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.     

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.     

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.     

Effect of cyclic AMP and cyclic GMP on the autophosphorylation of elongation factor 1 from wheat embryos

Extensively purified EF-1H (EF-1 alpha beta beta' gamma) from wheat embryos had a protein kinase activity and phosphorylated EF-1 beta which is one of the two EF-Ts-like factors (EF-1 beta and EF-1 beta'). In this reaction ATP and GTP were equally effective as phosphate donors, and threonine residue was phosphorylated. At 10(-7)M, 3', 5' cyclic GMP stimulated both the phosphorylation and phe-tRNA binding reactions, whereas 3', 5' cyclic AMP inhibited both reactions. These findings indicate that phosphorylation of EF-1H may play an important role in the translational control of protein biosynthesis at the elongation step.     

Molecular cloning,biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue

The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania. Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein [J. Biol. Chem. 277 (2002) 50190]. To characterize this apparent Leishmania virulence factor further, the cDNA encoding L. donovani EF-1 alpha was cloned and sequenced. Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L. donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes. Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion. Consistent with these structural differences, the sub-cellular distributions of L. donovani EF-1 alpha and host EF-1 alpha were strikingly different. Interestingly, infection of macrophages with L. donovani caused redistribution of host as well as pathogen EF-1 alpha. Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.     

Retropseudogenes constitute the major part of the human elongation factor 1 alpha gene family.

The elongation factor 1 alpha (EF-1 alpha) is a protein which promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes in the protein synthesis process. A human gene coding for EF-1 alpha has previously been cloned and sequenced along with a pseudo-gene. Here, we have further analyzed the family of human EF-1 alpha genes. Using an EF-1 alpha cDNA as probe twelve genomic EF-1 alpha-like clones were isolated and analyzed. Four of these were sequenced and found to contain EF-1 alpha retropseudogenes. A Southern blot analysis indicated that the remaining eight clones also contained retropseudogenes. Genomic Southern blot analysis revealed at least twenty loci in the human genome with sequence homology to the EF-1 alpha cDNA. Besides the already described active gene only one potentially active locus was found. The others appeared to be retropseudogenes. EF-1 alpha retropseudogenes were also found to be abundant in the mammalian species mouse and pig, while the chicken contained only one presumably active EF-1 alpha gene.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus.     

Purification and characterization of a germ cell-specific form of elongation factor 1 alpha (EF-1 alpha) from Xenopus laevis.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.     

Structure and expression of elongation factor 1 alpha in tomato.

A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues.     

Comparison of phosphorylation of elongation factor 1 from different species by casein kinase II

One subunit of EF-1 or EF-1 beta gamma from Artemia salina, wheat germ and rabbit reticulocytes is modified by casein kinase II. The subunit corresponds to the low Mr subunit of EF-1 (26,000-36,000) which functions along with a higher Mr subunit (46,000-48,000), to catalyze the exchange of GDP for GTP on EF-1 alpha. The factor from Artemia and wheat germ is phosphorylated directly on serine by casein kinase II whereas a modulatory compound is required for phosphorylation of EF-1 from reticulocytes. Polylysine increases the rate of phosphorylation of EF-1 from reticulocytes by 24-fold; both serine and threonine are modified. This suggests that polylysine may be substituting for a physiological regulatory compound which modulates phosphorylation in vivo.     

Developmental analysis of elongation factor-1 alpha expression in transgenic tobacco.

The developmental regulation of the translational elongation factor EF-1 alpha has been analyzed in tobacco. A gene fusion was constructed consisting of the 5' and 3' regions of the tomato genomic clone LeEF-A from the EF-1 alpha gene family and the beta-glucuronidase coding region. Analysis of the transgenic plants containing this chimeric gene demonstrated that the tomato LeEF-A flanking sequences were sufficient to confer expression patterns similar to those of the endogenous tobacco EF-1 alpha gene. The patterns of beta-glucuronidase activity in this system indicated that during plant growth and development EF-1 alpha is regulated with increased expression corresponding to regions of high protein synthesis, including meristems, rapidly growing tissues, and developing gametophytes. In addition, EF-1 alpha expression responds rapidly to changes in growth patterns induced by hormone treatment. Our results are in agreement with studies in animals indicating that EF-1 alpha expression may be rate limiting for protein synthesis and demonstrate that the analysis of EF-1 alpha is of value for studying interrelationships between protein synthesis and developmental control.     

Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.

We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.     

Elongation factor-1 alpha is a selective regulator of growth factor withdrawal and ER stress-induced apoptosis

To identify genes that contribute to apoptotic resistance, IL-3 dependent hematopoietic cells were transfected with a cDNA expression library and subjected to growth factor withdrawal. Transfected cells were enriched for survivors over two successive rounds of IL-3 withdrawal and reconstitution, resulting in the identification of a full-length elongation factor 1 alpha (EF-1alpha) cDNA. Ectopic EF-1alpha expression conferred protection from growth factor withdrawal and agents that induce endoplasmic reticulum stress, but not from nuclear damage or death receptor signaling. Overexpression of EF-1alpha did not lead to growth factor independent cell proliferation or global alterations in protein levels or rates of synthesis. These findings suggest that overexpression of EF-1alpha results in selective resistance to apoptosis induced by growth factor withdrawal and ER stress.