Nicholas T. Redpath

Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway.

It is well established that insulin and serum stimulate gene expression at the level of mRNA translation in animal cells, and previous studies have mainly focused on the initiation process. Here we show that, in Chinese hamster ovary cells expressing the human insulin receptor, insulin causes decreased phosphorylation of elongation factor eEF‐2 and that this is associated with stimulation of the rate of peptide‐chain elongation. eEF‐2 is phosphorylated by a very specific Ca 2+/calmodulin‐dependent protein kinase (eEF‐2 kinase) causing its complete inactivation. The decrease in eEF‐2 phosphorylation induced by insulin reflects a fall in eEF‐2 kinase activity. Rapamycin, a macrolide immunosuppressant which blocks the signalling pathway leading to the stimulation of the 70/85 kDa ribosomal protein S6 kinases, substantially blocks the activation of elongation, the fall in eEF‐2 phosphorylation and the decrease in eEF‐2 kinase activity, suggesting that p7O S6 kinase (p70s6k) and eEF‐2 kinase may tie on a common signalling pathway. Wortmannin, an inhibitor of phosphatidylinositide‐3‐OH kinase, had similar effects. eEF‐2 kinase was phosphorylated in vitro by purified p70s6k but this had no significant effect on the in vitro activity of eEF‐2 kinase.

Identification of the phosphorylation sites in elongation factor-2 from rabbit reticulocytes

The sites in eukaryotic elongation factor eEF‐2 phosphorylated by the Ca2+/calmodulin‐dependent eEF‐2 kinase in vitro have been identified. The kinase catalysed the rapid incorporation of one mol of phosphate per mol eEF‐2 and the slower incorporation of a second mol. All the phosphorylation sites in eEF‐2 are contained in the CNBr fragment corresponding to residues 22‐155. Tryptic digestion of phosphorylated eEF‐2 yielded 3 phosphopeptides, one being unique to monophosphorylated eEF‐2. The phosphorylation sites were identified as threonine residues 56 and 58, the former being more rapidly phosphorylated. Ala‐Gly‐Glu‐Thr‐Phe‐Thr14‐Asp‐Thr18‐Arg. The same sites are labelled in eEF‐2 isolated from reticulocyte lysates.

Cloning and Expression of cDNA Encoding Protein Synthesis Elongation Factor-2 Kinase

A cDNA from rat skeletal muscle encoding calcium/calmodulin-dependent eukaryotic elongation factor-2 kinase (eEF-2K) has been cloned and sequenced, and the amino acid sequence of the protein has been deduced. The kinase is composed of 724 amino acids and has a predicted molecular mass of 81,499 Da. The cDNA was judged to be full-length, as the protein, expressed in rabbit reticulocyte lysate or wheat germ extract, migrated upon SDS-PAGE with the same apparent molecular weight as the purified kinase and possessed eEF-2K activity. eEF-2K contains all of the 12 catalytic subdomains present in the majority of protein kinases, but they are atypical and display only limited homology with other kinases. A putative calmodulin-binding domain is present C-terminal to the catalytic domain as is a putative pseudosubstrate sequence. Two antipeptide antibodies raised against sequences derived from a partial rabbit cDNA clone, cross-reacted with purified eEF-2K, and one also immunoprecipitated eEF-2K activity from cell extracts. Northern blot analysis demonstrated that eEF-2K mRNA is expressed in a number of different tissues and that it may exist in multiple forms.

Analysis of the domain structure of elongation factor-2 kinase by mutagenesis

A number of elongation factor‐2 kinase (eEF‐2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF‐2K had no effect on activity, showing that such a lysine residue is not important in eEF‐2K activity. Mutation of a conserved pair of cysteine residues C‐terminal to the GXGXXG sequence, however, completely inactivated eEF‐2K. The eEF‐2K CaM binding domain was localised to residues 77–99 which reside N‐terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF‐2K activity. Removal of approximately 130 residues from the C‐terminus of eEF‐2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF‐2 kinase activity but not autokinase activity, suggesting that a short region at the C‐terminal end may be important in interacting with eEF‐2. Likewise, removal of between 75 and 100 residues from the N‐terminal end completely abolished eEF‐2K activity.

