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

Abstract

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.

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…HRI is a multidomain protein composed of five structural domains (Figure 2), namely, the N-terminal domain (NTD), the kinase I domain (K-I), the kinase insertion domain (KI), the kinase II domain (K-II) and the C-terminal domain (CTD) [56,[59][60][61]. The catalytic domain of HRI is divided in two halves, one on each side of the KI domain, and activation of HRI is thought to involve bringing residues on each of these two halves of the catalytic domain into close proximity [23,56,[59][60][61]. Two HRMs are present in HRI, one in the KI domain and the other in the CTD [23,56,[59][60][61].…”

“…The catalytic domain of HRI is divided in two halves, one on each side of the KI domain, and activation of HRI is thought to involve bringing residues on each of these two halves of the catalytic domain into close proximity [23,56,[59][60][61]. Two HRMs are present in HRI, one in the KI domain and the other in the CTD [23,56,[59][60][61]. The heme binding site in the KI domain is thought to reversibly bind heme and to play a role in the coordination of HRI activity with changes in heme concentration [61].…”

Phosphorylation of Eukaryotic Initiation Factor 2 by Heme-Regulated Inhibitor Kinase-Related Protein Kinases in Schizosaccharomyces pombe Is Important for Resistance to Environmental Stresses

“…For example, iron limitation and accompanying reductions in heme levels activate HRI through a mechanism involving autophosphorylation and an altered protein conformation (4,11,12). Distinct heme-binding sites were identified in the amino terminus of HRI and in the kinase insert region (58).…”

The Activities of the Yersinia Protein Kinase A (YpkA) and Outer Protein J (YopJ) Virulence Factors Converge on an eIF2α Kinase

“…Most pertinent to this study were the findings that HRI is necessary for macrophage maturation and function and contributes ϳ50% of the total eIF2␣ kinase activity in cultured macrophages following stimulation with lipopolysaccharide (40). HRI activity itself is controlled by multiple autophosphorylation sites that are responsive to heme levels (41)(42)(43). It will be of great interest to determine whether S. pombe YpkA and/or YopJ cause Hri2 to be phosphorylated at its normal sites or subjects it to novel modifications.…”