in eIF2 (27). The GST-eIF2 WT and mutant proteins indicated in Fig. 7 were purified from bacteria, phosphorylated by PKR in vitro, immobilized on glutathione-Sepharose beads, and then incubated with WCEs from yeast overexpressing all five eIF2B subunits. Western blot analysis of the bound proteins demonstrated the PKR (Ser51 phosphorylation)-enhanced binding of eIF2B subunits GCD1, GCD2, GCD6, and GCD7 to WT GST-eIF2 (Fig. 7B and C; compare lanes 3 and 5). In contrast, the eIF2 mutations L35A, I45N, L50S, K79A, Y81S, and D83E impaired the binding of eIF2B subunits to GST-eIF2 (Fig. 7B, lanes 6 to 9, and 7C, lanes 6 to 13). Western blot analyses using phospho-Ser51-specific antibodies confirmed that the eIF2 mutations did not interfere with phosphorylation (Fig. 7B and C). Since the L50S mutation disrupted the epitope for the phospho-Ser51 antibodies, kinase assays using [-³³P]ATP demonstrated efficient phosphorylation of this mutant (Fig. 7D). We conclude that the eIF2 mutations that abolish GCN4 regulation but not Ser51 phosphorylation weaken the interaction of phosphorylated eIF2 with eIF2B. Consistent with this conclusion, we propose that the mutations prevent the inhibition of eIF2B by phosphorylated eIF2.

View larger version (58K): [in this window] [in a new window]   FIG. 7. eIF2 mutations near and remote from Ser51 impair eIF2B binding to phosphorylated eIF2. (A) Effects of selected eIF2 mutations on amino acid analog sensitivity and growth rate in yeast expressing WT GCN3 (left panels) or the gcn3c-R104K mutant (right panel). (Left panels) Transformants of yeast strain H1643 expressing the indicated eIF2 alleles were grown to confluence on an SD plate and replica plated to a 3-AT plate (30 mM) and an SD plate. Plates were incubated for 3 days at 30°C. (Right panel) Derivatives of yeast strain H1658 (MAT ura3-52 leu2-3 leu2-112 ino– trp1-63 sui2 gcn3c-R104K p[SUI2, URA3] HIS4-lacZ, ura3-52) expressing the indicated eIF2 mutants were generated by plasmid shuffling, and the resulting strains were tested for growth on SD medium by spotting 10-fold serial dilutions of a saturated culture and incubating for 3 days at 30°C. As a control, WT GCN3 on a low-copy-number plasmid (GCN3+) was introduced into the strain bearing WT eIF2 to complement the recessive gcn3c-R104K mutation. (B and C) Mutations of eIF2 residues near and remote from Ser51 impair binding of eIF2B subunits to phosphorylated eIF2. WT GST-eIF2 and the indicated mutant derivatives were purified from bacteria and incubated with (+) or without (–) PKR in kinase buffer. Two different amounts of GST-eIF2 fusion proteins (0.1 and 1.0 µg) were immobilized on glutathione-Sepharose beads and then incubated with WCEs from yeast overexpressing all five eIF2B subunits. After extensive washing, bound proteins were resolved by SDS-PAGE and analyzed by Western blotting using antibodies against GCD1, GCD2, GCD6, GCD7, and Ser51-phosphorylated eIF2, as indicated. The eIF2 blot was stripped and reprobed with polyclonal antisera against total yeast eIF2. Input lanes contained 10% of the WCEs used in each reaction. (D) Efficient phosphorylation of eIF2-L50S by PKR in vitro. The indicated WT and mutant GST-eIF2 fusion proteins were purified from bacteria and incubated with recombinant PKR and [-33P]ATP. Reaction mixtures were resolved by SDS-PAGE, proteins were detected by Coomassie staining (lower panels), and eIF2 phosphorylation was analyzed by autoradiography (upper panels).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The eIF2 kinases recognize a large contiguous surface composed of residues near and remote from the Ser51 phosphorylation site. Biochemical studies revealed that PKA readily phosphorylated denatured substrates, proteolytic fragments derived from substrates, and model peptides derived from pyruvate kinase (Kemptide) with nearly equal efficiencies (see references 21, 23, and 24), indicating that the residues flanking the phosphorylation site, and not the context of a larger structural fold, are the principal determinants for substrate selection by PKA. In contrast to these findings, the eIF2 kinases PKR and HRI function inefficiently with peptide substrates compared with intact eIF2 (30), and our data demonstrate that residues remote from Ser51 are critical for kinase recognition. Previously, we found that both PKR and HRI phosphorylated Tyr in place of Ser51 in eIF2 (28). We propose that PKR and HRI interact with high affinity and stringency with the remote KGYID motif, which then orients eIF2 residue 51 into the kinase active site. A flexible active site enables any phosphoaccepting amino acid at residue 51 to be phosphorylated by the eIF2 kinases. In contrast to PKA, where denaturation of proteins enhanced phosphorylation (24), perhaps by exposing previously buried consensus sequence motifs, denaturation of eIF2 severely impaired phosphorylation (26). These results are consistent with the notion that the 3D structural integrity of eIF2 is important for kinase recognition.

