EIF-2 kinases

Mammalian cells contain at least one additional eIF-2-specific kinase, known as the RNA-specific eIF-2 kinase (abbreviated PKR). PKR is induced by interferon and can be activated by minimal concentrations of double-stranded RNA. Activated PKR phosphorylates eIF-2, which, according to the above mechanism, switches off protein biosynthesis. 

Fig. 1.56. Control of eIF-2 by phosphorylation. Phosphorylated eIF-2 GDP binds strongly to eIF-2B without nucleotide exchange occurring. Initiation of protein biosynthesis is not possible in this case.In reticulocytes, eIF-2 is subject to phosphorylation by the heme-regulated eIF-2-kinase (HRI). The activity of the dimeric HRI is regulated via the heme concentration. Another protein kinase that can phosphorylate and regulate eIF-2 is the RNA-dependent eIF2a-kinase (PKR). The latter is induced by interferons and activated by double stranded RNA.

Wek, R.C. eIF-2 kinases regulators of general and gene-spedfic translation initiation (1994) Trends Biochem. Sd. 19, 491-496... 

The protein kinase H RI (heme regulated eIF-2 kinase) was first identified in studies on the regulation of protein biosynthesis in erythroid cells. A decrease in the heme concentration in reticulocytes leads to inhibition of globin synthesis at the level of translation. This regulation mechanism ensures that only so much globin is produced as is heme available. If the level of heme drops, then HRI becomes activated. The activated HRI phosphorylates the eIF-2a subunit, which in turn shuts off protein biosynthesis (Fig. 1.48). The mechanism of regulation of HRI kinase by heme is not well understood. Heme binding sites have been identified on the N-terminus and the kinase domain of HRI. 

The protein kinase PKR (RNA-specific eIF-2 kinase) is regulated by binding of dsRNA and by interferon on the level of expression. PKR contains two dsRNA binding sites and it is thought that dsRNA binding disrupts inhibitory interactions in PKR leading to its activation. The activation of PKR by dsRNA and its induction by interferon identify PKR as a component of the cellular anit-viral defense. Consistent with this notion, a large number of viruses express inhibitors of PKR. 

Table 1). Two third of this eIF-2 kinase (PK-i) is in the cell sap and one third in ribosomal extracts, the latter containing most of protein 67 and 35 substrates (4) PK-i is not retained on DEAE-cellulose, elutes from phosphocellulose (pH6.7) at 330mMKCl, and from hydroxylapatite at 200mM phosphate overall purification was about 500- old. The endogenous protein 67 phosphorylation activity was still present in the purified PK-i (Figure 2) as well as a dsMA-dependent histone kinase selective for the arginine-rich... 

PsMA stimulates the rate, but not the extent of protein 67 phosphorylation, and is required for eIF-2 phosphorylation. This requirement can be partly explained by an activation of the eIF-2 kinase. 

The eIF-2-kinase of interferon-treated cells (PK-i) is not sensitive to cyclic nucleotides or to calcium, but is activated by pre-incubation with ATP and low dsMA. Study of the activation process is facilitated by the fact that high concentrations of dsRBA (20 xg/ml) inhibit the eIF-2-kinase unless it is pre-incubated with both ATP and low dsEITA (11) The activated protein kinase can therefore be defined as high-dsRBA resistant. 

The picture that emerges from these studies thus is that, in the absence of heme, an inactive eIF-2 kinase is converted to an active form that phosphorylates the a-subunit of eIF-2. The phosphorylated factor is normally active in initiation of translation, but the released eIF-2/GDP complex is unable to undergo GDP/GTP exchange that is catalyzed by eIF-2B. Consequently, recycling of eIF-2 is prevented, and initiation comes to a halt. 

A dsRNA-activated eIF-2 kinase that resembles the dsRNA-de-pendent kinase present in reticulocyte lysates (Farrell et al., 1977) has been purified from interferon-treated L and ascites cells (Sen et al., 1978 Kimchi et al., 1979 Samuel, 1979). The level of this kinase increases between three- and ten-fold after treatment with interferon interferons a, p, and 7 all induce the enzyme (Hovanessian et al., 1980 Lengyel, 1982a, ). Indeed, addition of dsRNA to intact, interferon-treated cells induces the phosphorylation of endogenous elF-2, while addition to control cells does not (Gupta, 1979). 

These observations can account for the greater sensitivity of translation to dsRNA in extracts from interferon-treated cells. Apparently, interferon treatment induces a rise in the level of dsRNA-dependent eIF-2-kinase, but since the enzyme is inactive in the absence of sufficient dsRNA, protein synthesis in interferon treated, uninfected cells continues normally. During infection, however, virus-generated dsRNA potentiates the kinase and the resulting extensive phosphorylation of eIF-2 leads to a general inhibition of initiation of translation. As noted for reticulocyte lysates, in extracts of interferon-treated cells, too, there is no good correlation between the extent of phosphorylation of eIF-2 and inhibition of translation (Jacobsen et al., 1983). 

In reticulocyte lysates, on the other hand, it is the activation of the eIF-2 kinase that seems primarily responsible for the observed inhibition, here at initiation of translation (Mizuno, 1977 Bonanou-Tzedaki et al., 1978 Ernst et al., 1982). 

Levin, D. H., Petryshyn, R., and London, I. M., 1981, Characterisation of purified double-stranded RNA-activated eIF-2 kinase from rabbit reticulocytes, J. Biol. Chem. 256 7638. 

