During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5 leader of the mouse ATF4 mRNA. The 5 proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4-coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals. During environmental stress conditions, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, to elicit apoptosis. Central to the early events in stress response pathways is a family of protein kinases that phosphorylate the ␣ subunit of eukaryotic initiation factor-2 (eIF2) (1-10). In mammals, four eIF2 kinases have been identified, and each recognizes distinct stress signals and modulate downstream response pathways via translational control. These eIF2 kinases include general control nonderepressible-2 (GCN2) that is activated by nutritional stresses, dsRNA induced protein kinase (PKR), important for an antiviral defense pathway mediated by IFN, heme regulated inhibitor (HRI) that couples protein synthesis to the availability of heme in erythroid cells, and pancreatic eIF2 kinase, PEK (also known as Perk), important for remedying protein misfolding in the endoplasmic reticulum (ER). Phosphorylation of the ␣ subunit of eIF2 reduces the levels of eIF2-GTP available for translation initiation, contributing to lowered global protein synthesis concurrent with induced translational expression of genes that function to alleviate stress damage in cells.We have been interested in the molecular mechanisms by which selected mRNAs are translated in response to eIF2 phosphorylation. A classic example of such a stress remedy pathway involves the transcriptional activator GCN4 in Saccharomyces cerevisiae (1, 2). In yeast starving for nutrients, GCN2 phosphorylation of eIF2 induces translation of GCN4 mRNA. Translational expression of GCN4 occurs by a mechanism that involves four upstream ORFs (uORFs) in the 5Ј noncoding portion of the GCN4 mRNA. Hinneb...

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF-2␣). In mammals, the phosphorylation was shown to be carried out by eIF-2␣ kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2␣ kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2␣ kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2␣ on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2␣ kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2␣. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2␣ kinase plays an important role in translational control from nematodes to mammals.

The GCN2 eIF2␣ 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 eIF2␣ 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. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2␣ and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2 ؊/؊ mice.

Phosphorylation of eukaryotic initiation factor-2 (eIF2) by pancreatic eIF2 kinase (PEK), induces a program of translational expression in response to accumulation of malfolded protein in the endoplasmic reticulum (ER). This study addresses the mechanisms activating PEK, also designated PERK or EIF2AK3. We describe the characterization of two regions in the ER luminal portion of the transmembrane PEK that carry out distinct functions in the regulation of this eIF2 kinase. The first region mediates oligomerization between PEK polypeptides, and deletion of this portion of PEK blocked induction of eIF2 kinase activity. The second characterized region of PEK facilitates interaction with ER chaperones. In the absence of stress, PEK associates with ER chaperones GRP78 (BiP) and GRP94, and this binding is released in response to ER stress. ER luminal sequences flanking the transmembrane domain are required for GRP78 interaction, and deletion of this portion of PEK led to its activation even in the absence of ER stress. These results suggest that this ER chaperone serves as a repressor of PEK activity, and release of ER chaperones from PEK when misfolded proteins accumulate in the ER induces gene expression required to enhance the protein folding capacity of the ER.

Wolcott-Rallison syndrome (WRS) is