Further support for the importance of the eIF2 tertiary structure for kinase recognition comes from comparison of the structures of eIF2 and the vaccinia virus K3L protein (Fig. 1B). The crystallographic structure of the K3L protein revealed an OB-fold composed of a five-stranded ß-barrel (4). The structure of the N-terminal two-thirds of yeast eIF2 revealed a similar OB-fold for residues 1 to 89, followed by a helical domain consisting of residues 90 to 175 (Fig. 8A) (10). Residues 47 to 63 in eIF2, including the phosphorylation site at Ser51, are in a large loop connecting strands ß3 and ß4. In agreement with our finding that mutations in the KGYID motif impaired Ser51 phosphorylation, mutations in the corresponding residues of the K3L protein, as well as the homologous C8L protein of swinepox virus, impaired PKR binding and kinase inhibition (4, 19, 20). These results indicate that the K3L protein and eIF2 bind in a similar manner to PKR with multiple contacts, and they support the idea that the 3D structure of these proteins is an important determinant for kinase recognition.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Locations of the mutations affecting Ser51 phosphorylation on the eIF2 structure, and model for eIF2 kinases and eIF2B recognizing overlapping surfaces on eIF2. (A) Ribbon diagram of yeast eIF2 (PDB code 1Q46; residues 2 to 175). Ser51 is shown in red; residues M44, K79, Y81, and D83, mutations of which impair Ser51 phosphorylation, are shown in green. (B) Ribbon diagram of a PKR protein kinase domain model. The PKR protein kinase domain homology model was generated with SWISS-MODEL (12) and is based on structures of the cyclic AMP-dependent protein kinase (PDB codes 2CPK, 1BKX, 1FMO, 1ATP, and 1BX6). The model coordinates (human PKR residues 268 to 330 and 360 to 524) were generated without refinement. Yellow, peptide substrate; blue, ATP; beige, helical residues of the PKA inhibitor PKI. (C) Surface representation of eIF2. Red, the Ser51 phosphorylation site; green, residues conserved amongeIF2 and K3L-like proteins. The arrow indicates the distance between the Ser51 phosphorylation site and Tyr81, at the center of the conserved surface region. (D) Surface representation of the PKR model. Residues in green are conserved among eIF2 kinases. A 20.5-Å vector projecting from the phosphoacceptor residue (red) within the peptide substrate (yellow) is shown. Blue, ATP; beige, PKI helical residues. (E) Surface representation of eIF2 highlighting residues (orange) that are critical for kinase recognition and Ser51 phosphorylation. Ser51 is shown in red. (F) Surface representation of eIF2 highlighting residues (purple) important for binding and inhibition of eIF2B by phosphorylated eIF2. All ribbon diagrams and surface models were generated with PyMOL, version 0.9 (6). (G and H) Model depicting eIF2 kinase (red) and eIF2B (blue) recognition of eIF2 (green). The eIF2 Ser51 and KGYID residues are labeled in magenta, with the flanking residues in black; phosphorylated Ser51 is indicated by the letter P, in red.

In analogy with the domain in c-Jun and the Cy motif in cyclin A-cdk2 substrates, the KGYID motif in eIF2 may function as an eIF2 kinase docking site. Two possibilities can be considered: either (i) the eIF2 kinases first bind to the KGYID motif and then translocate to Ser51 or (ii) the KGYID motif and Ser51 are part of a contiguous kinase recognition surface on eIF2. Several findings favor the latter model. First, the remarkable structural similarity between the K3L protein and eIF2 is consistent with recognition by the kinases of a particular 3D structure encompassing both Ser51 and the KGYID motif. If the KGYID motif were a docking site, then conservation of just this motif in the K3L protein might be expected to be sufficient for PKR inhibition. However, mutations in the region of the K3L protein corresponding to Ser51 in eIF2 have been shown to alter PKR binding and inhibition (4, 20). Second, the Ser51 region, the KGYID motif, and kinase determinants Gly30 and Ala31 in strand ß-2 and Met44 in strand ß-3 form a contiguous surface on one face of eIF2 (Fig. 8A and E), consistent with the idea that the kinases contact this entire face of eIF2. Third, in the K3L protein and eIF2 structures, the KGYID motif is located 21.5 Å from Ser51 (or its equivalent in the K3L protein) (4, 10). As described below, this spacing is compatible with the possibility that the kinases contact both regions simultaneously.

