Multiple Autophosphorylation Is Essential for the Formation of the Active and Stable Homodimer of Heme-Regulated eIF2α Kinase†

In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2α 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 t...