eIF2alpha Kinase Research Articles

Identification of spectrin-related peptides associated with the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 kinase and of Mr 95,000 peptide that appears to be the catalytic subunit.

An isolation procedure for the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 (eIF-2 alpha) kinase is described which yields different fractions with kinase activity. Each is associated with a different spectrin-related peptide as identified by anti-spectrin monoclonal antibodies. The most abundant of these peptides is the Mr 90,000 species characterized previously (Kudlicki, W., Fullilove, S., Kramer, G., and Hardesty, B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5332-5336). Association with the spectrin-related peptides appears to account for the heterogeneity of the enzyme during its isolation and for its highly asymmetric structure. Isolated alpha or beta spectrin subunits as well as the separated homogeneous Mr 90,000 peptide cause an increase in the initial rate of eIF-2 alpha phosphorylation that is related to a decrease in Km with little or no effect on Vmax for the phosphorylation reaction. Fractionation of highly purified eIF-2 alpha kinase preparations using affinity chromatography on monoclonal anti-spectrin antibodies has separated eIF-2 alpha kinase activity from the Mr 100,000 phosphopeptide which copurifies with the kinase during all other purification steps. A Mr 95,000 peptide, detectable only by photoaffinity labeling with 8-azido-[alpha 32P]ATP, is shown to be distinct from the Mr 100,000 phosphopeptide and appears to be the catalytic subunit of the eIF-2 alpha kinase.

A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.

The NH2-terminal sequence of the alpha and gamma subunits of eukaryotic initiation factor 2 and the phosphorylation site for the heme-regulated eIF-2 alpha kinase.

Rabbit reticulocyte eukaryotic initiation factor 2 was phosphorylated with the heme-regulated alpha subunit of eukaryotic initiation factor 2 kinase, and then the individual subunits were resolved by reversed-phase high performance liquid chromatography. Phosphorylated and unphosphorylated forms of the alpha subunit also were well resolved. The NH2-terminal sequences of intact alpha and gamma subunits were determined. No sequence was obtained from the beta subunit, suggesting that it may have a blocked NH2-terminus. Overlapping tryptic and chymotryptic phosphopeptides from the NH2-terminal sequence of the alpha subunit of eukaryotic initiation factor 2 were used to establish the order of amino acids 1-52 and localized the phosphorylation site within the sequence: -Leu-Leu-Ser48-Glu-Leu-Ser51-. Subdigestion of a tryptic fragment with chymotrypsin generated only phosphopeptides that appeared to terminate at leucine 50, indicating phosphorylation at serine 48.

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60 leukemic cells.

Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.

Fate of reversing factor during restoration of protein synthesis by hemin or GTP in heme-deficient reticulocyte lysates.

The inhibition of protein synthesis in hemedeficient reticulocyte lysates is reversed by the addition of hemin (20 microM) or MgGTP (2 mM). The rate of recovery is rapid and approaches control kinetics within a few minutes after the addition of either component. The restoration of protein synthesis is dependent upon the availability of functional reversing factor (RF). The fate of RF was monitored during recovery by using a method that measures RF activity in the lysate under physiological conditions. In the fully inhibited lysate, RF is sequestered in a nondissociable 15S [RF . eIF-2(alpha P)] complex (where eIF-2 indicates eukaryotic initiation factor 2) in which RF activity is not functional and cannot be assayed. The first step in the rescue of protein synthesis in inhibited lysates by hemin or MgGTP is the inhibition of heme-regulated eIF-2 alpha kinase, which enables endogenous phosphatase to dephosphorylate eIF-2(alpha P) and [RF . eIF-2(alpha P)]. The release of approximately 50% of the sequestered RF activity is sufficient to support optimal kinetics of recovery. Hemin and MgGTP both reverse inhibition by blocking the activation and/or activity of heme-regulated eIF-2 alpha kinase in the lysate. The conclusion that MgGTP exerts its effect on eIF-2 alpha kinase is supported by several in vitro findings: (i) 2 mM MgGTP inhibits the autophosphorylation of purified heme-regulated eIF-2 alpha kinase and abolishes its ability to phosphorylate eIF-2 alpha; (ii) 2 mM MgGTP cannot displace GDP in the binary complexes [eIF-2 . GDP] or [eIF-2(alpha P) . GDP] by mass action; and (iii) RF in the [RF . eIF-2(alpha P)] complex is not dissociated by 2 mM MgGTP.

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.

Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.

