Lead-induced upregulation of the heme-regulated eukaryotic initiation factor 2α kinase is compromised by hemin in human K562 cells Angshuman Sarkar a,1 , Abhijeet Kulkarni a , Samit Chattopadhyay b , Devraj Mogare b , Kiran K. Sharma a,2 , Kamini Singh b , Jayanta K. Pal a, ⁎ a Department of Biotechnology, University of Pune, Pune 411 007, India b National Centre for Cell Science, Ganeshkhind, Pune 411 007, India Received 12 April 2005; received in revised form 10 December 2005; accepted 19 December 2005 Available online 19 January 2006 Abstract Expression and kinase activity of the heme-regulated-eIF-2α kinase or -inhibitor (HRI) are induced during cytoplasmic stresses leading to inhibition of protein synthesis. Using a reporter construct with HRI promoter, we have determined the promoter activity during heat-shock and lead toxicity in human K562 cells. These two conditions induced HRI promoter activity by 2- to 3-fold. Contrary to this, hemin, a suppressor of HRI kinase activity, downregulated HRI promoter activity and stimulated hemoglobin synthesis. Interestingly, when hemin-treated cells were transfected and exposed to lead, hemin compromised lead-effect substantially by downregulating HRI promoter activity, HRI transcription and HRI kinase activity. These results together suggest that heme signaling in relation to translation regulation is not only restricted to the cytoplasm (modulating HRI kinase activity) alone but it also spans to the nucleus modulating HRI expression. Hemin may thus be useful for alleviation of stress-induced inhibition of protein synthesis. © 2005 Elsevier B.V. All rights reserved. Keywords: Heme-regulated inhibitor; Promoter activity; Expression; Kinase activity; Heat shock; Lead toxicity 1. Introduction Protein synthesis is regulated in response to environmental stimuli by covalent modifications, primarily phosphorylation, of components of the translational machinery. Several studies have suggested that phosphorylation of the subunit (α) of eukaryotic initiation factor 2 (eIF-2α) plays a central role in regulating the overall rate of protein synthesis in eukaryotes [1]. The phosphorylation of eIF-2α is caused by a family of eIF-2α kinases [2–7]. Among these, the role of the heme regulated eIF-2α kinase (also called the heme regulated inhibitor, HRI) in regulating initiation of protein synthesis in reticulocytes has been well established [4,8,9]. Upon activa- tion, HRI, a Ser/Thr protein kinase, undergoes autopho- sphorylation and subsequently phosphorylates the 38 kDa α subunit of eIF-2 at Ser-51 residue [8]. Phosphorylated eIF-2 [eIF-2α(P)] binds to eIF-2B, also called the guanine nucleotide exchange factor (GEF) or reversing factor (RF), and forms a stable complex that sequesters eIF-2B. Due to unavailability of eIF-2B, which is required for the exchange of GTP for GDP for recycling of eIF-2, initiation of protein synthesis ceases [4,8,10]. This kinase gets activated due to heme deficiency or a variety of other conditions, such as heat shock, heavy metal toxicity, low partial pressure of oxygen, degraded polypep- tides, reactive oxygen species, treatment with N-ethylmalei- mide (NEM) and oxidized glutathione (GSSG) [8–12]. HRI has been purified and extensively characterized from rabbit reticulocyte lysate. HRI is a dimer of 92 kDa polypeptide and it has a sedimentation co-efficient of 6.6S [8,10]. There are two heme binding domains in HRI: one at the N terminal region and the other in the insertion domain [13]. Rabbit HRI cDNA of 2.7 kb was cloned in 1991 [14]. In vitro translation of mRNA transcribed from HRI cDNA yielded a 90- kDa polypeptide which had eIF-2α kinase activity and specific Biochimica et Biophysica Acta 1732 (2005) 15 – 22 http://www.elsevier.com/locate/bba ⁎ Corresponding author. Tel.: +91 20 25692248; fax: +91 20 25691821. Email addresses: jkpal@unipune.ernet.in, jkpal@hotmail.com (J.K. Pal). 1 Present address: Cold Spring Harbor Laboratory, New York 11724, USA. 2 Present address: Department of Biochemistry and Biophysics, University of Rochester, NY 14642, USA. 0167-4781/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2005.12.003