High-resolution one-dimensional polyacrylamide gel isoelectric focusing of various forms of elongation factor-2

A system for analyzing covalent modifications of elongation factor-2 (eEF-2) by one-dimensional isoelectric focusing in slab polyacrylamide gels is described. Depending on the degree of phosphorylation, four species of eEF-2 could be resolved corresponding to the un-, mono-, bis-, and trisphosphorylated factor. Furthermore, the degree of ADP-ribosylation of the protein could also be assessed by this method. It was also shown that an acidic isoform of eEF-2 exists which appears not to be artifactual and that the relative level of this isoform appeared to vary between different cell types. By Western blotting the gels and using an antibody against eEF-2 it is possible to assess the state of phosphorylation of the factor in cells.

Regulation of polypeptide-chain initiation in rat skeletal muscle Starvation does not alter the activity or phosphorylation state of initiation factor eIF-2

In rats, 48‐h starvation causes a decrease in the rate of protein synthesis in skeletal (e.g. gastrocnemius) muscle, due largely to impairment of peptide‐chain initiation. In other cell types inhibition of initiation is associated with decreased activity and recycling of initiation factor eIF‐2, and increased phosphorylation of its α‐subunit. However, 48‐h starvation has no effect on the activity or recycling of eIF‐2 measured in extracts of gastrocnemius muscle, or on the level of α‐subunit phosphorylation. The effects of starvation on peptide‐chain initiation in skeletal muscle must therefore involve alterations in other components of the translational machinery.

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.

Heterogeneity of cardiac rat and human elongation factor 2

Elongation factor 2 (EF‐2) catalyses the last step of the elongation cycle, translocation, in the course of protein biosynthesis. A system for analyzing post‐translational modifications of EF‐2, which is a single polypeptide of 857 amino acids, is reported and its application to cytosolic extracts of cultured neonatal rat heart myocytes, neonatal and adult rat cardiac tissue, and extracts of human left ventricular myocardium is described. Comparing different pH ranges in immobilized pH gradient‐isoelectric focusing (IPG‐IEF), a range of pH 3 — 10 and 4 — 9 resulted in a highly defined and reproducible resolution of six different EF‐2 variants of all extracts in the first dimension. These six variants were detected by the „imaging plate”︁ (phosphor radiation image sensor) after specific labeling with Pseudomonas exotoxin A catalyzed [32P]ADP‐ribosylation. This finding could be confirmed in Western blot analysis with a specific polyclonal rabbit antibody. Using two‐dimensional polyacrylamide gel electrophoresis (2‐D‐PAGE), five to six EF‐2 variants could be demonstrated in all extracts. By application of a second IPG indicator strip to the 2‐D gel, they could be aligned with corresponding spots in a silver‐stained 2‐D separation of human myocardial tissue, revealing that the EF‐2 variants belong to the group of low‐abundance proteins.

Phosphorylation of Initiation and Elongation Factors and the Control of Translation

Several protein components of the translational machinery of eukaryotic cells are subject to phosphorylation in vitro and/or in vivo. They include initiation and elongation factors, and the ribosomal protein S6. Changes in the phosphorylation states of these proteins accompany alterations in rates of translation under a variety of conditions, suggesting that their phosphorylation provides a regulatory mechanism. However, in only a few cases is there clear evidence that phosphorylation actually affects translational activity (reviewed by Hershey [1]), i.e. initiation factor eIF-2 and elongation factor EF-2, for which effects of phosphorylation on activity are now well established, and the cap-binding initiation factor eIF-4E and ribosomal protein S6, for which circumstantial evidence for regulation by phosphorylation is available. The work described here concerns the phosphorylation of eIF-2 and EF-2, and in particular the identification of the phosphorylation sites in these proteins, the characterisation of the kinases which phosphorylate them and the identification of the protein phosphatases responsible for their dephosphorylation.