Kinetics of dephosphorylation of eIF-2(alpha P) and reutilization of mRNA.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) causes mRNA to accumulate in 48 S complexes containing Met-tRNAf and eIF-2(alpha P). When the eIF-2 alpha kinase is inhibited by 2-aminopurine, the mRNA is slowly transferred from 48 to 80 S initiation complexes after an initial lag. The cause of this lag was examined by investigating whether mRNA and Met-tRNAf dissociated from 48 S complexes before binding to 80 S. Both compounds were quantitatively transferred from 48 to 80 S complexes after addition of 2-aminopurine and the eIF-2(alpha P) bound to 48 S complexes was dephosphorylated after an initial lag more slowly than unbound eIF-2(alpha P), which was rapidly dephosphorylated. the eIF-2(alpha P) in isolated 48 S complexes was slowly dephosphorylated by partially purified lysate phosphatases, whereas free eIF-2(alpha P) was readily dephosphorylated. These results indicated that 48 S complexes could directly join to a 60 S ribosomal subunit after eIF-2(alpha P) dephosphorylation. The lag and slow kinetics of dephosphorylation of eIF-2(alpha P) bound to 48 S complexes accounted for the slow transfer of mRNA from 48 to 80 S complexes. Moreover, the mRNA bound to 48 S complexes was more susceptible to cleavage by an endonuclease than mRNA in polyribosomes, as shown by activating the (2'-5')oligo(A)-dependent endonuclease. This finding is discussed in view of the possible role of eIF-2 alpha kinase and endonuclease in the inhibition of viral mRNA translation in interferon-treated cells.

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells.

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.

Indirect inactivation of rabbit reticulocyte initiation factor eIF-2 by helenalin and bis(helenalinyl) malonate.

Helenalin and bis(helenalinyl) malonate, sesquiterpene lactones that react primarily with exposed sulfhydryl groups, were shown to be equally effective inhibitors of endogenous protein synthesis in rabbit reticulocyte lysates. By use of partially fractionated systems, it was possible to show that helenalin preferentially inhibited the conversion of the ternary initiation complex to the 48S preinitiation complex. Previous experiments have shown that this preferential inhibition is due to selective inactivation of eIF-3 [Williams, W. L., Chaney, S. G., Willingham, W., Considine, R. T., Hall, I. H., & Lee, K.-H. (1983) Biochim. Biophys. Acta 740, 152-162]. Bis(helenalinyl) malonate was much less active as an inhibitor of 48S complex formation than helenalin and clearly did not possess sufficient activity in that assay to explain its effectiveness as a protein synthesis inhibitor in whole lysates. Kinetic studies also showed a clear difference between the mechanism of action of these two drugs. Bis(helenalinyl) malonate inactivated protein synthesis in reticulocyte lysates only after a lag of 10 min, and the inhibition of protein synthesis could be completely reversed by the addition of 5 mM cAMP. Helenalin showed more complex kinetics. While full inhibition only occurred after a lag of 10-15 min, a partial inhibition was observed from very early times. cAMP at 5 mM was only partially able to reverse inhibition by helenalin. Phosphorylation studies showed that both helenalin and bis(helenalinyl) malonate were equally effective at activating eIF-2 alpha kinase and indirectly causing phosphorylation of eIF-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.

Effects of glucose 6-phosphate and hemin on activation of heme-regulated eIF-2 alpha kinase in gel-filtered reticulocyte lysates.

In heme-deficient reticulocyte lysates, protein synthesis initiation is inhibited due to the activation of a heme-regulated protein kinase which blocks protein synthesis by the specific phosphorylation of the alpha-sub-unit of eukaryotic initiation factor 2 (eIF-2 alpha). The restoration of synthesis requires both hemin and glucose-6-P (Ernst, V., Levin, D. H., and London, I. M. (1978) J. Biol. Chem. 253, 7163-7172). The sugar phosphate fulfills two functions in initiation: (i) the generation of NADPH, and (ii) an effector function in some step in initiation. This latter effect is readily demonstrated in lysates depleted of low molecular weight components by filtration in dextran gels. In gel-filtered lysates, linear protein synthesis is sustained only by the addition of both hemin (20 microM) and glucose-6-P (or 2-deoxyglucose-6-P) (50-500 microM). The omission of either component gives rise to inhibitions which are characterized by the activation of heme-regulated eIF-2 alpha kinase and the concomitant phosphorylation of both endogenous heme-regulated eIF-2 alpha kinase and endogenous eIF-2 alpha, indicating that glucose-6-P is involved in the regulation of heme-regulated eIF-2 alpha kinase. In support of this, we find (a) that gel-filtered lysates incubated with hemin but depleted of glucose-6-P produce sufficient heme-regulated eIF-2 alpha kinase to inhibit protein synthesis when mixed with normal hemin-supplemented lysates; (b) the inhibitions of protein synthesis produced by heme-regulated eIF-2 alpha kinase generated either in glucose-6-P-depleted lysates or heme-deficient lysates are reversed by added eIF-2; and (c) the eIF-2 alpha kinase activities formed in the absence of either hemin or glucose-6-P are both neutralized by an anti-heme-regulated eIF-2 alpha kinase antiserum. We conclude that the physiological activation of heme-regulated eIF-2 alpha kinase is controlled by both hemin and glucose-6-P.

