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. 2002 Oct;22(19):6681-8.
doi: 10.1128/MCB.22.19.6681-6688.2002.

The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

Peichuan Zhang  1 , Barbara C McGrath, Jamie Reinert, DeAnne S Olsen, Li Lei, Sangeeta Gill, Sheree A Wek, Krishna M Vattem, Ronald C Wek, Scot R Kimball, Leonard S Jefferson, Douglas R Cavener
Affiliations

"VSports最新版本" The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

Peichuan Zhang et al. Mol Cell Biol. 2002 Oct.

Abstract

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted VSports手机版. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice. .

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"V体育ios版" Figures

FIG. 1.
FIG. 1.
Structure and expression of the mouse Gcn2 gene. (A) Three isoforms of mouse GCN2 RNA transcripts are encoded within a 75-kb region of the mouse. The first exon of the α isoform (denoted 6a) is inclusive of exon 6b present in the β and γ isoforms and shares a common 3′ end with exon 6b. The first exon of the β isoform (denoted 1a) is inclusive of exon 1b present in the γ isoform and shares a common 3′ end with exon 1b. Each of the three isoforms contains a unique translation initiation site (34), but terminates translation at a common UGA stop codon in exon 39. All of the exon-intron junctions conform to the eukaryotic consensus splice sites. Restriction sites mapped in genomic DNA: X, XbaI; and R, EcoRI. (B) Expression of mGCN2 mRNA isoforms. Primers specific for each of the three GCN2 mRNA isoforms were used in RT-PCRs with RNA isolated from various mouse tissues to detect their relative tissue-specific expression. Further quantitative RT-PCR experiments indicated that the β isoform is considerably more abundant than the α and γ isoforms. Diagrams depicting the domains included in each isoform are shown to the right.
FIG. 2.
FIG. 2.
Gcn2 targeting knockout vector. The approximate locations of the mouse Gcn2 exons 10 to 15 are shown below a restriction endonuclease map of genomic DNA. Xb, XbaI; Sac, SacI; Bam, BamHI; R, EcoRI; H, HindIII; and Xh, XhoI. The left arm of the target vector included a 3.0-kb HindIII fragment including a small part of exon 12, whereas the right arm included a 5.0-kb XhoI-HindIII fragment. The Neor gene was inserted between the right arm and left arm of the targeting vector, resulting in a deletion and substitution of most of exon 12.
FIG. 3.
FIG. 3.
Targeted disruption of the mouse Gcn2 gene. (A) Southern hybridization was performed on SacI-restricted genomic DNA to verify the targeted disruption. The Southern blot was probed with a combination of mGCN2 cDNA derived from exons 9 to 12 and a BamHI 1.3-kb genomic fragment corresponding to the 3′ end of the left arm of the targeting vector. The sizes of the fragments are indicated. +/+, wild type; +/−, heterozygote; −/−, homozygous mutant. (B) Transgenic animals were verified by PCR amplification of genomic DNA. The wild-type mGCN2 allele was detected as a 550-bp fragment from exon 12, whereas the mutant allele was detected as an 890-bp product from Neor gene substitution. (C) RT-PCR products (exons 11 to 15) derived from liver RNA were analyzed by Southern hybridization with Neor gene or a fragment of exon 12 as the probe. Gcn2−/− and Gcn2+/− mice display an 812-bp fragment detected by the Neor probe. Sequence analysis of this fragment revealed that it corresponded to an RNA generated from a cryptic splice located on the antisense strand of the Neor gene. As expected, the Gcn2−/− mice completely lacked the RT-PCR product (742 bp) containing exon 12 coding sequences. (D) Western blot analysis of mouse GCN2 with antisera prepared against the carboxyl terminus of mouse GCN2 indicated the absence of GCN2 in the homozygous mutant liver and kidney.
FIG. 4.
FIG. 4.
Effect of histidinol treatment on relative eIF2α phosphorylation, eIF2B activity, and protein synthesis. (A) Mouse livers were perfused with histidinol in situ as described in Materials and Methods. Total eIF2α and eIF2α[P] were then detected by Western blot analysis. (B) Quantitative analysis of eIF2α[P] relative to total eIF2α is shown as the mean of seven livers per condition. (C) The guanine nucleotide exchange activity of eIF2B in mouse liver homogenates was measured as described in Materials and Methods. The results represent the mean ± standard error of three to seven livers per condition. (D) The eIF2α phosphorylation and eIF2B activity data presented in panels B and C were subjected to linear regression analysis and are presented in graphic form. (E) Rates of protein synthesis were determined by the incorporation of [3H]leucine into protein during the final 10 min of the perfusion and are expressed as milligrams of protein synthesized per gram of tissue protein per hour. The values represent the mean ± standard error for three to six livers per condition: a complete mixture of amino acids (open bars) or medium containing all amino acids except histidine and additionally containing 4 mM histidinol (solid bars). *, P < 0.01 versus complete amino acid condition. The perfusate contained amino acids present at 10 times the concentrations typically found in arterial plasma (10X control medium) or 10 times the concentration found in arterial plasma except for histidine and additionally containing 4 mM histidinol (Hisol medium).
FIG. 5.
FIG. 5.
Phosphorylation of eIF2α in leucine-deprived ES cell is dependent upon GCN2. Gcn2+/+ (A) and Gcn2−/− (B) ES cells were grown in leucine-replete (lane C) or leucine-deficient (−Leu) media as described in Materials and Methods. Cells were harvested at 1, 3, 6, 9, and 12 h and analyzed by immunoblotting with antisera directed against either eIF2α or eIF2α[P]. The ratio of eIF2α[P] to eIF2α is indicated at the bottom of each panel. For comparison, Gcn2+/+ and Gcn2−/− ES cells were also treated for 1 or 3 h with 1 μM thapsigargin (Tg) to induce eIF2α phosphorylation via the PERK-dependent unfolded protein response.
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