Autophosphorylation of heme-regulated eukaryotic initiation factor 2[alpha] kinase and the role of the modification in catalysis

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme shortage in reticulocytes and aids in the maintenance of a heme:globin ratio of 1:1. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein and autophosphorylation subsequently occurs. Autophosphorylation comprises a preliminary critical step before the execution of the intrinsic function of HRI; specifically, phosphorylation of Ser-51 of eIF2α to inhibit translation of the globin protein. The present study indicates that dephosphorylated mouse HRI exhibits strong intramolecular interactions (between the N-terminal and C-terminal domains) compared to phosphorylated HRI. It is therefore suggested that autophosphorylation reduces the intramolecular interaction, which induces irreversible catalytic flow to the intrinsic eIF2α kinase activity after heme dissociates from the protein. With the aid of MS, we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichiacoli. Phosphorylated sites at Ser, Thr and Tyr were predominantly localized within the kinase insertion region (16 sites) and kinase domain (12 sites), whereas the N-terminal domain contained five sites. We further generated 30 enzymes with mutations at the phosphorylated residues and examined their catalytic activities. The activities of Y193F, T485A and T490A mutants were significantly lower than that of wild-type protein, whereas the other mutant proteins displayed essentially similar activity. Accordingly, we suggest that Tyr193, Thr485 and Thr490 are essential residues in the catalysis. Heme association/dissociation at the heme-sensing site of heme regulated inhibitor (HRI) regulates the eukaryotic intitiation factor2α (eIF2α) kinase reaction. Heme association with full-length HRI blocks catalysis, whereas heme dissociation exposes the active site and permits catalysis. HRI is autophosphorylated at key residues, including Tyr-193, Thr-485, and Thr-490. HRI autophosphorylates multiple residues. HRI becomes an active eIF2α kinase, phosphorylating the substrate at Ser-51. [PUBLICATION ABSTRACT]