Phosphorylation of initiation factor eIF-2 alpha, binding of mRNA to 48 S complexes, and its reutilization in initiation of protein synthesis.

The formation of 80 S initiation complexes containing labeled viral mRNA was drastically inhibited when mRNA binding assays were carried out with reticulocyte lysate preincubated with double-stranded RNA (dsRNA). When the assays were analyzed by centrifugation on sucrose gradients, the mRNA incubated with lysate pretreated with dsRNA sedimented as a 48 S complex. Met-tRNA, GDP, and phosphorylated initiation factor eIF-2(alpha P) were shown to co-sediment with the 48 S complex. Therefore, the formation of this complex was attributed to the phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase. These observations suggested that mRNA could bind to a 40 S ribosomal subunit containing Met-tRNAf, GDP, and eIF-2(alpha P), but the joining of a 60 S ribosomal subunit was inhibited. When the 48 S complex was isolated and incubated with lysate without added dsRNA, the mRNA could form 80 S initiation complexes. The shift of mRNA from 48 S to 80 S complexes was also observed when the eIF-2 alpha kinase activity was inhibited by the addition of 2-aminopurine. This shift was quite slow, however, when compared to the rate of binding of free mRNA to 80 S initiation complexes. The 2-aminopurine was effective in reversing the inhibition of protein synthesis by dsRNA and in maintaining a linear rate of protein synthesis for 3 h in lysates. Without added 2-aminopurine, protein synthesis was inhibited after 90 min even in lysates supplemented with hemin and eIF-2(alpha P) was detected in these lysates. This finding indicated that eIF-2 alpha phosphorylation could be in part responsible for limiting the duration of protein synthesis in mammalian cell-free systems.

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.

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

A procedure is described by which the 38,000-dalton alpha subunit of native eukaryotic peptide initiation factor 2 (eIF-2) can be cleaved by trypsin to yield a 34,000-dalton fragment and a peptide of about 4,000 daltons after elimination of the beta subunit. Under nondenaturing conditions the 4,000-dalton peptide remains bound to the modified eIF-2 and still can be phosphorylated by the heme-controlled eIF-2 alpha kinase from reticulocytes. All of the phosphorylation sites for this protein kinase are located on the 4,000-dalton peptide. The ability of eIF-2 to form a ternary complex with GTP and Met-tRNAf and the ability to promote binding of Met-tRNAf to 40S ribosomal subunits are lost differentially during the proteolysis. Loss of te latter activity occurs rapidly and appears to be correlated with loss of the beta subunit. Loss of activity for ternary complex formation is correlated with the appearance of the 4,000-dalton peptide.

Activation and partial characterization of a human reticulocyte heme-dependent eIF-Z alpha kinase.

An inhibitor of globin translation initiation that is activated under heme-deficient conditions and whose activity is associated with phosphorylation of the 38,000 dalton (alpha) subunit of the initiation factor eIF-2 has been previously described in rabbit reticulocytes. We describe here an analogous enzyme in human reticulocytes. The human enzyme can be activated in intact reticulocytes under conditions of heme synthesis inhibition or by incubation of lysates in the absence of hemin. Addition of hemin to lysates prevents activation of the eIF-2 alpha kinase. The partially purified kinase is associated with inhibition of globin synthesis in a rabbit cell-free system and phosphorylates the small subunit of highly purified rabbit eIF-2. We conclude that human reticulocytes contain a protein kinase that is analogous to the rabbit HCR and that may play a role in clinical heme deficiency states. l

Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes.

Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes.

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase.

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.