Regulation of protein synthesis in rabbit reticulocyte lysates: the hemeregulated protein kinase (HRI) and double stranded RNA induced protein kinase (dRI) phosphorylate the same site(s) on initiation factor eIF-2

Double stranded RNA (dsRNA) induced inhibitor (dRI) has been partially purified (80–100 fold). The dRI inhibits protein synthesis in rabbit reticulocyte lysates; the inhibition is overcome by the initiation factor eIF-2. The dRI preparations phosphorylate the 38,000-dalton subunit of eIF-2. Heme-deficiency in rabbit reticulocyte lysates also induces a translational inhibitor (HRI) which inhibits protein chain initiation by specifically phosphorylating the 38,000-dalton subunit of eIF-2. To establish correlation of the mechanism of inhibition of protein synthesis by dRI and HRI, the phosphopeptide patterns of eIF-2 phosphorylated by using HRI or dRI are compared. Treatment with various proteases of eIF-2 phosphorylated by HRI or dRI yield identical phosphopeptide patterns. This finding suggests that HRI and dRI phosphorylate the same site(s) of the 38,000-dalton subunit of eIF-2 and raises the possibility that dRI may also inhibit protein chain initiation by the mechanism similar to that of HRI.     

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Separation of heme regulated protein kinase into a high and a low molecular weight species

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14–15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) $\text{Proc}\text{.}\text{Natl}\text{.}\text{Acad}\text{.}\text{Sci}\text{.}\text{USA}$ 73, 4349–4353). The 14–15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5–9S. The 5.8S HRI, on weight basis, is about 5–10 times more active than the 14–15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant ot the mechanism of activation and inactivation of HRI in the absence and presence of heme $\text{in}\text{situ}$.     

Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

GTP (2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited by the activation of specific adenosine 3′:5′ cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the $\text{in}\text{,}\text{situ}$ state of eIF-2 phosphorylation. The results show that GTP (2 mM) blocks eIF-2 phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protin chain initiation cycle.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

Purification and properties of eIF-2 phosphatase

Eukaryotic initiation factor 2 (eIF-2) phosphatase has been purified 840-fold to apparent homogeneity from rabbit reticulocyte lysate. Native eIF-2 phosphatase has a Mr = 98,000, pI = 6.1, s20,w = 5.1, and a Stokes radius = 38 A. A subunit composition of one 60,000-dalton polypeptide and one 38,000-dalton polypeptide is indicated. The Km for [32P]eIF-2 is 30 microM and the Vmax = 1.1 nmol of phosphate released/min/microgram. The 38,000-dalton subunit of eIF-2 phosphatase does not co-electrophorese with the catalytic subunit of liver phosphorylase phosphatase, a type 1 protein phosphatase. The specificity of eIF-2 phosphatase for phosphorylation sites on th alpha- and beta-subunits of eIF-2 appears to be determined by the environment of the phosphatase and substrate. Both the alpha- and beta-subunits of [32P]eIF-2 are rapidly dephosphorylated by the purified phosphatase. In unfractionated lysate and in unfractionated lysate supplemented with an equivalent activity of the purified phosphatase, only the alpha-subunit of eIF-2 is dephosphorylated. This indicates other factors are present in the lysate which govern the dephosphorylation of eIF-2.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Heparan sulphate is a potent inhibitor of DNA synthesis in vitro

The nature and the role of eIF-2 phosphoprotein phosphatase in rabbit reticulocyte lysates have been examined. The eIF-2 phosphoprotein phosphatase is inhibited by a variety of divalent metal ions (Cd⁺⁺>Ag⁺⁺> Cu⁺⁺>Pb⁺⁺>Zn⁺⁺>Co⁺⁺>Sr⁺⁺>Mo⁺⁺) in lysates $\text{in situ}$. In addition, PPi, EDTA and NaF inhibit this enzyme. The eIF-2 phosphoprotein phosphatase is also inhibited by NaHSO₃ and Na₂S₂O₅. Na₂S₂O₅ is, however, more effective. Na₂S₂O₅ has been found to be a potent inhibitor of protein synthesis in lysates. This inhibition is associated with the phosphorylation of the 38,000-dalton subunit of initiation factor eIF-2. eIF-2 overcomes this inhibition. These findings suggest that under optimum conditions of protein synthesis the phosphorylation and dephosphorylation of eIF-2 are in a dynamic state of equilibrium in which dephosphorylation is favored. The inhibition of eIF-2 phosphoprotein phosphatase by Na₂S₂O₅ shifts this equilibrium in favor of eIF-2 phosphorylation, consequently, protein synthesis is inhibited. The sulfhydryl nature of eIF-2 phosphoprotein phosphatase has been established.     

Regulation of protein synthesis in rabbit reticulocyte lysates. Requirement of initiation factor eIF-2 holoprotein for substrate specificity of heme-regulated protein kinase

The specificity of the heme-regulated protein kinase (HRI) was investigated further by utilizing the isolated 38,000 Da subunit (alpha subunit) polypeptide of eIF-2 as the substrate. For this purpose, the three subunit polypeptides of eIF-2 (38,000 Da, alpha; 50,000 Da, beta; and 52,000 Da, gamma) were resolved by reversed-phase high performance liquid chromatography (HPLC). Results show that HRI is incapable of phosphorylating the 38,000 Da subunit separated from the other two eIF-2 polypeptides. Data suggest that the substrate specificity of HRI is determined by the quaternary structure assumed by the alpha subunit in association with the other two subunits in the eIF-2 holoprotein.     

