Although the physiological role of tissue‐specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme‐regulated eIF2α kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both α‐ and β‐globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron‐deficient HRI−/− mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.

Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of eukaryotic translation initiation factor 2 at Ser-51 on the ␣ subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2␣, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2␣, inhibition of protein synthesis, stress granule formation, and survival.

Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) by eIF2␣ kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2␣ kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2␣ kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2␣ phosphorylation upon arsenite treatment. HRI is also the major eIF2␣ kinase responsible for the increased eIF2␣ phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. In heme deficiency, protein synthesis is inhibited, with a marked decrease in the formation of 40S-eukaryotic initiation factor 2 (eIF2)-MettRNA f GTP (43S preinitiation complex) and with increased phosphorylation of the ␣ subunit of eIF2. eIF2 is an initiation factor which binds GTP and Met-tRNA f and is required for the formation of the 43S initiation complex. The phosphorylation of eIF2␣ in heme deficiency is the result of the activation of heme-regulated inhibitor (HRI), which is a heme-regulated eIF2␣ kinase (8, 9). The mechanism of inhibition of translation initiation by the phosphorylation of eIF2␣ has been studied extensively (18,19). In brief, the recycling of eIF2 in initiation requires the exchange of bound GDP for GTP. Under physiological conditions, eIF2 has a 400-fold-greater affinity for GDP than for GTP. The exchange of tightly bound GDP for GTP requires eIF2B, which is rate limiting and is present at 15 to 25% of the amount of eIF2. When eIF2␣ is phosphorylated, the binding of eIF2(␣P)-GDP to the regulatory subcomplex of eIF2B is much tighter than the binding of eIF2-GDP to eIF2B (25). This tighter interaction of phosphorylated eIF2 with eIF2B prevents the GDP-GTP exchange activity of eIF2B. In this manner, once the amount of phosphorylated eIF2 exceeds the amount of eIF2B, the shutoff of protein synthesis occurs.Phosphorylation of eIF2␣ was later found to be a common regulatory mechanism of protein tr...