Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2

To investigate the effect of Mn2+ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [gamma-32P]ATP. Analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography revealed a single protein (p98), with an Mr of 98,000 and a pI of 6.4-6.5, which was phosphorylated in a dose-dependent manner by Mn2+. A threshold effect was observed at 35 microM, and maximal effect at 1.1 mM Mn2+. Ca2+ and calmodulin (CaM) did not cause p98 phosphorylation, but Mg2+ (10 mM) caused faint non-specific phosphorylation of p98. Ca2+ (0.03-3 mM) and CaM (1-10 micrograms/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg2+ inhibited Mn(2+)-stimulated p98 phosphorylation. Under the above incubation conditions, Mn(2+)-stimulated protein phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. Partial purification of p98 and amino acid sequencing of the protein band corresponding to p98 indicated complete sequence homology with rat elongation factor 2 (EF-2). Furthermore, the combination of Ca2+, Mg2+ and CaM, which is known to induce the phosphorylation of EF-2, mimicked the actions of Mn2+. Inasmuch as EF-2 is the major substrate for CaM-dependent protein kinase III (CaM-PK III), these studies suggest that in the pancreatic acinar cell Mn2+/CaM protein kinase activity is mediated via CaM-PK III and the Mn2+ participates in the regulation of this enzyme in the pancreas.     

Ribosome-bound elongation factor 2 escapes phosphorylation by Ca2+/calmodulin-dependent protein kinase III.

The activity of eukaryotic elongation factor 2 is regulated by phosphorylation catalysed by a highly specific Ca2+/calmodulin-dependent protein kinase. Phosphorylated EF2 binds to ribosomes with decreased affinity. The present evidence indicates that EF2 prebound to ribosomes is protected from phosphorylation, just as earlier evidence indicated that ribosome-bound EF2 is protected from ADP-ribosylation catalysed by diphtheria toxin. Ribosome-inactivating proteins ricin and gelonin, by interfering with the EF2-ribosome interaction, allow full phosphorylation of EF2.     

Regulation of the phosphorylation of elongation factor 2 by MEK-dependent signalling in adult rat cardiomyocytes

The Gq-coupled agonists phenylephrine and endothelin-1 each activate protein synthesis in cardiomyocytes as part of the programme that leads to cardiac hypertrophy. Here we show that they each induce the dephosphorylation of elongation factor (eEF) 2, a protein that in its dephosphorylated state mediates the translocation step of elongation. The ability of both agonists to induce dephosphorylation of eEF2 requires signalling via the mTOR and MEK/Erk signalling pathways, but is independent of phosphoinositide 3-kinase. Expression of an activated form of MEK leads to dephosphorylation of eEF2, in an mTOR independent manner, indicating that signalling via MEK/Erk suffices to cause dephosphorylation of eEF2.     

Regulation of the activity of eukaryotic peptide elongation factor 1 by autocatalytic phosphorylation

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [gamma-32P]GTP and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-1 alpha subunit of the factor (Mr 53 000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [gamma-32P]GTP with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32PO4, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21-0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80S ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 X GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.     

Interaction of Mycobacterium tuberculosis elongation factor Tu with GTP is regulated by phosphorylation

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the Mycobacterium tuberculosis phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of M. tuberculosis Ef-Tu (MtbEf-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in Mycobacterium smegmatis indeed reduced the level of protein synthesis. MtbEf-Tu was found to be phosphorylated by PknB on multiple sites, including Thr118, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated MtbEf-Tu but not on the phosphorylated protein. Our results show that the modulation of the MtbEf-Tu-GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.     

The phosphorylation of an actin depolymerizing factor by a calcium-dependent protein kinase regulates cotton fiber elongation

A cotton fiber is a single and highly elongated ovule epidermal cell. However, the mechanism that governs the development of fiber traits remains unclear. In this study, we cloned a calcium-dependent protein kinase (GhCPK1) and an actin depolymerizing factor (GhADF1) from Gossypium hirsutum. Real-time PCR analyses indicated that the expression of GhCPK1 and GhADF1 correlated with the elongation pattern of cotton fibers. Yeast two-hybrid assays using full-length GhCPK1 and truncated forms of GhCPK1 as baits identified GhADF1 as an interactor of GhCPK1. Furthermore, GhCPK1 is capable of phosphorylating GhADF1 in vitro in a calcium-dependent manner, and the phosphorylation of GhADF1 by GhCPK1 occurs on the Ser-6 of GhADF1. In addition, we observed that the heterologous expression of the GhCPK1 gene induced longitudinal growth of the host cells by 3.18-fold, with no apparent effect on other aspects of the host cells. The results strongly suggest that GhCPK1 may regulate the function of GhADF1 by phosphorylating this protein during cotton fiber elongation.     