IFN-a, -P and -y are all known to induce the enzyme in various animal cells. However, in human epithelial cells the kinase is induced only by type I interferons, whereas none of the interferons seem capable of inducing synthesis of the enzyme in human fibroblasts. The purified kinase is highly selective for initiation factor eIF-2, which it phosphorylates at a specific serine residue. 

The ability of interferons (especially type I interferons) to induce an antiviral state is unlikely to be solely dependent upon the enzymatic mechanisms discussed above. Furthermore the 2 -5 A synthetase and eIF-2a kinase systems may play important roles in mediating additional interferon actions. The ability of such systems to stall protein synthesis in cells may play a role in interferon-induced alterations of cellular differentiation or cell cycle progression. They may also be involved in mediating interferon-induced anti-proliferative effects on various transformed cells. 

Inactivation of eukaryotic translation factors by covalent modification is one of the few mechanisms known to regulate the rate of translation. Specific protein kinases have been identified that phosphorylate and inactive both eIF-1 and EF-2. The significance of the phosphorylation of EF-2 as a regulatory mechanism of the elongation rate is still not clear, but the phosphorylation of eIF-2 appears to be a general mechanism for controlling translation initiation in many cells. 

The regulation of translation through the phosphorylation of eIF-2 is best understood as it operates in the rabbit reticulocyte. Two protein kinases specific for the a subunit of eIF-2 have been purified from reticulocytes. One of these kinases, termed the heme-regulated inhibitor repressor (HRI), serves to coordinate the rate of hemoglobin synthesis (more than 90% of the total protein synthesized in the reticulocyte is hemoglobin) with the availability of hemin (the... 

The second eIF-2-specific kinase appears to be present at low levels in most mammalian cells. This kinase is activated by double-stranded RNA and for this reason has been named the double-stranded RNA-activated inhibitor (DAI). DAI may play a role in defending cells against invasion by viruses. 

HRI and DAI are different proteins, but both phos-phorylate the same amino acid residue in a subunit of eIF-2, and, in consequence, both kinases inhibit protein synthesis by the same mechanism. Phosphorylated eIF-2 is capable of... 

Regulation of protein synthesis in the rabbit reticulocyte. The vast majority of the protein synthesized in the rabbit reticulocyte is hemoglobin. The gross rate of protein synthesis in the reticulocyte is controlled indirectly by the concentration of heme. Heme inactivates a kinase that would otherwise inactivate the initiation complex involving eIF-2 and eIF-2B. The kinase phosphorylates the eIF-2 factor, making it impossible for the eIF-2-eIF-2B complex to exchange GDP for GTP. 

Initiation mRNAs with AUG initiation code fMet-tRNAi IF-1, IF-2, and IF-3 initiation factors, GTP, Mg2+ 30 and 50 S mRNAs with AUG initiation code Met-tRNA eIF3, eIF2, eIF4, eIF5 initiation factors GTP, Mg2+, eIF-2 protein kinase 40 and 60 S ribosomal subunits... 

High concentrations of hemin inhibit the transport of ALA synthase into the mitochondria, where one of the substrates, succinyl-CoA, is formed. Thus, heme synthesis is inhibited until enough globin is made to react with any heme already formed. Low concentrations, or the absence, of hemin is the signal that globin is not needed this protein (and, therefore, globin) synthesis is inhibited. In the absence of hemin, a protein kinase is activated this phosphorylates an initiation factor of (eukaryotic) protein synthesis, eIF-2, which then inhibits polypeptide chain initiation (Chap. 17) and hence inhibits globin synthesis. 

Chen JJ, London IM. Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase. Trends Biochenu Sci. 1995 20 105-108. 

Type I (IFN-a/P) and type II (IFN-y) IFNs are major lines of defense against viral infection. IFNs mediate direct antiviral effector mechanisms that inhibit multiple steps of viral replication (Samuel 1991 Vilcek and Sen 1996). For example, 2, 5 -oligoadenylate synthetase (2, 5 -OAS) activates ribonuclease L, which degrades mRNA and limits the accumulation of viral transcripts. Protein kinase R blocks translation of viral transcripts by phosphorylating translation initiation factor eIF-2. Mx proteins block influenza, vesicular stomatitis virus, and herpes simplex virus replication by an unknown mechanism. 

Kitajewski J, Schneider R, Safer B, Munemitsu S, Samuel C, Thimmappaya B, Shenk T (1986) Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase. Cell 45 195-200 Korner H, Burgert H-G (1994) Down-regulation of HLA antigens by the adenovirus type 2 E3/19K protein in a T-lymphoma cell line. J Virol 68 1442-1448 Korner H, Fritzsche U, Burgert H-G (1992) Tumor necrosis factor alpha stimulates expression of adenovirus early region 3 proteins implications for viral persistence. Proc Natl Acad Sci USA 89 11857-11861... 

Fig. 3. The effect of eRF on ternary complex formation. (A) The assay for ternary complex formation was performed according to Gupta et al. (12). A typical reaction mixture of 25 /nl contained 20 mM HEPES-KOH, pH 7.6,120 mM KAc, 2 mM MgAc, 1 mM ATP, 0.4 mM GTP, 5 mM creatine phosphate, 0.05 unit of creatine kinase, 1 mM dithiothreitol, 5 pmole PH]Met-tRNA, and 1.5 pmole eIF-2 and eRF as indicated. Incubation was for 10 minutes at 37°C. The reaction was stopped by adding buffer with 0 mM Mg, followed by filtration through cellulose-nitrate filters. The filters were washed twice, dried, and counted. (B) Temaiy complex formation with 2.5 pmole eIF-2 (O) or 3 pmole eIF-2 eRF ( ). The filters were washed with 0 mM Mg because the ternary complex is most stable at this Mg concentration.

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