cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells. (1/305)

Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.  (+info)

The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus. (2/305)

A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed.  (+info)

Interaction of elongation factor eEF-2 with ribosomal P proteins. (3/305)

The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosome-dependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (Kd = 3.8 x 10-8 m) than for P2 (Kd = 2.2 x 10-6 m). Phosphorylation resulted in a moderate increase (two- to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.  (+info)

Does prothymosin alpha affect the phosphorylation of elongation factor 2? (4/305)

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Dominguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.  (+info)

Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid. (5/305)

Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 from Candida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[(3)H]puromycin on polysomes from Candida albicans.  (+info)

Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elongation factor binding. (6/305)

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl(2), 0.5 M NH(4)Cl, at 30 degrees C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.  (+info)

Nutrients differentially regulate multiple translation factors and their control by insulin. (7/305)

Eukaryotic initiation factor eIF2B and eukaryotic elongation factor eEF2 each mediate regulatory steps important for the overall regulation of mRNA translation in mammalian cells and are activated by insulin. Here, we demonstrate that their activation by insulin requires the presence, in the medium in which the cells are maintained, of both amino acids and glucose: insulin only induced activation of eIF2B and the dephosphorylation of eEF2 when cells were exposed to both types of nutrient. Other translational regulators, e.g. the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the eIF4E binding protein 1, 4E-BP1, are also regulated by insulin but their control does not require glucose, only amino acids. The effects of nutrients on the activation of eIF2B do not reflect changes in the phosphorylation of eIF2 (and, by inference, operation of a kinase analogous to yeast Gcn2p), or a requirement for nutrients for inactivation of glycogen synthase kinase-3 or dephosphorylation of eIF2B. Nutrients did not affect the ability of insulin to activate protein kinase B. These data show that activation by insulin of p70 S6 kinase, which modulates the translation of specific mRNAs, depends on the availability of amino acids whereas regulation of factors involved in overall activation of translation (eIF2B, eEF2) requires both amino acids and glucose. These results add substantially to the emerging evidence that nutrients themselves modulate functions of mammalian cells and indicate that (i) nutrients modulate the activation of eIF2B and eEF2 through as-yet unidentified mechanisms and (ii) regulation of p70 S6 kinase and 4E-BP1 by insulin requires other inputs in addition to protein kinase B.  (+info)

Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin. (8/305)

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.  (+info)