Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Phosphorylation of the elongation factor 2: the fifth Ca2+/calmodulin-dependent system of protein phosphorylation

Elongation factor 2 (EF-2) has been recently shown to be extensively phosphorylated in a Ca2+/calmodulin-dependent manner in extracts of mammalian cells (A. G. Ryazanov (1987) FEBS Lett. 214, 331-334). In the present study, we partially purified the protein kinase which phosphorylates EF-2 from rabbit reticulocytes. The molecular weight of the enzyme determined by gel filtration was about 140,000. Unlike the substrate, the EF-2 kinase had no affinity for RNA and therefore could be separated from EF-2 by chromatography on RNA-Sepharose. After chromatography on hydroxyapatite, the kinase activity became calmodulin-dependent. Two-dimensional separation of the phosphorylated EF-2 according to O'Farrell's technique revealed that there were two phosphorylation sites within the EF-2 molecule; in both cases, the phosphorylated amino acid was threonine. The EF-2 kinase differed from the four known types of Ca2+/calmodulin-dependent protein kinases. Thus, the system of EF-2 phosphorylation represents the novel (fifth) Ca2+/calmodulin-dependent system of protein phosphorylation. This system is supposed to be responsible for the regulation of the elongation rate of protein biosynthesis in eukaryotic cells.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.     

Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.     

Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.     

Identification of Nsp100 as elongation factor 2 (EF-2)

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.     

cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.     

A poly(U)-binding factor stimulating EF-1 activity in the wheat-germ soluble fraction

A poly(U)-binding activity is present in the high-speed supernatant fraction of embryo homogenates from wheat seeds. The factor responsible for such activity was found to have a stimulatory effect on the elongation factor 1 (EF-1). It copurifies with EF-1L, the lighter form of EF-1, through Sephadex G-200, DEAE-cellulose, hydroxyapatite and poly(U)-Sepharose 4B column chromatography. The two factors could be separated only through a heating step which destroyed EF-1 activity whilst leaving most of the poly(U)-binding activity unaltered.     

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1 gamma and EF-1 beta

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF-1 beta and EF-1 gamma. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.     

Phosphorylation of Xenopus elongation factor-1 gamma by cdc2 protein kinase: identification of the phosphorylation site.

The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.     

Heterogeneity of native rat liver elongation factor 2

The high heterogeneity of native rat liver EF-2 prepared from either 105000 x g supernatant or microsome high-salt extract was detected by two-dimensional equilibrium isoelectric focusing-SDS-polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of Mr 95,000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin-dependent protein kinase (most likely Ca2+/calmodulin-dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299-17303; (1988) Nature 334, 170-173]). Results of ADP-ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP-ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF-2. Phosphorylated and ADP-ribosylated forms of EF-2 could be ADP-ribosylated and phosphorylated, respectively, but a native form both ADP-ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF-2 and of its phosphorylated and ADP-ribosylated derivatives.     

The effect of phosphorylation of the EF-2 isolated from rat liver cells on protein biosynthesis in vitro.

The activity of EF-2 was distinctly decreased after phosphorylation catalysed by a partly purified calmodulin and Ca2+ dependent protein kinase III. At the same time 32P from [gamma-32P]ATP was incorporated into EF-2 molecule. After dephosphorylation of EF-2 catalysed by alkaline phosphatase the activity of this factor was increased. This suggests that the phosphorylation-dephosphorylation of EF-2 is the regulatory process in the elongation step of the translation. Preliminary purification of the kinase III from rat liver resulted in 8-fold purified enzyme with a recovery of 60%.     

Thrombin and histamine stimulate the phosphorylation of elongation factor 2 in human umbilical vein endothelial cells

The effects of thrombin and histamine on protein phosphorylation in intact cultured human umbilical vein endothelial cells (HUVEC) prelabeled with 32PO4 were investigated. Incubation of HUVEC with either thrombin or histamine, agonists known to induce rapid transient increases in intracellular calcium levels in HUVEC, caused a rapid reversible increase in the phosphorylation of a protein with a Mr = 100,000 independent of the presence of extracellular calcium. Immunological and biochemical studies demonstrated that this Mr = 100,000 protein is elongation factor 2 (EF-2), a substrate previously shown to be phosphorylated by calcium/calmodulin-dependent protein kinase III (Nairn, A. C., and Palfrey, H. C. (1987) J. Biol. Chem. 262, 17299-17303). EF-2 is crucial for protein synthesis because it catalyzes the translocation of peptidyl-tRNA on the ribosome. Phosphoamino acid analysis of the EF-2 immunoprecipitated from HUVEC revealed that all of the thrombin-stimulated phosphorylation occurred on threonine. EF-2 was also phosphorylated when HUVEC were treated with the calcium ionophore, ionomycin. Phosphorylation of EF-2 was not increased by treatment with D-Phe-Pro-Arg-chloromethyl ketone thrombin, phorbol dibutyrate, forskolin, or 8-bromo-cGMP. The transient nature of the phosphorylation of EF-2 is consistent with it having a role in mediating some of the transient effects of thrombin and histamine on endothelial cell protein synthesis and functional capabilities.     

