Heme-regulated eIF-2 Alpha Kinase Research Articles

Heme-regulated eIF2α kinase modifies the phenotypic severity of murine models of erythropoietic protoporphyria and β-thalassemia

Heme-regulated eIF2alpha kinase (HRI) controls protein synthesis by phosphorylating the alpha-subunit of eukaryotic translational initiation factor 2 (eIF2alpha). In heme deficiency, HRI is essential for translational regulation of alpha- and beta-globins and for the survival of erythroid progenitors. HRI is also activated by a number of cytoplasmic stresses other than heme deficiency, including oxidative stress and heat shock. However, to date, HRI has not been implicated in the pathogenesis of any known human disease or mouse phenotype. Here we report the essential role of HRI in 2 mouse models of human rbc disorders, namely erythropoietic protoporphyria (EPP) and beta-thalassemia. In both cases, lack of HRI adversely modifies the phenotype: HRI deficiency exacerbates EPP and renders beta-thalassemia embryonically lethal. This study establishes the protective function of HRI in inherited rbc diseases in mice and suggests that HRI may be a significant modifier of many rbc disorders in humans. Our findings also demonstrate that translational regulation could play a critical role in the clinical manifestation of rbc diseases.

Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding.

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.

Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI.

In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.

Differential Inhibition of Hsc70 Activities by Two Hsc70-Binding Peptides

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency.

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.

Multiple Autophosphorylation Is Essential for the Formation of the Active and Stable Homodimer of Heme-Regulated eIF2α Kinase

In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2alpha kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme.

Brain ischemia and reperfusion activates the eukaryotic initiation factor 2alpha kinase, PERK.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha). To identify kinases responsible for eIF2alpha phosphorylation [eIF2alpha(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2alpha(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2alpha kinase (HRI), or the amino acid-regulated eIF2alpha kinase (GCN2). A 10-fold increase in eIF2alpha(P) was observed in reperfused wild-type mice and in the HRI-/- or GCN2-/- mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2alpha kinase (PERK) exhibited an isoform mobility shift on SDS-PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2alpha(P), and in conjunction with our previous report that eIF2alpha(P) is produced in the brain of reperfused PKR-/- mice, provides evidence that PERK is the kinase responsible for eIF2alpha phosphorylation in the early post-ischemic brain.

Hsp90 Regulates p50 cdc37 Function during the Biogenesis of the Active Conformation of the Heme-regulated eIF2α Kinase

Recent studies indicate that p50(cdc37) facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50(cdc37) is required for the biogenesis of the heme-regulated eIF2 alpha kinase (HRI) in reticulocyte lysate. p50(cdc37) interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50(cdc37) stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50(cdc37) function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50(cdc37) interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50(cdc37) interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50(cdc37) to bind HRI and promote its activation, but did not disrupt the native association of p50(cdc37) with Hsp90. A C-terminal truncated mutant of p50(cdc37) inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50(cdc37) were detected in cultured K562 erythroleukemia cells. These results suggest that p50(cdc37) provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.

The Heme-regulated Eukaryotic Initiation Factor 2α Kinase: A POTENTIAL REGULATORY TARGET FOR CONTROL OF PROTEIN SYNTHESIS BY DIFFUSIBLE GASES

Nitric oxide (NO) has been reported to inhibit protein synthesis in eukaryotic cells by increasing the phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF) 2. However, the mechanism through which this increase occurs has not been characterized. In this report, we examined the effect of the diffusible gases nitric oxide (NO) and carbon monoxide (CO) on the activation of the heme-regulated eIF2alpha kinase (HRI) in rabbit reticulocyte lysate. Spectral analysis indicated that both NO and CO bind to the N-terminal heme-binding domain of HRI. Although NO was a very potent activator of HRI, CO markedly suppressed NO-induced HRI activation. The NO-induced activation of HRI was transduced through the interaction of NO with the N-terminal heme-binding domain of HRI and not through S-nitrosylation of HRI. We postulate that the regulation of HRI activity by diffusible gases may be of wider physiological significance, as we further demonstrate that NO generators increase eIF2alpha phosphorylation levels in NT2 neuroepithelial and C2C12 myoblast cells and activate HRI immunoadsorbed from extracts of these non-erythroid cell lines.

