Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.     

Site-specific phosphorylation of the α subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2α kinases from rabbit reticulocyte lysates

The site specificity of phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) by the heme-regulated and double-stranded RNA-activated eIF-2α kinases were compared by phosphopeptide mapping. eIF-2α was maximally phosphorylated in vitro with [γ-³²P]ATP and either crude or partially purified preparations of the kinases. ³²P-Labeled eIF-2α was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [³²P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [³²P]eIF-2α contained in fixed, dried, and stained gel slices was achieved with Staphylococcus aureus protease V8, chymotrypsin, or subtilisin, and the partial ³²P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [³²P]phosphopeptide profiles after partial or exhaustive proteolysis of [³²P]eIF-2α. With a given protease, identical [³²P]phosphopeptide patterns were obtained whether eIF-2α was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that, in vitro, the kinases phosphorylate sites on eIF-2α that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2α kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2α that are similar, if not identical, to the sites phosphorylated in vitro with partially purified eIF-2α kinase(s) and eIF-2.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Additional initiation factor required for formation of ternary complex (eIF-2·GTP·Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.     

Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation.

The eukaryotic initiation factor eIF-2 forms a ternary complex with Met-tRNAf and GTP. This complex binds to the 40S ribosomal subunit in the absence of mRNA and mRNA binding factors. Highly purified eIF-2 from rabbit reticulocytes was labeled with 125I by using the Bolton-Hunter reagent or with [gamma-32P]ATP by using the heme-regulated translational inhibitor protein kinase. The labeled eIF-2 was bound, together with equimolar amounts of Met-tRNAf and GTP, to the 40S subunit. In the presence of mRNA, mRNA binding factors, and 60S ribosomal subunits (complete initiation assay), eIF-2 was released from the 40S initiation complex in the subunit joining reaction. GTP also was released in this step and probably was hydrolyzed in the reaction that is dependent upon eIF-5 and the 60S subunit. The function of phosphorylated eIF-2 in initiation of protein synthesis is discussed.     

Characterization of double-stranded-RNA-activated kinase that phosphorylates α subunit of eukaryotic initiation factor 2 (eIF-2α) in reticulocyte lysates

Incubation of reticulocyte lysates with low levels of double-stranded (ds) RNA (1-20 ng/ml) activates a cAMP-independent protein kinase (dsI) that phosphorylates the α-subunit (M_r 38,000) of initiation factor 2 (eIF-2) and produces an inhibition of protein chain initiation similar to that caused by heme deficiency. Activation of dsI from its latent precursor takes place on the ribosomes and requires ATP. dsI can also be activated in ribosomal salt washes and in partially purified preparations of the latent precursor of dsI. In all preparations, activation is accompanied by the ds RNA-dependent phosphorylation of a polypeptide doublet that migrates as bands of 67 and 68.5 kilodaltons (67/68.5) in NaDodSO₄/acrylamide gels. The rate of phosphorylation of these components in a ribosome salt wash is more rapid than the ds RNA-dependent phosphorylation of eIF-2α. Other polypeptides in the salt wash also undergo ds RNA-dependent phosphorylation, but their significance is not clear. All of these phosphorylations are prevented by high concentrations of poly(I)·poly(C)(20 μg/ml), but not by an antiserum specific for the heme-regulated eIF-2α kinase. Both the latent and activated forms of dsI have been partially purified from a 0.5 M KCl wash of reticulocyte ribosomes. The two species have similar M_rs (≈120,000) and sedimentation coefficients (≈3.75 S), which suggests that activation of dsI probably does not involve extensive changes. By comparison, the heme-regulated eIF-2α kinase has an M_r of ≈160,000 and sediments at ≈6.6 S. However, in vitro, dsI and HRI both phosphorylate the same site(s) of eIF-2α. Purified dsI inhibits protein synthesis in hemin-supplemented lysates with the same kinetics induced by the addition of ds RNA; both inhibitions are reversed by eIF-2. dsI that has been activated in the salt wash and then purified does not require ds RNA for expression and no longer displays phosphorylation of the 68.5/67 doublet, which appears to occur only during activation. The data support the view that this component(s) may be the eIF-2α kinase activated by ds RNA.     

Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNA(f) binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: (i) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; (ii) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition-i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; (iii) both inhibitions are prevented or reversed by eIF-2; (iv) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; (v) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of elF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.     

Regulation of double-stranded RNA-activated eukaryotic initiation factor 2α kinase by type 2 protein phosphatase in reticulocyte lysates

Protein synthesis initiation in reticulocyte lysates is inhibited by low concentrations (1-20 ng/ml) of double-stranded RNA (ds RNA) due to the activation of a ds RNA-dependent cAMP-independent protein kinase (ds I) that phosphorylates the α subunit of the eukaryotic initiation factor eIF-2. In lysates, ds I is present in the latent inactive form and is associated with the ribosome complement. Latent ds I is solubilized by extraction with high-salt buffers and can be purified in its latent form. Activation of purified latent ds I requires ds RNA and ATP and is accompanied by the ds RNA-dependent autophosphorylation of a polypeptide doublet of 70,000 and 72,000 daltons (“70k/72k”), which represent different phosphorylated states of the same polypeptide. These are phosphorylated in the sequence 70k→72k; increased phosphorylation of 72k is associated with increased ds I activation. Lysates (or Sepharose 6B ribosomes) treated with ds RNA display a similar ds I phosphoprotein profile, and this is accompanied by the phosphorylation of endogenous eIF-2α (38,000 daltons). Delayed ³²P pulses in ds RNA-inhibited lysates indicate that the phosphates on ds I and eIF-2α turn over. Under defined conditions, activated ds I in lysates is selectively dephosphorylated by endogenous protein phosphatase(s), and this is accompanied by the dephosphorylation of eIF-2α. Similarly, purified activated ds I is rapidly dephosphorylated by unfractionated lysate protein phosphatase(s) and by type 2 protein phosphatase but not by type 1 protein phosphatase. The dephosphorylation of ds I occurs in the sequence 72k→70k and is correlated with ds I inactivation. The heat-stable protein phosphatase inhibitor-2, which selectively blocks type 1 protein phosphatase, does not significantly affect the dephosphorylation of ds I by type 2 protein phosphatase or by unfractionated lysate phosphatases. The data support the conclusion that a ds I phosphatase activity with type 2 characteristics is involved in the regulation of ds I activity.     

Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates.

Studies in intact rabbit reticulocytes and reticulocyte lysates provide further evidence of a functional role for the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) in the regulation of initiation of protein synthesis in eukaryotic cells. In intact reticulocytes treated with isonicotinic acid hydrazide to inhibit heme synthesis, the phosphorylation of eIF-2 alpha was significantly greater than in control cells. In heme-deficient reticulocyte lysates and in lysates treated with double-stranded RNA, significant phosphorylation of eIF-2 alpha occurred prior to the onset of inhibition of protein synthesis; a large proportion, however, of the total eIF-2 alpha remained unphosphorylated. These findings indicate that a modest concentration of phosphorylated eIF-2 alpha can suffice to inhibit initiation, and they suggest that one of the factors with which eIF-2 must interact may be rate limiting, especially when eIF-2 alpha is phosphorylated.     

The 90-kDa component of reticulocyte heme-regulated eIF-2 alpha (initiation factor 2 alpha-subunit) kinase is derived from the beta subunit of spectrin.

