Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation.

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2a) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2a phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2at subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2ao kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2ot. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2ot 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2a, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2oa impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2a and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2ot inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.In the initiation step of protein synthesis in eukaryotic cells, the joining of the 48S preinitiation complex with the 60S subunit to form the 80S initiation complex is accompanied by the hydrolysis of GTP and the formation of a binary complex of eukaryotic initiation factor 2 (eIF-2) and GDP (eIF-2 GDP) (reviewed in references 15 and 29). The exchange of GTP for GDP in the eIF-2 * GDP complex is catalyzed by a multimeric protein factor called eIF-2B (previously called reversing factor or GEF) (1,22,26,33,41). The guanine nucleotide exchange is a prerequisite for eIF-2 to enter another initiation cycle and bind the initiator tRNA, MettRNAi. eIF-2 is a heterotrimer composed of the a, P, and y subunits. The activity of eIF-2 is an important control point in the regulation of polypeptide chain initiation and is regulated at least i...

Section: Resultsmentioning

confidence: 99%

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

Konakanchi¹,

Davies²,

Chen³

et al. 1994

“…This finding suggests that the GCN2-independent pathway accessed by tRNA al* derepresses GCN4 translation by decreasing eIF-2 function. This could involve phosphorylation of the P or y subunit of eIF-2, or of one of the subunits of eIF-2B itself, for which there is some evidence from animal systems (10,53). Another possibility is that some other initiation factor that interacts with eIF-2 and is required for reinitiation on GCN4 mRNA is down-regulated by the GCN2-independent derepression mechanism in the presence of uncharged tRNA.…”

Section: Discussionmentioning

confidence: 99%

Multicopy tRNA genes functionally suppress mutations in yeast eIF-2 alpha kinase GCN2: evidence for separate pathways coupling GCN4 expression to unchanged tRNA.

Aldana¹,

Wek²,

Segundo³

et al. 1994

GCN2 is a protein kinase that stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating the a subunit of translation initiation factor 2 (eIF-2). We isolated multicopy plasmids that overcome the defective derepression of GCN4 and its target genes caused by the leaky mutation gcn2-507. One class of plasmids contained tRNAHIS genes and conferred efficient suppression only when cells were starved for histidine; these plasmids suppressed a gcn2 deletion much less efficiently than they suppressed gcn2-507. This finding indicates that the reduction in GCN4 expression caused by gcn2-507 can be overcome by elevating tRNAHIS expression under conditions in which the excess tRNA cannot be fully aminoacylated. The second class of suppressor plasmids all carried the same gene encoding a mutant form of tRNAVal(AAC) with an A-to-G transition at the 3' encoded nucleotide, a mutation shown previously to reduce aminoacylation of tRNAVU' in vitro. In contrast to the wild-type tRNAHiS genes, the mutant tRNAVaI gene efficiently suppressed a gcn2 deletion, and this suppression was independent of the phosphorylation site on eIF-2a (Ser-51). Overexpression of the mutant tRNAVaI did, however, stimulate GCN4 expression at the translational level. We propose that the multicopy mutant tRNAVal construct leads to an accumulation of uncharged tRNAV2I that derepresses GCN4 translation through a pathway that does not involve GCN2 or eIF-2a phosphorylation. This GCN2-independent pathway was also stimulated to a lesser extent by the multicopy tRNAHIS constructs in histidine-deprived cells. Because the mutant tRNAval exacerbated the slow-growth phenotype associated with eIF-2a hyperphosphorylation by an activated GCN2C kinase, we suggest that the GCN2-independent derepression mechanism involves down-regulation of eIF-2 activity.

“…This leads to tight binding of eIF-2B in an inactive complex with phosphorylated eIF-2 (29,32,44). Interestingly, it was also reported that the guanine nucleotide exchange activity of eIF-2B decreases in response to amino acid starvation in rabbit reticulocytes (16) and that changes in the phosphorylation state of one of the subunits of eIF-2B alters its function (10). Given that GCN2, one of the positive regulators of GCN4 translation, is a protein kinase (51), perhaps moderate phosphorylation of eIF-2ot or one of the subunits of the GCD1-containing complex is responsible for reducing the rate of reinitiation at uORFs 3 and 4 by one of the mechanisms described.…”

Section: Figmentioning

confidence: 99%

Complex formation by positive and negative translational regulators of GCN4.

Cigan¹,

Foiani²,

Hannig³

et al. 1991

117

108

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2, and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCI. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2a, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.The GCN4 protein of the yeast Saccharomyces cerevisiae is a transcriptional activator of amino acid biosynthetic genes that are subject to general amino acid control. Transcription of these genes is stimulated by GCN4 in response to starvation for any amino acid. The expression of GCN4 itself is regulated by amino acid availability, but at the translational level. Four short open reading frames (uORFs) in the long leader sequence of GCN4 mRNA function as cis-acting regulatory elements that couple the rate of GCN4 translation to amino acid levels. Under nonstarvation conditions, the uORFs restrict scanning ribosomes from reaching the GCN4 start codon; in amino acid-starved cells, this translational barrier is overcome, leading to increased GCN4 protein synthesis (reviewed in reference 26).Multiple trans-acting factors have also been implicated in translational control of GCN4 expression. GCD genes were defined genetically as negative regulatory factors that are r...