Heme-regulated eIF-2alpha kinase purifies as a hemoprotein.

The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme-regulated eIF-2alpha kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF-2alpha kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent Ki of 0.5 microM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of heme-binding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.

Characterization of the Hemin-sensitive Eukaryotic Initiation Factor 2α Kinase from Mouse Nonerythroid Cells

The heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme 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 eIF2alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2alpha 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 eIF2alpha 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 eIF2alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.

Evidence that Hsc70 negatively modulates the activation of the heme-regulated eIF-2alpha kinase in rabbit reticulocyte lysate.

The role of the heat-shock cognate protein, Hsc70, in regulating the activity of the heme-regulated eIF-2alpha kinase (HRI) in hemin-supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF-2alpha phosphorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42 degrees C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg2+. The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein-synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg2+. Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF-4 E from heat-induced dephosphorylation, and its inability to protect translational elongation from GSSG-induced inhibition, respectively. Activation of HRI in heat-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss of the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.

The role of the 90-kDa heat-shock protein and its associated cohorts in stabilizing the heme-regulated eIF-2alpha kinase in reticulocyte lysates during heat stress.

The heme-regulated eIF-2alpha kinase (HRI) is activated not only in heme-deficient rabbit reticulocyte lysates (RRL), but also in hemin-supplemented RRL treated with heat-shock, N-ethylmaleimide (MalNEt) or heavy metal ions. We have demonstrated previously that heat-shock proteins, Hsp90, Hsp70 and FKBP52, are associated with HRI in RRL; the association of HRI with Hsp90 and FKBP52, but not Hsp70, is enhanced by hemin. To study the role of Hsp90 and its associated cohorts in the regulation of HRI, we examined the interaction of these proteins with HRI in hemin-supplemented RRLs during heat or oxidative stress. The association of HRI with Hsp90, FKBP52 and p23 was maintained in heat-, MalNEt- or Hg2(+)-treated hemin-supplemented RRL. Glycerol gradient centrifugation and gel filtration on Sephacryl S-300 indicated that neither heat shock nor MalNEt-treatment affected the apparent molecular mass of HRI in hemin supplemented RRL. In addition, active HRI was coimmunoprecipitated with 8D3 anti-Hsp90 from both heme-deficient and MalNEt-treated hemin-supplemented RRL. These results demonstrate that activation of HRI in response to heat stress and oxidative stress does not require dissociation of Hsp90 from HRI. Furthermore, HRI activity was inhibited upon addition of hemin to Hsp90-depleted heme-deficient RRL, indicating that inhibition of HRI activity by hemin is not mediated by the reassociation of Hsp90 with HRI. We also examined the dynamics of the interaction of Hsp90 with HRI. Reconstitution of the interaction of Hsp90 with HRI was stimulated by elevated temperature and required both Mg2+ and ATP. Addition of purified Hsp90 to hemin-supplemented RRL which had been treated with MalNEt to inactivate its capacity to chaperone protein renaturation, protected HRI from irreversible denaturation and aggregation upon incubation at 41 degrees C. Our results suggest that Hsp90 interacts with HRI primarily in its capacity as a molecular chaperone, stabilizing HRI from denaturation under conditions of heat stress and oxidative stress.

Inhibition of Protein Synthesis by the Heme‐Controlled Eif‐2α kinase Leads to the Appearance of mRNA‐Containing 48S Complexes that Contain eIF‐4E but Lack Methionyl‐tRNAf

