Ian W. Jeffrey

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 α subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2α 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. Cell Death and Differentiation (2000) 7, 603–615

The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

Regulation of the interferon-inducible eIF-2α protein kinase by small RNAs

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

Differential requirements for caspase‐8 activity in the mechanism of phosphorylation of eIF2α, 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 eIF2α 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 eIF2α 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 p70S6K, the enhanced binding of 4E‐BP1 to eIF4E, and, at later times, the cleavage of eIF2α. 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.

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 eIF2α. 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.