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.     

Regulation of phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight core complex in reticulocytes

Five aminoacyl-tRNA synthetases found in the high molecular weight core complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The synthetases were isolated by affinity chromatography on tRNA-Sepharose followed by immunoprecipitation. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, and aspartyl-tRNA synthetases and, to a lesser extent, the methionyl-tRNA synthetase. In addition, a 37,000-dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and 3-isobutyl-1-methylxanthine resulted in a 6-fold increase in phosphorylation of the glutaminyl-tRNA synthetase and a 2-fold increase in phosphorylation of the aspartyl-tRNA synthetase. When the high molecular weight core complex was isolated by gel filtration/affinity chromatography, the profile of phosphorylation was similar to that observed by immunoprecipitation with a 9- and 3-fold stimulation of the glutaminyl- and aspartyl tRNA-synthetase, respectively. From this data it was concluded that the increased phosphorylation of the glutaminyl- and aspartyl-tRNA synthetases obtained with 8-bromo-cAMP did not appear to be involved in dissociation of the high molecular weight core complex.     

Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites.

Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.     

Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.     

Protein kinase C phosphorylates glutamyl-tRNA synthetase in rabbit reticulocytes stimulated by tumor promoting phorbol esters

A high Mr synthetase core complex isolated from higher eukaryotes contains aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. Previously, five of the synthetases were shown to be phosphorylated in reticulocytes, and the glutaminyl- and aspartyl-tRNA synthetases were shown to be selectively phosphorylated in response to 8-bromo cAMP (Pendergast, A. M., Venema, R. C., and Traugh, J. A. (1987) J. Biol. Chem. 262, 5939-5942). Exposure of reticulocytes to phorbol 12-myristate 13-acetate stimulates the selective phosphorylation of one synthetase in the complex, glutamyl-tRNA synthetase. Only the glutamyl-tRNA synthetase is modified to a significant extent when the purified complex is phosphorylated in vitro by protein kinase C; up to 0.7 mol of phosphate is incorporated per mol of synthetase. Two-dimensional phosphopeptide mapping shows a single tryptic phosphopeptide, which is identical for the enzyme modified in vitro by protein kinase C or in phorbol 12-myristate 13-acetate-stimulated cells. Phosphorylation in vivo is reproducibly accompanied by a 38 +/- 10% reduction in aminoacylation activity of partially purified glutamyl-tRNA synthetase assayed in vitro. Phosphorylation in vitro has no detectable effect on aminoacylation. This difference may be due to the absence of a required effector molecule which alters activity by interaction with the phosphorylated synthetase. Glutamyl-tRNA synthetase is one of a growing number of translational components, including initiation factors, which are coordinately modified by protein kinase C in response to phorbol 12-myristate 13-acetate.     

Diadenosine oligophosphates: peculiarities of synthesis by phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8.

Temperature and other factors affecting synthesis of bis(5'-adenosyl) tetraphosphate (Ap4A) and bis(5'-adenosyl)triphosphate (Ap3A) catalyzed by phenylalanyl-tRNA synthetases (PheRSs) from Escherichia coli MRE-600 and Thermus thermophilus HB8 have been investigated. Those two synthetases exhibited different temperature-dependent rates of the Ap4A and Ap3A synthesis. However, with respect to the effects of such effectors of the Ap4A synthesis as Zn2+, Mg2+, tRNA and Ap4A phosphonate analogues, as well as some inhibitors of aminoacyl-tRNA synthetase, those two enzymes were apparently undistinguishable.     

P1,P4-Di(adenosine-5')tetraphosphate inhibits phosphorylation of immunoglobulin G by Rous sarcoma virus pp60src

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 2-6) inhibited phosphorylation of immunoglobulin G from tumor-bearing rabbits (TBR IgG) by pp60src protein kinase purified from Rous sarcoma virus-transformed rat tumor cells. Ap4A, a nucleotide associated with eukaryotic cell proliferation, was one of the most effective inhibitors in the series, causing 50% inhibition of TBR IgG phosphorylation at 15 microM. Ap4A inhibited pp60src-dependent phosphorylation of TBR IgG in solution and immunoprecipitates, as well as the phosphorylation of tubulin, microtubule-associated proteins, and vinculin. Under similar assay conditions, Ap4A did not inhibit phosphorylation of histone H2b by cAMP- or cGMP-dependent protein kinases. Ap4A appears to interact noncovalently with the enzyme, because removal of pp60src by immunoprecipitation from solutions containing Ap4A restored activity to uninhibited levels. A 100-fold increase in ATP (4-400 nM) caused a 13-fold increase in the 50% inhibitory concentration of Ap4A (2.5-33 microM), consistent with the interpretation that Ap4A competes for an ATP-binding site on the pp60src molecule. The simplest explanation of these results is that Ap4A binds to the phosphodonor site for ATP.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Variation in intracellular P1P4-bis(5''-adenosyl) tetraphosphate (Ap4A) in virus-infected cells.

The effect of virus infection on the intracellular concentration of the proposed stress alarmone P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) has been examined in Vero cells. Compared with exposure to 0.8 mM-Cd2+, which causes a 30-fold increase in Ap4A, infection with simian virus 40 and poliovirus causes only a 2-fold increase, whereas herpes simplex virus type 1 results in a decrease in Ap4A during the course of the infection.     

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.     

Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells

In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.     

Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.     

Threonyl-tRNA synthase from rabbit reticulocytes: a reversible transition from the RNA-non-binding to the RNA-binding form

A simple three-step procedure was used to isolate threonyl-tRNA synthetase of rabbit reticulocytes which is in a ribosome-free extract in the RNA-non-binding form. According to SDS electrophoresis, the enzyme has a molecular weight of 86 000 Da and is heterogeneous by isoelectric point; pI of the major component is near 6.2. Threonyl-tRNA synthetase is capable of interacting with a high molecular weight RNA (E. coli rRNA). Thus, in the course of purification threonyl-tRNA synthetase passes from the RNA-non-binding to the RNA-binding form. This transition was shown to be reversible.     

Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl-tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.     

Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.     

Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A).

The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant.     

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by organellar and cytoplasmic phenylalanyl-tRNA synthetases of Euglena gracilis

Purified phenylalanyl-tRNA synthetases present in chloroplasts, mitochondria and cytoplasm of green and bleached Euglena gracilis strains, respectively, are able to synthesize diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A). Ap4A synthesis is strictly dependent on zinc ions. This is the first evidence that chloroplasts should be able to synthesize Ap4A. Synthesis of Ap4A by phenylalanyl-tRNA synthetases of the three compartments of a plant cell or by other enzymes such as Ap4A phosphorylase is discussed.     

Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser(462) and Thr(455) were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.     

Synthesis of diadenosine 5',5' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.