The role of heme-regulated eIF-2a kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2o kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. In heme deficiency, inhibition of protein synthesis occurs as a result of the activation of the heme-regulated inhibitor HRI (5,21,23,31). HRI is a cyclic AMP-independent protein kinase which specifically phosphorylates the 38-kDa ot subunit (eIF-2a) of eukaryotic initiation factor 2 (eIF-2) (18,26,28,46). Phosphorylation of eIF-2ao in reticulocyte lysates results in the binding and sequestration of eIF-2B in an eIF2(cP)-eIF-2B complex. The unavailability of eIF-2B, which is required for the exchange of GTP for GDP in the recycling of eIF-2 and the formation of the ternary complex (eIF-2/GTP/Met-tRNAf), results in the cessation of the initiation of protein synthesis (reviewed in references 21, 23, and 31).HRI is present in an inactive form in hemin-supplemented reticulocyte lysates (33, 34) and becomes active under conditions of heme deficiency (33,34) Ehrlich ascites cells which they believe to be distinct from HRI and the double-stranded RNA-dependent eIF-2ot kinase (PKR). Furthermore, increased eIF-2ot phosphorylation has been observed in HeLa and Ehrlich ascites cells under conditions of heat shock and nutrient starvation (11,13,14,39,50), in GH3 pituitary cells treated with calcium-mobilizing agents (45), and in vasopressin-treated rat liver (24). It has not yet been determined whether the eIF-2ot kinase activities measured in these cells are mediated by HRI or one or more as yet uncharacterized eIF-2a kinases.Previous studies using a monoclonal ant...

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Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2α kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2α. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.