In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2alpha kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme.

Protein synthesis is regulated by the phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣) in response to different environmental stresses. One member of the eIF2␣ kinase family, hemeregulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells. We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI. The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2␣, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI. Overexpression of Hri1p, Hri2p, or the human eIF2␣ kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S. pombe due to elevated phosphorylation of eIF2␣. Cells from strains with deletions of the hri1 ؉ and hri2 ؉ genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds. Measurements of in vivo phosphorylation of eIF2␣ suggest that Hri1p and Hri2p differentially phosphorylate eIF2␣ in response to these stress conditions. These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2␣ kinases are important participants in diverse stress response pathways in some lower eukaryotes.