Roles of the translationally controlled tumour protein (TCTP) and the double-stranded RNA-dependent protein kinase, PKR, in cellular stress responses.

Translationally controlled tumour protein (TCTP) is a highly conserved protein present in all eukaryotic organisms. Various cellular functions and molecular interactions have been ascribed to this protein, many related to its growth-promoting and antiapoptotic properties. TCTP levels are highly regulated in response to various cellular stimuli and stresses. We have shown recently that the double-stranded RNA-dependent protein kinase, PKR, is involved in translational regulation of TCTP. Here we extend these studies by demonstrating that TCTP is downregulated in response to various proapoptotic treatments, in particular agents that induce Ca(++) stress, in a PKR-dependent manner. This regulation requires phosphorylation of protein synthesis factor eIF2alpha. Since TCTP has been characterized as an antiapoptotic and Ca(++)-binding protein, we asked whether it is involved in protecting cells from Ca(++)-stress-induced apoptosis. Overexpression of TCTP partially protects cells against thapsigargin-induced apoptosis, as measured using caspase-3 activation assays, a nuclear fragmentation assay, using fluorescence-activated cell sorting analysis, and time-lapse video microscopy. TCTP also protects cells against the proapoptotic effects of tunicamycin and etoposide, but not against those of arsenite. Our results imply that cellular TCTP levels influence sensitivity to apoptosis and that PKR may exert its proapoptotic effects at least in part through downregulation of TCTP via eIF2alpha phosphorylation.

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1.

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m(7)GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.

Regulation of translation factors eIF4GI and 4E-BP1 during recovery of protein synthesis from inhibition by p53.

Activation of the tumour suppressor protein p53 rapidly inhibits protein synthesis. This is associated with dephosphorylation and cleavage of initiation factor eIF4GI and the eIF4E-binding protein 4E-BP1. When the activation of p53 is reversed within 16 h 4E-BP1 becomes rephosphorylated, the level of intact eIF4GI slowly increases and protein synthesis gradually recovers. The recovery of protein synthesis is partially blocked by rapamycin and wortmannin but not by the protein kinase inhibitors PD98059 and CGP74514A. Both rapamycin and wortmannin, but not PD98059 or CGP74514A, delay the reappearance of eIF4GI. In contrast, full-length 4E-BP1 rapidly becomes rephosphorylated and this process is partially inhibited by rapamycin, PD98059 and CGP74514A. Thus, activation of p53 results in the inhibition of distinct rapamycin- and wortmannin-sensitive pathways that target eIF4GI, and rapamycin-sensitive and -insensitive pathways that target 4E-BP1. Following inactivation of p53 the gradual recovery is determined largely by the kinetics of restoration of eIF4GI rather than by the rephosphorylation of full-length 4E-BP1. These findings suggest that the ability of cells to rephosphorylate 4E-BP1, resynthesise eIF4GI and restore the rate of protein synthesis after inactivation of p53 is an important aspect of recovery following the relief of physiological stress.

Initiation factor modifications in the preapoptotic phase.

Recent studies have identified several mechanistic links between the regulation of translation and the process of apoptosis. Rates of protein synthesis are controlled by a wide range of agents that induce cell death, and in many instances, the changes that occur to the translational machinery precede overt apoptosis and loss of cell viability. The two principal ways in which factors required for translational activity are modified prior to and during apoptosis involve (i) changes in protein phosphorylation and (ii) specific proteolytic cleavages. In this review, we summarise the principal targets for such regulation, with particular emphasis on polypeptide chain initiation factors eIF2 and eIF4G and the eIF4E-binding proteins. We indicate how the functions of these factors and of other proteins with which they interact may be altered as a result of activation of apoptosis and we discuss the potential significance of such changes for translational control and cell growth regulation.

Initiation factor eIF2 alpha phosphorylation in stress responses and apoptosis.

The alpha subunit of polypeptide chain initiation factor eIF2 can be phosphorylated by a number of related protein kinases which are activated in response to cellular stresses. Physiological conditions which result in eIF2 alpha phosphorylation include virus infection, heat shock, iron deficiency, nutrient deprivation, changes in intracellular calcium, accumulation of unfolded or denatured proteins and the induction of apoptosis. Phosphorylated eIF2 acts as a dominant inhibitor of the guanine nucleotide exchange factor eIF2B and prevents the recycling of eIF2 between successive rounds of protein synthesis. Extensive phosphorylation of eIF2 alpha and strong inhibition of eIF2B activity can result in the downregulation of the overall rate of protein synthesis; less marked changes may lead to alterations in the selective translation of alternative open reading frames in polycistronic mRNAs, as demonstrated in yeast. These mechanisms can provide a signal transduction pathway linking eukaryotic cellular stress responses to alterations in the control of gene expression at the translational level.

Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages.

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding protein in vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.

The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation.

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.

Translational regulation in cell stress and apoptosis. Roles of the eIF4E binding proteins.

Several mechanisms have been identified by which protein synthesis may be regulated during the response of mammalian cells to physiological stresses and conditions that induce apoptotic cell death (reviewed in Clemens et al., Cell Death and Differentiation 7, 603-615, 2000). Recent developments allow us to up-date this analysis and in this article I concentrate on one particular aspect of this regulation that has not previously been reviewed in depth in relation to apoptosis, viz. the control of the initiation of protein synthesis by eukaryotic initiation factor eIF4E and the eIF4E binding proteins (4E-BPs). Changes in the state of phosphorylation of the 4E-BPs and in the extent of their association with eIF4E occur at an early stage in the response of cells to apoptotic inducers. The review discusses the mechanisms by which these events are regulated and the significance of the changes for the control of protein synthesis, cell proliferation and cell survival.

