Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses

Abstract

Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by eIF2alpha kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2alpha kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2alpha kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2alpha phosphorylation upon arsenite treatment. HRI is also the major eIF2alpha kinase responsible for the increased eIF2alpha phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.

FIG. 1

Activation of HRI by arsenite in mouse reticulocytes. (A) Effect of arsenite concentration on activation of HRI. Mouse reticulocytes were isolated as described in Materials and Methods. After recovery, the cells were treated with 100, 200, or 400 μM arsenite (As) or 1 μM Tg for 30 min and then labeled with [³⁵S]methionine for an additional 30 min. Total cellular proteins were then separated by SDS-PAGE. The incorporation of [³⁵S]methionine into globins was detected by exposing the nitrocellulose membrane to X-ray film. The extents of HRI activation and eIF2α phosphorylation were detected with anti-mouse HRI antibody and anti-eIF2αP antibody. Total eIF2α was determined with anti-eIF2α monoclonal antibody. The upshifted species of HRI are indicated by an arrow. C, control. (B) Time course of HRI activation by arsenite. Mouse reticulocytes were treated with 200 μM arsenite for different time periods as indicated. (C) Kinase activity of HRI. HRI in reticulocytes was immunoprecipitated from lysates of arsenite-treated, Tg-treated, or control reticulocytes with anti-HRI antibody, and the kinase activities were assayed in vitro by using recombinant yeast eIF2α as a substrate. The incorporation of ³²P into eIF2α was detected by autoradiography. In lanes 7 and 8, the reticulocyte lysates from the control cells were either treated in vitro with 50 μM arsenite or heat shocked (hs) at 42°C for 30 min prior to immunoprecipitation. (D) Heme-independent activation of HRI by arsenite. Reticulocytes were treated with or without hemin (H, 40 μM) for 1 h prior to arsenite treatment (200 μM, 1 h).

FIG. 2

Differential activation of HRI by different stresses in reticulocytes. (A) Activation of HRI by osmotic stress and heat shock. Mouse reticulocytes were subjected to osmotic stress (Os) and heat shock (hs) for 30 min and then labeled with [³⁵S]methionine for an additional 30 min. The extents of HRI activation and eIF2α phosphorylation and the incorporation of [³⁵S]methionine into globins were detected as described in the legend to Fig. 1. C, control. (B) Effects of cytoplasmic stresses on HRI activation. Mouse reticulocytes were subjected to stresses as described in Materials and Methods. The extents of HRI activation and eIF2α phosphorylation were examined by Western blot analysis. The amounts of eIF2αP under various stresses were quantitated by densitometry scans of Western blots from three separate experiments and are shown here. As, arsenite; AA−, amino acid starvation; S−, serum starvation. (C) eIF2α kinase activity of HRI. The activity of HRI in either stressed or nonstressed reticulocytes, as indicated, was assayed by IP-coupled protein kinase assays as described in Materials and Methods and the legend to Fig. 1C. The incorporation of ³²P into eIF2α was detected by autoradiography. NI, nonimmune serum control. (D) Effect of alkaline phosphatase (AP) on the electrophoretic mobility of HRI. Cell lysates from either stressed or nonstressed reticulocytes were treated with or without 2 U of calf intestine alkaline phosphatase at 37°C for 30 min. The phosphorylation status of HRI was analyzed by SDS-PAGE and Western blot analysis. (E) Autophosphorylation is required for the slower electrophoretic mobility of the HRI activated by arsenite treatment. Wild-type (Wt) HRI and its inactive mutant K196R HRI were expressed in CHO-TRX cells by induction with tetracycline (Tet) overnight as described in Materials and Methods. Cells were treated with arsenite for 1 h. The expression and activation of HRI and its mutant in CHO cells were examined as described above.

FIG. 3

Activation of HRI by various cytoplasmic stresses in fetal liver erythroid progenitors. (A) Effect of arsenite. Fetal liver cells from both wild-type and HRI^−/− mice were isolated as described in Materials and Methods. Cells were treated with different concentrations of arsenite (As) for 1 h. C, control. (B) Effect of osmotic stress (Os), heat shock (hs), and Tg. Fetal liver cells were treated with different cytoplasmic stresses: osmotic stress by NaCl (0.5 M, 1 h), heat shock (42°C, 30 min), and ER stress by Tg (1 μM, 1 h). (C) Effect of amino acid starvation and serum starvation. Starvation of cells was performed by incubation in either methionine (Met)-free or serum-free medium for 1 h. (D) Effects of arsenite treatment on protein synthesis in wild-type and HRI^−/− fetal liver cells. Fetal liver cells were subjected to arsenite treatment for 90 min. [³⁵S]methionine was added at time zero. Cells were harvested at different time periods as indicated. The total cellular proteins were separated by SDS-PAGE. The incorporation of [³⁵S]methionine into globins was detected by autoradiography and was quantitated by scintillation counting of nitrocellulose strips containing globin chains.

FIG. 4

Cytoplasmic stresses of wild-type, HRI^−/−, and PKR^−/− reticulocytes. (A) eIF2α phosphorylation of wild-type, HRI^−/−, and PKR^−/− and reticulocytes upon different stresses. Reticulocytes from wild-type, HRI^−/−, or PKR^−/− mice were subjected to different stress conditions as described in the legend to Fig. 2A. As, arsenite; Os, osmotic stress; hs, heat shock; C, control. (B) eIF2α kinase activity of PKR. PKR from either stressed or nonstressed reticulocytes was immunoprecipitated with anti-PKR antibody. The kinase activity of PKR was determined by in vitro kinase assays as described in Materials and Methods. The incorporation of ³²P into eIF2α was detected by autoradiography. NI, nonimmune serum control.

FIG. 5

Role of ROS in HRI activation. Mouse reticulocytes were pretreated with different concentrations of the reducing reagent NAC as indicated for 30 min before being subjected to different stresses: no stress treatment (C), arsenite treatment (200 μM) for 1 h (As), heat shock at 42°C for 30 min, and osmotic stress with 0.5 M NaCl for 1 h. The activation of HRI and eIF2aP was examined.

FIG. 6

Role of chaperones in activation of HRI by arsenite. Reticulocytes were pretreated with different concentrations of GA (4, 8, 20, and 40 μg/ml) (B) and CIA (5, 10, 20, and 40 mM) (A) for 30 min and then subjected to a 60-min incubation with (As) or without (C) arsenite treatment at 200 μM. The activation of HRI and eIF2αP was examined.

FIG. 7

Model of the role of HRI in the stress response of erythroid cells. HRI is a major stress-activated eIF2α kinase in erythroid cells and can be activated by several different stresses in addition to heme deficiency. HRI is the only eIF2α kinase activated by arsenite. It is also the main eIF2α kinase activated by heat shock. The activation of HRI contributes to the regulation of cell proliferation, survival, and/or differentiation under stress challenges. AA, amino acid.