Antibodies from three different lines of monoclonal hybridomas crossreact with both the beta subunit of spectrin and the 90-kDa peptide present in highly purified preparations of the heme-controlled eIF-2 alpha (initiation factor 2 alpha-subunit) kinase from rabbit reticulocytes. Antibodies from two of the three lines enhance the enzymatic activity of the kinase preparation for phosphorylation of the alpha subunit of eukaryotic translational initiation factor 2 (eIF-2) and for phosphorylation of the 100-kDa peptide thought to be a peptide of the kinase that is phosphorylated during its activation. Also, it is shown that both the beta subunit of spectrin and the 90-kDa peptide can be phosphorylated by two protein kinases from reticulocytes, the catalytic subunit of cAMP-dependent protein kinase and a cAMP-independent protein kinase similar to casein kinase II. Furthermore, a phosphorylated 90-kDa peptide can be derived from phosphorylated beta subunit of spectrin by tryptic proteolysis. We conclude that the 90-kDa peptide is derived by proteolysis from the beta subunit of spectrin, probably from its carboxyl terminus, and suggest that the heme-sensitive eIF-2 alpha kinase, like the 56-kDa phosphatase [Wollny, E., Watkins, K., Kramer, G. & Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492], is associated with an element of the membrane skeleton in intact reticulocytes.     

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.     

Regulation of protein synthesis: Activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2

Incubation of reticulocyte lysates or isolated crude ribosomes with low levels of double-stranded RNA (0.1-10 ng/ml) induces the formation of an inhibitor of protein synthesis initiation similar to that observed in heme deficiency. The inhibitor is associated with a cyclic AMP-independent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylates the small polypeptide (38,000 daltons) of the eukaryotic initiation factor eIF-2. Activation of the inhibitor requires ATP in addition to double-stranded RNA and is accompanied by the phosphorylation of a 67,000-dalton polypeptide of unknown function. The inhibitor remains associated with the ribosomes during high-speed sedimentation. Once formed, the ribosome-associated inhibitor phosphorylates eIF-2 and inhibits protein synthesis in the absence of double-stranded RNA. Inhibition is prevented by exogenous eIF-2. The bound inhibitor can be solubilized by extraction with 0.5 M KCl. The soluble inhibitor preparation retains the ability to phosphorylate the small polypeptide of eIF-2 and to inhibit protein synthesis. Untreated crude ribosomes also contain cyclic AMP-independent protein kinase activities that phosphorylate the middle polypeptide (49,000 daltons) of eIF-2 and several polypeptide subunits of eIF-3 (160,000, 125,000, and 65,000 daltons); these kinase activities are not affected by double-stranded RNA and do not inhibit protein synthesis.     

Protein synthesis in rabbit reticulocytes: a study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates.

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.     

Regulation of protein synthesis in rabbit reticulocyte lysates: characteristics of inhibition of protein synthesis by a translational inhibitor from heme-deficient lysates and its relationship to the initiation factor which binds Met-tRNAf.

In heme-deficient reticulocyte lysates a translational inhibitor which regulates protein synthesis is formed or activated. To define the mechanism of action of the translational inhibitor (RI), RI was partially purified. We have utilized the isolated RI to examine its relationship to the translational inhibitor formed in situ in heme-deficiency, some quantitative aspects of inhibition of protein synthesis, and the relationship of RI concentration to the initiation factor (IF-MP) which forms a ternary complex with Met-tRNAf and GTP (IF-MP-Met-tRNAf-GTP). The results demonstrate that the activity of isolated RI is related to the in situ heme-deficiency inhibitor by several criteria: (a) the biphasic kinetics of inhibition manifested by RI in lysates containing optimal levels of hemin are very similar to those observed in heme-deficiency, i.e., an initial period in which several rounds of protein synthesis proceed at the control rate followed by an abrupt decline in the rate of protein synthesis. (b) Both inhibitions are accompanied by the disaggreagation of polyribosomes with a concomitant increase in 80S ribosomes. (c) Both inhibitions are reversed by IF-MP. The isolated RI blocked protein synthesis in lysates at temperatures ranging from 15 degrees to 30 degrees. Although the rate of protein synthesis was a function of the temperature of incubation, the number of rounds of protein synthesis prior to shut-off was essentially the same at various temperatures. When RI was added to lysates, at increasing intervals after the start of incubation, the period of synthesis before shut-off (lag) progressively decreased. The inhibition of protein synthesis by RI was immediately reversed by the addition of IF-MP. The extent of reversal increased with increasing concentrations of IF-MP; at low levels of RI almost complete reversal of inhibition by IF-MP was obtained. However, at high levels of RI which did not appreciably increase the degree of inhibition of protein synthesis, equivalent amounts of IF-MP were less effective in reversing inhibition. These results suggest that the inhibition of protein synthesis by the isolated inhibitor involves the initiation factor IF-MP.     