Differential requirements for caspase-8 activity in the mechanism of phosphorylation of eIF2alpha, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells.

Previously we have reported that induction of apoptosis in Jurkat cells results in an inhibition of overall protein synthesis with the selective and rapid cleavage of eukaryotic initiation factor (eIF) 4GI. For the cleavage of eIF4GI, caspase-3 activity is both necessary and sufficient in vivo, in a process which does not require signaling through the p38 MAP kinase pathway. We now show that activation of the Fas/CD95 receptor promotes an early, transient increase in the level of eIF2alpha phosphorylation, which is temporally correlated with the onset of the inhibition of translation. This is associated with a modest increase in the autophosphorylation of the protein kinase activated by double-stranded RNA. Using a Jurkat cell line that is deficient in caspase-8 and resistant to anti-Fas-induced apoptosis, we show that whilst the cleavage of eIF4GI is caspase-8-dependent, the enhancement of eIF2alpha phosphorylation does not require caspase-8 activity and occurs prior to the cleavage of eIF4GI. In addition, activation of the Fas/CD95 receptor results in the caspase-8-dependent dephosphorylation and degradation of p70(S6K), the enhanced binding of 4E-BP1 to eIF4E, and, at later times, the cleavage of eIF2alpha. These data suggest that apoptosis impinges upon the activity of several polypeptides which are central to the regulation of protein synthesis and that multiple signaling pathways are involved in vivo.

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.

Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis.

Induction of apoptosis results in inhibition of the rate of overall protein synthesis in a variety of cell types. We have shown previously that polypeptide chain initiation factor eIF4GI is rapidly cleaved by caspase-3, whereas other components of the eIF4F complex are much more stable during apoptosis in BJAB and Jurkat cells. We have now extended our analysis to other factors involved in the initiation of protein synthesis and we report here that eIF4B, the p35 subunit of eIF3, and minor proportions of the alpha subunit of eIF2 and the eIF4E-binding protein 4E-BP1 are also cleaved to give rise to discrete fragments. These cleavages occur with delayed kinetics relative to that seen for eIF4GI, and eIF2beta and eIF2gamma levels also decrease at a relatively late stage of apoptosis. In contrast, the second form of eIF4G described recently, eIF4GII, is cleaved as rapidly as eIF4GI under the same conditions. Purified recombinant caspase-3 is able to degrade eIF4B and eIF3(p35) in vitro, producing fragments of the same sizes as those seen in intact cells. Induction of apoptosis also results in a biphasic change in the association of 4E-BP1 with eIF4E. Thus the progress of apoptosis is characterized by a complex programme of changes in several initiation factors, including the specific fragmentation or complete degradation of some and alterations in the association status of others. These events are likely to contribute to the inhibition of protein synthesis seen under these conditions.

Translational control by the La antigen. Structure requirements for rescue of the double-stranded RNA-mediated inhibition of protein synthesis.

The La antigen is a protein which can bind both single-stranded and double-stranded forms of RNA and has regulatory effects on gene expression at the levels of transcription and translation. It was previously shown to inhibit the activation of the dsRNA-dependent protein kinase PKR by sequestering and/or unwinding double-stranded RNA. Here, we demonstrate that, as predicted by these properties, the La antigen can rescue protein synthesis in the reticulocyte lysate system from inhibition by low concentrations of dsRNA. This effect is reversed by higher concentrations of dsRNA. Using a series of deletion mutants we have investigated the structural features of the La antigen that are required for these effects. The ability to bind dsRNA is influenced by regions within both the previously characterized N-terminal RNP motif and the C-terminal half of the protein. La mutants with either N-terminal or C-terminal deletions retain the ability to inhibit the protein kinase activity of PKR and to rescue protein synthesis from inhibition by dsRNA. It is notable that sequences in the C-terminal half of the La antigen, including a phosphorylation site at Ser366, which are needed for other regulatory effects of the protein on gene expression are dispensable for the effects of La on PKR. We suggest that La regulates PKR activity solely as a result of its ability to act as an RNA-binding protein that can compete with PKR for limiting amounts of dsRNA.

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.

Activation of the interferon-inducible (2'-5') oligoadenylate synthetase by the Epstein-Barr virus RNA, EBER-1.

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.

Translational control: the cancer connection.

There is now a growing body of evidence which suggests links between the regulation of protein synthesis and the disruption of cell behaviour that typifies cancer. This directed issue of the International Journal of Biochemistry and Cell Biology presents several review articles of relevance to this field. The topics covered include the significance of the regulation and overexpression of polypeptide chain initiation factors for cell transformation and malignancy, the role of mRNA structure in the control of synthesis of key growth regulatory proteins, the actions of the eIF2 alpha-specific protein kinase PKR in the control cell growth and apoptosis, and the involvement of the elongation factor eEF1 in oncogenesis. The purpose of this article is to give an overview of the field and to indicate where we may expect developments to occur in the next few years.

Inhibition of the double-stranded RNA-dependent protein kinase PKR by mammalian ribosomes.

Previous evidence has shown that the majority of the interferon-inducible, double-stranded RNA-dependent protein kinase PKR is associated with ribosomes in vivo. Here we show that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent the phosphorylation of the PKR substrate eIF2alpha. We suggest that ribosomes constitute a reservoir of inactive PKR and that the protein kinase must be displaced from the ribosome by dsRNA in order to become activated.