Studies on rat liver catalase. X. Effect of hemin and an inhibitor on the translation of catalase messenger RNA1.

Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.     

Differential effect of Mn2+ on the hemin-controlled translational repressor and the double-stranded RNA-activated inhibitor

The inhibition of protein synthesis that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin is due to the activation of a protein kinase termed the hemin-controlled translational repressor, and that occurring when reticulocyte lysate is incubated with a low level of double-stranded RNA is mediated by the activation of a separate protein kinase termed the double-stranded RNA-activated inhibitor. Both the hemin-controlled translational repressor and the double-stranded RNA-activated inhibitor act by phosphorylating the M_r = 35 000 (α) subunit of eIF-2. MnCl₂ (0.5 mM) partly reverses the inhibition of protein synthesis produced by hemin deficiency but not that induced by double-stranded RNA. In addition, Mn²⁺ reverses the inhibition of binding of [³⁵S]Met-tRNA_f to reticulocyte ribosomal components, isolated on Sepharose 6B, produced by the hemin-controlled translational repressor but not by the double-stranded RNA-activated inhibitor. The effect of Mn²⁺ is mediated at the level of activation and eIF-2α kinase activity of these two regulatory protein kinases. Specifically, Mn²⁺ inhibits activation of the hemin-controlled translational repressor in the absence of hemin and the phosphorylation of eIF-2α by pre-activated translational repressor. In contrast, the phosphorylation of eIF-2α by the double-stranded RNA-activated inhibitor is not suppressed by Mn²⁺, and the activation and autophosphorylation of this inhibitor is enhanced by Mn²⁺. Finally, while the activation and inactivation of the hemin-controlled translational repressor does not appear to be mediated by autophosphorylation and dephosphorylation, the activation of the double-stranded RNA-activated inhibitor does appear to require autophosphorylation.     

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.     

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.     

Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.

Inhibition of protein synthesis in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this report, immunoadsorption with monoclonal antibodies (mAbs) and Western blot analysis were used to investigate the interaction of HRI, the 90-kDa heat shock protein (hsp 90), hsp 70, and the EC1 antigen in rabbit reticulocyte lysates under protein synthesizing conditions. The data indicate that hsp 90, hsp 70, and the EC1 antigen interact with HRI in rabbit reticulocyte lysate. The EC1 antigen is a protein that has been demonstrated to be associated with several steroid hormone receptor-hsp 90 complexes and reacts with the KN 382/EC1 mAb (EC1). The association of HRI with hsp 90 and the EC1 antigen in the reticulocyte lysate was found to be dependent on the presence of hemin at a concentration of 5 microM or higher; little HRI was coadsorbed by the 8D3 anti-hsp 90 mAb or the EC1 mAb in the absence of hemin. Hsp 70 remains associated with HRI in the absence of hemin, suggesting that hsp 90 and 70 may bind to HRI at different sites. The immunological properties of the hsp 70 associated with HRI indicate that it may be the constitutively express heat shock cognate protein (hsc 73). The results suggest that the association of HRI with hsp 90 and the EC1 antigen may be in a dynamic equilibrium, in which complex formation is either facilitated or stabilized by the presence of hemin, and supports the notion that these proteins in conjunction with hsp 70 may play a role in regulating HRI activity or activation in situ.     

Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II

In contrast to reticulocyte polypeptide chain initiation factor 2 (eIF-2), the Artemia factor retains activity in the presence of Mg2+ or after phosphorylation of its alpha-subunit by rabbit reticulocyte heme-controlled repressor (Mehta, H. B., Woodley, C. L., and Wahba, A. J. (1983) J. Biol. Chem. 258, 3438-3441). Furthermore, we have so far been unable to demonstrate a requirement for a GDP/GTP nucleotide exchange factor with Artemia eIF-2. In order to explain these differences we compared the structure of eIF-2 from Artemia and rabbit reticulocytes by using one- and two-dimensional phosphopeptide and iodopeptide maps. Partial trypsin digestion of the alpha-subunit of Artemia eIF-2 after phosphorylation by the heme-controlled repressor generates a 4000 Mr phosphopeptide. Upon extensive trypsin digestion, the two-dimensional phosphopeptide maps of the alpha-subunits for the reticulocyte and Artemia factors are indistinguishable, whereas the iodopeptide maps are different. In addition, immunoblotting indicates that there is no consistent cross-reactivity of the reticulocyte subunits with antibodies prepared in rabbits against the Artemia eIF-2 subunits. A casein kinase II activity was isolated from Artemia embryos that phosphorylates the beta-subunit of reticulocyte eIF-2, but specifically phosphorylates the alpha-subunit of eIF-2 preparations from several non-mammalian sources, including Artemia, yeast, and wheat germ embryos. Since this kinase phosphorylates a site distinct from that recognized by the heme-controlled repressor, and this phosphorylation does not alter the ability of Artemia eIF-2 to undergo nucleotide exchange, caution must be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in non-mammalian cells.     

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

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.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.