Phosphorylation of the initiation factor eIF-2 by the heme-regulated eIF-2 alpha kinase (HCR) results in pronounced inhibition of protein synthesis and of binding of Met-tRNA(f) to 40S subunits in reticulocyte lysates. This inhibition is associated with the appearance of a more rapidly sedimenting 48S complex; this contains mRNA detectable by poly(U) hybridization, but not Met-tRNA(f). In contrast, 48S complexes that accumulate in the presence of the initiation inhibitor edeine contain both Met-tRNA(f) and mRNA. We have compared the composition of the particles that accumulate in the presence of HCR with those seen in the presence of edeine and find that both particles contain the cap binding protein, eIF-4E. Moreover, both particles exhibit a buoyant density of 1.40 g/ml in CsCl equilibrium density gradients. This is consistent with the presence of 500-700 kDa of protein additional to ribosomal structural protein, and suggests the presence of eIF-3 on both types of 48S complex. Lysates pre-treated with HCR and then treated with edeine show the ability to accumulate 48S complexes containing Met-tRNA(f), though at a slower rate than control lysates. These observations are discussed in the light of mechanisms previously suggested for the appearance of 48S particles following HCR treatment. In addition, we have observed association of eIF-4E with polysomes and 80S monosomes in reticulocyte lysates, suggesting that this factor may not be released immediately following the binding of the 40S ribosomal subunit to the 5' end of the mRNA.

Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2 alpha kinase.

To study further the regulation of the heme-regulated eIF-2 alpha kinase (HRI), we have produced functional wild type HRI using the baculovirus expression system. The amount of recombinant HRI protein expressed in insect cells is approximately 10 times higher than levels in reticulocytes. Baculovirus-expressed HRI (BV-HRI) is indistinguishable from HRI purified from rabbit reticulocytes. It is active both as an autokinase and an eIF-2 alpha kinase. BV-HRI is regulated by heme in vitro as well as in intact insect cells. Coexpression of the wild type HRI with the inactive K199R HRI, S51A eIF-2 alpha, or interleukin-1 beta (IL-1 beta) results in diminished expression of these proteins. Expression of wild type HRI also results in severe inhibition of general protein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-beta. In addition, the guanine nucleotide exchange activity of eIF-2B is suppressed in Sf9 cells expressing wild type HRI but not in cells expressing the K199R HRI or IL-1 beta. Furthermore, expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2 alpha or by the addition of hemin, which inhibits HRI activity. These results provide evidence that translational regulation by phosphorylation of eIF-2 alpha and sequestration of eIF-2B can operate in insect cells.

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Erythroid Expression of the Heme-Regulated eIF-2α Kinase

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase.

The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp) and a 56-kDa protein in hemin-supplemented lysates [Matts, R.L., Xu, Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267m 18160-18167]. In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-2. This stimulation of eIF-2 alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed with denatured BSA, but not with not with native BSA. Hsp70 was co-adsorbed with HRI from lysate in the presence of native BSA, but not in the presence of denatured BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hemin and porphyrin compounds on intersubunit disulfide formation of heme-regulated eIF-2 alpha kinase and the regulation of protein synthesis in reticulocyte lysates.

To study the mechanism by which heme regulates the heme-regulated eIF-2 alpha kinase (HRI), the effects of various protoporphyrin IX (PP) compounds on the kinase activities and intersubunit disulfide formation of HRI and on protein synthesis in reticulocyte lysates were examined. Hemin and cobalt protoporphyrin (CoPP) are more effective than ZnPP, NiPP, SnPP, and metal-free PP in promoting intersubunit disulfide bond formation in HRI, in inhibiting the autokinase and eIF-2 alpha kinase activities of HRI, in inhibiting phosphorylation of eIF-2 alpha in rabbit reticulocytes, in maintaining protein synthesis, and in reversing the inhibition of protein synthesis in heme deficiency. There is an apparent correlation of in vitro intersubunit disulfide formation of HRI and the regulation of HRI kinase activities and protein synthesis by these porphyrin compounds. HRI in the reticulocyte lysate can be cross-linked by 1,6-bismaleimidohexane (bis-NEM). The formation of bis-NEM cross-linked dimers in lysates is prevented completely by N-ethylmaleimide (NEM) which alkylates free sulfhydryl groups and is diminished by hemin and CoPP. These results support the view that HRI in hemin-supplemented lysates is in equilibrium between the noncovalently linked dimer and the disulfide-linked dimer. The molecular size of HRI in control, hemin-supplemented, or NEM-treated hemin-supplemented lysates is identical to that of purified HRI; activation of HRI and changes in its thiol status do not significantly affect its molecular size.

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.