Structures of PKA (Fig. 8B and D), phosphorylase kinase (PhK), and the insulin receptor kinase (IRK) bound to a peptide substrate or pseudosubstrate inhibitor revealed that the substrate residues flanking the phosphorylation site interacted with the activation segment located between subdomains VII and VIII (between helices E and F) in the C-terminal lobe of each kinase (see references 18 and 22) (Fig. 8B). This may represent a general mode of substrate recognition by protein kinases, wherein the residues flanking the phosphorylation site are recognized by specific interactions with the residues in the activation loop of the kinase. The structure of PKA bound to its inhibitor peptide, PKI (25, 44), revealed additional interactions between an -helical region in PKI and a binding groove in the C-terminal lobe of the kinase (Fig. 8B) (see reference 22). Interestingly, the distance between the -helix in PKI and the phosphorylation site is about the same as the distance between the KGYID motif and Ser51 in eIF2 (Fig. 8C and D), suggesting a potential binding site for the KGYID motif on PKR. The OB-fold of eIF2 can be easily slid into the position of PKI in the PKA-PKI structure, and in this superposition the KGYID motif of eIF2 overlies the -helix of PKI and Ser51 is positioned in the active site of the kinase. Thus, we propose that PKR and the other eIF2 kinases recognize a contiguous surface on eIF2 extending from the KGYID motif to the vicinity of the Ser51 phosphorylation site (Fig. 8E and G).

Overlapping surfaces on eIF2 mediate eIF2 kinase recognition and eIF2B inhibition. The identification of eIF2 mutations that block translational regulation but do not impair Ser51 phosphorylation indicates that these mutations likely prevent inhibition of the GEF eIF2B by phosphorylated eIF2. The regulatory subcomplex of eIF2B composed of the , ß, and subunits (GCN3, GCD7, and GCD2 in yeast) binds to intact eIF2 as well as the isolated eIF2 subunit (27, 33). Binding of the regulatory subcomplex to eIF2 is enhanced by phosphorylation of Ser51 (27). The eIF2 mutations I58M, L84F, R88C, and V89I alleviate the slow-growth phenotype associated with hyperactive GCN2^c alleles in yeast (41). The GCN2^c kinases phosphorylate a large fraction of the eIF2 in the cell, resulting in elevated GCN4 expression and impaired cell growth due to inhibition of general protein synthesis. Whereas the R88C mutation appeared to impair Ser51 phosphorylation (41), the other eIF2 mutations apparently blocked the ability of phosphorylated eIF2 to inhibit eIF2B. In agreement with the latter model, Krishnamoorthy et al. (27) showed that SUI2 mutations (including the E49N, E49Q, K79A, G80A, and R88T mutations described in this report) that suppress the inhibition of eIF2B by phosphorylated eIF2 weaken the binding of GST-eIF2P (GST-eIF2 phosphorylated on Ser51) to the eIF2Bß regulatory subcomplex. In this report we extend these findings and demonstrate that the eIF2 mutations L35A, I45N, L50S, Y81A, and D83E impair eIF2B binding to phosphorylated eIF2 (Fig. 7B and C). We propose that the eIF2 mutations weaken the interaction between phosphorylated eIF2 and the eIF2Bß regulatory subcomplex, enabling a productive interaction between eIF2ß and the eIF2B catalytic subcomplex that triggers GTP-GDP exchange.

Of particular note regarding the two classes of eIF2 mutations identified in this report is the finding that both types of mutations have been found at the same residue in eIF2. For example, the D83A mutation impaired Ser51 phosphorylation (Fig. 5 and 6), whereas the D83E mutation did not prevent Ser51 phosphorylation but instead blocked eIF2B binding (Fig. 5 to 7). Thus, the same residues in eIF2 contribute to both eIF2 kinase recognition and eIF2B inhibition. As described above, the Ser51 region and the KGYID motif are located on the same face of eIF2, and as illustrated in Fig. 8E to H, we propose that this face forms the docking site for both the kinases and eIF2B. Future experiments will be aimed at defining the residues in the eIF2 kinases responsible for eIF2 recognition and Ser51 phosphorylation, as well as the residues in the eIF2Bß regulatory subcomplex that recognize phospho-Ser51 on eIF2.

	ACKNOWLEDGMENTS

 
We thank Weimin Yang for the GST-eIF2 plasmid. We also thank present and past members of the Dever and Hinnebusch labs for assistance and helpful discussions, and Alan Hinnebusch for comments on the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: National Institutes of Health, 6 Center Dr., Bethesda, MD 20892-2427. Phone: (301) 496-4519. Fax: (301) 496-8576. E-mail: tdever{at}box-t.nih.gov.

M.D. and B.T. contributed equally to this work.

Present address: Durham College, Oshawa, Ontario L1H 7L7, Canada.

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