Phosphorylation of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities

Valyl-tRNA synthetase from mammalian cells is isolated in a high Mr complex with elongation factor 1 (EF-1). This complex, which represents all of the valyl-tRNA synthetase activity and a significant portion of the EF-1 activity in rabbit reticulocytes, contains five polypeptides identified as valyl-tRNA synthetase and the four subunits of EF-1. In this study, we have examined the potential for regulation of the complex by phosphorylation of these components. The valyl-tRNA synthetase.EF-1 complex has been purified by gel filtration and tRNA-Sepharose chromatography from 32P-labeled rabbit reticulocytes stimulated by phorbol 12-myristate 13-acetate (PMA) and compared to the complex purified from control cells. One- and two-dimensional polyacrylamide gel electrophoresis and autoradiography show that valyl-tRNA synthetase and the alpha, beta and delta subunits of EF-1 are phosphorylated in vivo. Phosphorylation of each of the four proteins is increased 2-4-fold in response to PMA. Phosphorylation of valyl-tRNA synthetase in response to PMA is reproducibly accompanied by a 1.7-fold increase in aminoacylation activity, whereas phosphorylation of EF-1 is associated with a 2.0-2.2-fold stimulation of activity, as measured by poly(U)-directed polyphenylalanine synthesis. These data suggest that stimulation of translational rates in response to PMA is mediated, at least in part, by phosphorylation of valyl-tRNA synthetase and EF-1.     

The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.     

Phosphorylation of elongation factor 1 beta by an endogenous kinase affects its catalytic nucleotide exchange activity

Elongation factor 1 beta (EF-1 beta) from Artemia is phosphorylated to a high percentage at serine 89 by an endogenous kinase present in EF-1 beta gamma. Protein sequencing of EF-1 beta revealed that this serine residue is located N-terminally of an acidic cluster of amino acids, (formula; see text) which is critical for casein kinase II-type substrate recognition. A number of compounds known to influence casein kinases were studied, revealing that the kinase activity as present in EF-1 beta gamma belongs to the class of casein kinase II. The rate of nucleotide exchange on EF-1 alpha as catalyzed by EF-1 beta was found to be affected reversibly by the state of phosphorylation of EF-1 beta. In the presence of dephosphorylated EF-1 beta, the exchange rate is almost twice as large compared to the rate in the presence of phosphorylated EF-1 beta. Rephosphorylation of dephosphorylated EF-1 beta diminishes the activity of the protein again. The role of casein kinase II-type enzymes in modulating the function of proteins involved in polypeptide synthesis is discussed.     

Distribution of elongation factors EF-1 and EF-2 among components of rabbit reticulocyte lysate

The distribution of activity of the elongation factors EF-1 and EF-2 among the components of rabbit reticulocyte lysate separated by sucrose density gradient centrifugation was studied. At low ionic strength (0.01 M KCl) about 30% of the EF-1 activity was found in polyribosomes. At moderate ionic strength (0.1 M KCl) the EF-1 activity was absent in the polyribosomes. An addition of RNA excess to the lysate prior to centrifugation at low ionic strength resulted in elimination of the EF-1 activity from the polyribosomes. This indicates that EF-1 is reversibly bound to the polyribosomes and that EF-1 may be retained on them due to interaction with RNA of polysomes mediated by its RNA-binding site. After dissociation of polyribosomes containing EF-1 in the presence of EDTA and subsequent fractionation of the dissociation products at low ionic strength (0.01 M KCl) the EF-1 activity was revealed in the ribosomal subparticles (predominantly in 60S). At 0.1 M KCl EF-1 mainly sedimented in the zone of distribution of polyribosomal informosomes. The elongation factor EF-2 was not revealed in polyribosomes during lysate centrifugation even at low ionic strength which corresponds to its lower affinity for RNA.