Altered sensitivity of eukaryotic elongation factor 2 for trypsin after phosphorylation and ribosomal binding

We have used variations in the trypsin sensitivity of eukaryotic protein synthesis elongation factor 2 (eEF-2) to probe for structural alterations induced by phosphorylation, ribosomal binding, or guanosine nucleotides. We could not detect any nucleotide-related effect on the tryptic cleavage rate of Arg66. However, eEF-2 was protected from trypsin after ribosomal binding. Also, phosphorylation of eEF-2 led to a protection of Arg66. This indicates that phosphorylation leads to a structural rearrangement that could explain the reduced affinity of the phosphorylated factor for ribosomes (Carlberg, U., Nilsson, A., and Nyg?rd, O. (1990) Eur. J. Biochem. 191, 639-645). Cleavage of Arg66 led to a complete loss of the ability of the factor to be phosphorylated. Furthermore, ribosome-bound eEF-2 was found to be inaccessible for phosphorylation. Based on these findings and previously published data, we suggest that the region around the sites of phosphorylation and trypsin cleavage is vitally important for the factor function and ribosomal binding.     

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.     

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.     

beta-Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium-independent eEF2 kinase activity

The beta-adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca(2+)/calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca(2+)/calmodulin-independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP-dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase.     

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4.The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.     

The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2.

Okadaic acid, a tumour promoter which potently inhibits protein phosphatases, inhibited translation in the reticulocyte-lysate cell-free system. Inhibition was dose-dependent, with half-maximal effects occurring at 20-40 nM-okadaic acid. Inhibition of translation by okadaic acid resulted in the accumulation of polyribosomes, indicating that it was due to a decrease in the rate of elongation relative to initiation. Okadaic acid (at concentrations which inhibited translation) caused increased phosphorylation of a number of proteins in the lysate. Prominent among these was a protein of Mr 100,000, which has previously been identified as elongation factor 2 (EF-2). EF-2 is a specific substrate for a Ca2+/calmodulin-dependent protein kinase, which phosphorylates EF-2 on threonine residues. The Mr-100,000 band was phosphorylated exclusively on threonine residues, and its degree of 32P labelling was decreased by the Ca2+ chelator EGTA and by the calmodulin antagonist trifluoperazine. These agents attenuated the effects of okadaic acid on EF-2 phosphorylation and translation. When ranges of concentrations of each agent were tested, their effects on EF-2 labelling correlated well with their ability to reverse the okadaic acid-induced inhibition of translation. These findings demonstrate that increased phosphorylation of EF-2 results in an impairment of peptide-chain elongation when natural mRNA is used. The possible physiological role of EF-2 phosphorylation in the control of translation is discussed.     

Effect of ADP-ribosylation and phosphorylation on the interaction of elongation factor 2 with guanylic nucleotides.

Samples of unmodified EF-2, EF-2 ADP-ribosylated with diphtheria toxin and NAD, and/or phosphorylated using ATP and the Ca(2+)-calmodulin dependent kinase III partially purified, were irradiated at 254 nm with 32P-labeled GDP or GTP, and analyzed by one- and two-dimensional gel electrophoresis. By this method we showed that unmodified EF-2 formed a stable complex with GDP but not with GTP, whereas phosphorylated EF-2 and ADP-ribosylated + phosphorylated EF-2 formed stable complexes even in the absence of irradiation, with GTP but not GDP. ADP-ribosylated EF-2 did not form stable complexes with either GDP or GTP. Prior ADP-ribosylation of EF-2 increased its ability to the phosphorylated. These results show that the structures of the two domains containing diphtamide 715 and the phosphorylatable threonines (between Ala 51 and Arg 60) are interdependent; modifications of these residues induce different conformational changes of EF-2 which alter the interactions of the factor with guanylic nucleotides as well with ribosomes.     

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.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.