Phosphorylation of a Heme-Regulated Eukaryotic Initiation Factor 2αkinase Enhances the Interaction with Heat-Shock Protein 90 and Substantially Upregulates kinase Activity

Kentaro      Mukai; Toru      Shimizu; Jotaro      Igarashi

Abstract

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with δ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.

Keywords: Heme, eIF2α kinase, phosphorylation, heat shock protein 90, HRI, thalassemia, eIF2a, GCN2, PERK, PKR, RNA, ATP, Coomassie Brilliant Blue, Hsp90, SDS-PAGEHeme, eIF2α kinase, phosphorylation, heat shock protein 90, HRI, thalassemia, eIF2a, GCN2, PERK, PKR, RNA, ATP, Coomassie Brilliant Blue, Hsp90, SDS-PAGE