17.1. Importance of Translational Control in Eukaryotes
Generally, the protein production of the eukaryotic cell can be regulated at several levels: (1) issuing an encoded genetic information in the form of RNA, i.e. transcription; (2) processing of the RNA and its intracellular transport (mostly from the nucleus to the cytoplasm); (3) reading the messenger RNA formed, i.e. translation; (4) degradation of the product of translation; (5) degradation of messenger RNA. Ribosomes may be involved in some of them, such as control of mRNA degradation, but the translational regulation of protein production is the main one which directly concerns ribosomes. How important is this level of regulation of protein synthesis in Eukaryotes?
The relative metabolic stability of most eukaryotic mRNAs makes translational control particularly important in the general pattern of protein synthesis regulation. Specifically, along with the signals for mRNA activation, i.e. for the initiation of translation, the signals for the arrest of translation become necessary. Hormonal regulation of translation provides examples of both the switching on and the shutting down of translation of certain mRNA species. Heat shock triggers the synthesis of a few special proteins, while translation of most of the pre-existed cellular mRNAs becomes ceased or reduced; cell recovery at a normal temperature is accompanied by reactivation of major mRNA translation and cessation of heat-shock protein synthesis. Oogenesis and spermatogenesis, as well as plant seed ripening, are accompanied by inactivation and storage of mRNA which further exists in oocytes, spermatocytes or seeds in a non-translatable - masked - form. Fertilisation, as well as seed germination, results in general and selective activation of translation of the stored mRNA. In processes of embryonic development and cell differentiation the synthesis of mRNA and the accumulation of mRNA in the cytoplasm may take place long before this RNA is used in translation; specific signals selectively activate the corresponding mRNA species at proper stages.
Several systems of translational regulation are known to exist. They can be subdivided, rather conditionally, into two groups: the systems for non-selective regulation of total level of translation, and the systems where the control is selective and mRNA-specific.
17.2. Total Translational Regulation 17.2.1.Regulation by Modifications of Initiation Factors
220.127.116.11.Phosphorylation of Met-tRNAj/GTP-binding Factor (eIF2)
Phosphorylation of eIF2, and specifically of its a-subunit, is one of the most used mechanisms of global translational regulation in mammalian cells, as well as in yeast (for reviews, see Jackson, 1991; Chen, 1993; Kramer et al., 1993; Clemens, 1996). Two special protein kinases both phosphorylating serine residue(s) at the N-terminal part (Ser-51, sometimes also Ser-48) of the a-subunit of eIF2 are known in mammalian cells. One called "heme-controlled repressor" (HCR) or "heme-regulated inhibitor" (HRI) is a 90 kDa (625 aa) protein present in a soluble (ribosome-unbound) form in the cytoplasm of reticulocytes and, possibly, some other mammalian cells. The other called "double-stranded RNA-activated inhibitor" (DAI or PKR) is a 68 kDa (550 aa) protein inducible in mammalian cells by interferon and sticking to ribosomes. Both kinases have some sequence homology. An interesting feature of both kinases is their capability of multiple phosphorylation (by casein kinase II) and autophosphorylation at serine and threonine residues in response to some signals this resulting in their activation. For example, the DAI is autophosphorylated and therefore activated by double-stranded RNAs or extended elements of RNA secondary structure. On the other hand, there exists a protein in mammalian cells which interacts with eIF2 and protects it from phosphorylation by the kinases; this is a p67 glycoprotein containing multiple O-linked GlcNAc residues. Also phosphatases can dephosphorylate eIF2. It is likely that all mammalian cells have some level of the eIF2 kinase activities, but at the same time they have anti-phosphorylation protection mechanisms.
In reticulocytes, both the kinase activities are quite noticeable and can be further enhanced by heme deficiency (HCR stimulation) or by double-stranded RNA (PKR stimulation). Heat shock, serum deprivation, amino acid starvation and viral infections are known to promote the kinase activities in a variety of mammalian cells. Fungi (yeast) also possess an analogous eIF2-specific kinase called GCN2 which is activated in response to similar environmental factors. When eIF2 becomes phosphorylated due to the enhancement of the kinase activities the protein synthesis is inhibited.