Non-specific stimulation of cell-free protein synthesis by a dialyzable factor isolated from reticulocyte initiation factors ("iRNA").

The specificity of a dialyzable component, isolated from rabbit reticulocyte initiation factors, in stimulating protein synthesis was examined. It appeard that his factor (hereafter designated as "iRNA") was able to restore the activity of initiation factor preparations that were inactivated by dialysis. The "iRNA" from reticulocytes stimulated polypeptide synthesis directed by the homologous globin mRNA as well as heterologous lens crystallin mRNAs. No selectivity in its stimulating action with regard to the type of mRNA studied could be observed. The source of ribosomes or supernatant enzymes did not influence the effect of "iRNA". However, in an ascites lysate that was dependent on the addition of initiation factors, "iRNA" increased polypeptide formation only marginally, suggesting that in this lysate a similar factor was already present in an active form. It is concluded that "iRNA" may regulate protein synthesis, but without exhibiting specificity towards mRNAs, at least those tested so far.     

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet·40S ribosomal subunit complexes

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF.We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.     

Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2alpha kinase

Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2alpha kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2alpha. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.     

Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.     

Phosphorylation of proteoglycans from human articular cartilage by a camp-dependent protein kinase

Purified proteoglycan subunits from human articular, bovine articular and nasal cartilages, and a rat chondrosarcoma were phosphorylated in vitro by beef heart cAMP-dependent protein kinase in the presence of γ³²P-ATP. In these experiments, a maximum of 1.7 moles of ³²P were incorporated per mole of proteoglycan from human cartilage. Phosphorylation was dependent on the presence of cAMP. Analysis by autoradiography revealed that serine residues in the core protein of the proteoglycan were the sites of phosphorylation. Treatment of proteoglycan subunits with chondroitinase ABC and alkaline phosphatase prior to reaction with cAMP-dependent protein kinase increased the incorporation of ³²P by 12–30% when compared with untreated proteoglycans. These data indicate that proteoglycans in cartilage can be phosphorylated by cAMP-dependent protein kinase.     

Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells.

The double-stranded RNAs (I)n X (C)n and (A)n X (dUfl)n (dUfl is 2'-fluoro-2'-deoxyuridylic acid) have been compared as inhibitors of translation in cell-free systems from interferon-treated mouse L cells and from rabbit reticulocytes. In the interferon-treated mouse L-cell system, both double-stranded RNAs stimulated kinase activity, leading to phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha (eIF-2 alpha), but only (1)n X (C)n activated the (2'-5')-oligoadenylate synthetase. Moreover, in this system, (I)n X (C)n, but not (A)n X (dUfl)n, inhibited translation. Both (A)n X (dUfl)n and (I)n X (C)n also activated the rabbit reticulocyte kinase to phosphorylate protein P1 and eIF-2 alpha, but, in contrast to mouse L-cell systems, both (A)n X (dUfl)n and (I)n X (C)n were potent inhibitors of translation in reticulocyte lysates. These results indicate that protein P1 and eIF-2 alpha phosphorylation are not sufficient to cause inhibition of protein synthesis in interferon-treated mouse L-cell extracts. They further suggest that protein synthesis inhibition by (I)n X (C)n in extracts of interferon-treated L cells correlates better with activation of (2'-5')-oligoadenylate synthetase than with activation of the protein P1 and eIF-2 alpha kinase.