Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

Abstract

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the a subunit of translation initiation factor 2 (eIF-2a) in amino acid-starved cells. Phosphorylation of eIF-2a reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN27 alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2' alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class H aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2C alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2c for their effects on GCN4 expression and to increase the level of eIF-2a phosphorylation in the absence of amino acid starvation. Synthetic GCN2" double mutations show greater derepression ofGCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotpes of the GCN2C alleles are dependent on GCNI and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2am kinase GCN2.Phosphorylation of the a subunit of translation initiation factor 2 (eIF-2a) in mammalian cells leads to inhibition of protein synthesis at the initiation step. Two different mammalian (eIF-2a) kinases have been identified: the doublestranded-RNA-activated inhibitor of translation (DAI) that is activated in response to viral infections and the hemecontrolled repressor (HCR) that is activated in reticulocytes by heme deficiency (for a review, see reference 26). Both kinases phosphorylate eIF-2a on the serine residue at position 51 (Ser-51) (9, 47). The phosphorylation of mammalian eIF-2a inhibits translation initiation by impairing the conversion of eIF-2-GDP to eIF-2-GTP at the completion of each initiation cycle, a reaction carried out by the guanine nucleotide exchange factor eIF-2B. Only the GTP-bound form of eIF-2 is able to form a ternary complex with the initiator tRNAMet and catalyze new rounds of translation initiation (41).GCN2 to increased synthesis of GCN4, a transcriptional activator of numerous genes encoding enzymes involved in amino acid biosynthesis. The kinase activity of GCN2 is required f...

YIH1 Is an Actin-binding Protein That Inhibits Protein Kinase GCN2 and Impairs General Amino Acid Control When Overexpressed

“…In this report, we provided several lines of evidence demonstrating that the 3AT s phenotype conferred by YIH1 overexpression results from dissociation of the GCN1-GCN2 complex and consequent impaired activation of GCN2. First, we showed that overexpression of YIH1 can suppress the growth defect conferred by a constitutively activated form of GCN2 that inhibits general translation initiation by high level phosphorylation of eIF2␣ (33). This result provides strong genetic evidence that YIH1 overexpression specifically inhibits GCN2 function.…”

“…The Slg Ϫ phenotype of GCN2 c -E601K-E1591K is suppressed by deletion of GCN1 (33), suggesting that GCN2 c -E601K-E1591K requires GCN1 binding for its activation. Therefore, if overexpressed YIH1 disrupts GCN1-GCN2 interaction, it should reverse the Slg Ϫ phenotype associated with the GCN2 c allele.…”

“…If so, then deletion of YIH1 should lead to constitutive activation of GCN2. To test this possibility, we asked whether deletion of YIH1 confers resistance to the tryptophan and histidine analogs 5-fluoro-DL-tryptophan (5FT) and triazolealanine (TRA), respectively, a phenotype conferred by known GCN2 c mutations that lead to constitutive induction of GCN4 and the tryptophan and histidine biosynthetic genes under GAAC (Gcd Ϫ phenotype) (33). Surprisingly, the yih1⌬ strain showed the same sensitivity to 5FT and TRA as the isogenic wild-type strain (Fig.…”

Conserved Intermolecular Salt Bridge Required for Activation of Protein Kinases PKR, GCN2, and PERK

“…Plasmids pC2707, pC2708, pC2709, and pC2710 expressing PERK-R598E, PERK-D603R, the double mutant PERK-R598E,E603R, and PERK-K633R, respectively, were generated by using fusion PCR to introduce the appropriate mutations in pC2706. Plasmid p722 is a low copy number URA3 vector that expresses GCN2 from its native promoter (7). Site-directed mutagenesis of p722 was used to generate the plasmids pC2745, pC2746, pC2747, and pC2748 expressing GCN2-K628R, GCN2-R594D, GCN2-D598R, and GCN2-R594D,D598R, respectively.…”

“…Thus, growth on medium containing 3-AT is a simple test for eIF2␣ kinase activity in yeast. In contrast, high level phosphorylation of eIF2␣ by mutationally activated forms of GCN2 impairs general translation initiation in yeast and results in a slow growth phenotype (3,7). Whereas yeast cells lacking GCN2 are unable to grow on medium containing 3-AT, low level expression of heterologous PKR or PERK kinases confers a 3-AT-resistant phenotype (8,9).…”