What is the mechanism of the inhibition? It is found that the eIF2 with phosphorylated a-subunit is quite functional in the formation of the ternary Met-tRNA:GTP:eIF2 complex and in the interactions with ribosomal particles, including ribosome-induced GTP cleavage. However, when the eIF2:GDP complex of the phosphorylated eIF2 with GDP (eIF2aP:GDP) is released from the ribosome and interacts with eIF2B (see Fig. 15.17), a very stable, unexchangeable pair of eIF2aP:eIF2B is formed. As a result, the eIF2B which is present in the cell in a limiting amount becomes sequestered by the eIF2aP. Under the conditions of eIF2B shortage the exchange of GDP for GTP on eIF2 is decelerated and the rate of initiation declines. In other words, the down-regulation of the recycling of eIF2 takes place. Hence, mammalian cells react to heme deficiency, growth factors deprivation, amino acid starvation, heat shock, or virus infection by reducing the total protein synthesis via this mechanism of the initiation rate inhibition.
The inhibition of protein synthesis in reticulocytes in response to heme deficiency is the best studied example. Several models of inducing phosphorylation of eIF2 by the heme-controlled protein kinase (HCR) have been reported. In any case, the depletion of heme is known to lead to reduction of intramolecular disulfide bridges in latent HCR, its multiple phosphorylation and autophosphorylation, and therefore to its activation. There are indications that HCR can directly bind heme. According to one of the models, the inactive HCR contains heme and is assembled into inactive homodimer through the formation of disulphide bridges (Fig. 17.1 A). The depletion of heme could be responsible for inducing reduction of the bridges, accompanying phosphorylation and autophosphorylation of the protein and dissociation into active monomers. The phosphorylated active kinase further phosphorylates eIF2.
Another model is based on the observation that HCR can interact with the heat shock protein HSP90 and the interaction is directly involved in the process of activation of HCR under stress conditions. This model suggests that the heme-bound HCR is associated with HSP90 into inactive heterodimer, with intramolecular disulphide bonds in each subunit (Fig. 17.1 B). Upon removal of heme, the heterodimer reversibly dissociates, and the subsequent phosphorylation of the monomeric HCR and HSP90 results in irreversible dissociation and activation of HCR.
An alternative model implies that the heme-controlled kinase (HCR) is already autophosphorylated and active in normal reticulocyte cytoplasm, but the glycoprotein p67 protects eIF2, by binding to it, from the enzymatic attack (Gupta et al., 1993). In this case, the absence of heme induces deglycosylation of p67 and its subsequent degradation, thus permitting the attack of HCR on eIF2. All these models may be not mutually exclusive, and it could be possible that each mechanism works in reticulocyte to some extent.
It is interesting that p67 seems to be present in all mammalian cells and everywhere protects eIF2 from phosphorylation. It is also deglycosylated and subsequently degraded, e.g., in response to growth factors deprivation; this leads to phosphorylation of eIF2 which can contribute to the protein synthesis reduction under these conditions. On the contrary, mitogens induce an increase of p67, and this correlates with the increase of total protein synthesis. It is not clear yet which of the two kinases, HCR or PKR, takes the main part in the phosphorylation of eIF2 as a result of growth factors deprivation, amino acid starvation or heat shock.
The inhibition of total protein synthesis by the double-stranded RNA-activated kinase (DAI or PKR) during viral infection is another well studied example of global translational regulation (for reviews, see Mathews, 1996; Schneider, 1996; Katze, 1996). First of all, many viruses induce the production of interferon in mammalian cells. In its turn, the interferon stimulates the synthesis of the p68-kinase (PKR) in the targeted cells. The cells acquire the so-called "antiviral state". The penetration of a virus into such a cell and the appearance of long double-stranded RNA regions in the cell immediately activate the pre-synthesised PKR, seemingly as a result of direct interaction of a double-stranded region or fragment with the kinase. This leads to the phosphorylation of eIF2 and, thus, the reduction of the initiation rate. This event may be significant to the inhibition of synthesis of viral components, among other events induced by interferon.
As in the case of HCR, the dsRNA-activated kinase (PKR) is inactive until phosphorylated. In contrast to HRC, however, the inactive PKR is monomeric. Its N-terminal part contains two dsRNA-binding domains. When two PKR molecules are bound with dsRNA side-by-side they seem to interact with each other resulting in their dimerization and mutual phosphorylation or autophosphorylation. This makes them active PKR molecules and induce their dissociation (Fig. 17.2). Once PKR is phosphorylated its kinase activity becomes independent of dsRNA. The active (phosphorylated) monomeric PKR attacks eIF2.
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