Characterization of the hemin-sensitive eukaryotic initiation factor 2α kinase from mouse nonerythroid cells

Abstract

The heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (heme-regulated inhibitor (HRI)) is activated by heine deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2α kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme- sensitive eIF2α kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2α kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2α kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2α kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.