F-actin bundling activity of Tetrahymena elongation factor 1 alpha is regulated by Ca2+/calmodulin

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome. Previously, Tetrahymena 14-nm filament-associated protein was identified as EF-1 alpha [Kurasawa et al. (1992) Exp. Cell Res. 203, 251-258]. This and several other studies suggest that EF-1 alpha functions not only in translation but also in regulation of some part of the cytoskeleton. Tetrahymena EF-1 alpha bound to F-actin and induced bundling of F-actin. We investigated the effects of GTP/GDP and Ca2+/calmodulin on F-actin bundling activity of EF-1alpha. The presence of GTP, GDP, or guanylyl-imidodiphosphate (GMP-PNP) slightly decreased the amount of EF-1 alpha which bound to F-actin, but each had virtually no effect on the F-actin bundling activity. The formation of F-actin bundles by EF-1 alpha was Ca(2+)-insensitive. In the absence of Ca2+, calmodulin did not bind to EF-1 alpha and F-actin. On the other hand, in the presence of Ca2+, calmodulin directly bound to EF-1 alpha but did not have any serious influence on EF-1 alpha/F-actin binding. Under the conditions, electron microscopy demonstrated that Ca2+/calmodulin completely inhibited the F-actin bundling by EF-1 alpha. These results indicate that CA2+/calmodulin regulates the F-actin bundling activity of EF-1 alpha without inhibition of the binding between Ef-1 alpha and F-actin.     

The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A

Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1alpha), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 10(-3) s-1. When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.     

Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.     

Antibiotics MDL 62,879 and kirromycin bind to distinct and independent sites of elongation factor Tu (EF-Tu)

Antibiotic MDL 62,879 inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). In this study we show that the inhibition of protein synthesis by MDL 62,879 in an Escherichia coli cell-free system was fully reversed by addition of stoichiometric amounts of EF-Tu but not by large excesses of EF-Ts, ribosomes, or aa-tRNA. MDL 62,879 bound tightly to EF-Tu and formed a stable 1:1 MDL 62,879:EF-Tu (M:EF-Tu) complex. We show that binding of MDL 62,879 to EF-Tu strongly affects the interaction of EF-Tu with aa-tRNA and causes rapid dissociation of preformed EF-Tu.aa-tRNA complex, suggesting that inhibition of aa-tRNA binding is due to a conformational change in EF-Tu rather than competition for the aa-tRNA binding site. Indication of a conformational change in EF-Tu induced by MDL 62,879 is further confirmed by proteolytic cleavage experiments: MDL 62,879 binding strongly protects EF-Tu against trypsin cleavage. The observed effects of MDL 62,879 appear to be different from those of the kirromycin class of antibiotics, which also inhibit protein synthesis by binding to EF-Tu, suggesting two distinct binding sites. Indeed, the M:EF-Tu complex was able to bind stoichiometric amounts of kirromycin to form a 1:1:1 M:EF-Tu:kirromycin (M:EF-Tu:K) complex, providing direct evidence that the two antibiotics bind to independent and distinct sites on the EF-Tu molecule. The interaction of the M:EF-Tu:K complex with aa-tRNA and other co-factors suggest that the contemporary binding of the two antibiotics locks EF-Tu into an intermediate conformation in which neither antibiotic exhibits complete dominance.     

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.     

Factors influencing the functional outcome of patients with acute epidural hematomas: analysis of 200 patients undergoing surgery

An actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actin-depolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.     

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.     

Upregulation of the elongation factor-1alpha gene by p53 in association with death of an erythroleukemic cell line

Genes upregulated by p53 were screened using an erythroleukemic cell line (1-2-3) that expresses only the temperature-sensitive p53 by the mRNA differential display method. One of the upregulated genes was identified as the elongation factor-1alpha (EF-1alpha) gene, an essential component of the eukaryotic translation apparatus. Three p53-responsive elements were found in the mouse EF-1alpha gene and in the corresponding human, rat, and frog genes. These elements conferred the capacity for induction by p53. EF-1alpha is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with upregulation of EF-1alpha by p53 may be a cause of the cell death.     

General construction pattern of histamine H3-receptor antagonists: change of a paradigm

The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha). EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex?

Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia. Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1. Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.     

Lymphocyte-specific protein 1 expression in eukaryotic cells reproduces the morphologic and motile abnormality of NAD 47/89 neutrophils

Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, "hairlike" F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.     

A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction

The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416-1880) bound F-actin with a Kd of 14.2 +/- 5.2 microM and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030-1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.     

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.     

The effect of a caldesmon fragment with a mol. weight of 38 kDa and calponin on the capacity of actin to form a "strong" form of binding with myosin heads

Effect of calponin and 38 kD actin-binding proteolytic fragment of caldesmon on actin structure alterations, initiated by decoration of thin filaments by N-ethylmaleimide-modified skeletal myosin subfragment-1 (NEM-S1) and by phosphorylated smooth heavy meromyosin (pHMM), has been studied by polarized fluorimetry. F-actin of myosin-free ghost fiber was labeled with fluorescent probe fluoroscein-5-maleimide. Both the actin-binding regulatory proteins have been demonstrated to inhibit conformational changes of actin typical for the "strong" binding of myosin head to actin. Tropomyosin weakens the inhibitory effect of calponin and markedly increases the effect of the 38 kD fragment of caldesmon. The results indicate similarity of molecular mechanisms of the regulation of muscle contraction by calponin and the actin-binding fragment of caldesmon. It is proposed that the regulation of smooth muscle contraction by calponin and caldesmon is carried out via the inhibition of the formation of the stage AM in ATP hydrolysis cycle.     

The role of the GLUT 4 transporter in regulating rat myoblast glucose transport processes

Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.     

Increase in transforming growth factor beta1 in ovarian follicular fluid following ovarian stimulation and in-vitro fertilization correlates to pregnancy

Talin, an actin-binding protein from smooth muscle, is shown to bind to myosin in such a way that it stimulates the ATPase activity of myosin irrespective of the phosphorylation state of myosin. The binding site is shown to be localized at the N-terminal, 47 KDa fragment. The position of the actin-binding site at the C terminal suggests that talin may work as a crosslinker between myosin and actin.     

Actin stabilization by jasplakinolide enhances apoptosis induced by cytokine deprivation

Participation of the actin cytoskeleton in the transduction of proliferative signals has been established through the use of compounds that disrupt the cytoskeleton. To address the possibility that actin also participates in the transduction of an apoptotic signal, we have studied the response of the murine interleukin 2 (IL-2)-dependent T cell line CTLL-20 to treatment with the actin-binding compound jasplakinolide upon IL-2 deprivation. Like phalloidin, jasplakinolide stabilizes F-actin and promotes actin polymerization. Treatment of CTLL-20 cells with jasplakinolide, in the presence or absence of recombinant human IL-2, altered actin morphology as assessed by confocal fluorescence microscopy. Jasplakinolide was not toxic to CTLL-20 cells, nor was apoptosis induced in the presence of exogenous recombinant human IL-2. However, actin stabilization at the time of IL-2 deprivation enhanced apoptosis by changing the time at which CTLL-20 cells committed to the apoptotic pathway. This effect of jasplakinolide correlated with its ability to stabilize polymerized actin, as treatment with a synthetic analog of jasplakinolide with a greatly reduced ability to bind actin, jasplakinolide B, did not enhance apoptosis. The enhancement occurred upstream of the induction of caspase-3-like activity and could be inhibited by the overexpression of the anti-apoptotic protein Bcl-xL. These data suggest that the actin cytoskeleton plays an active role in modulating lymphocyte apoptosis induced by cytokine deprivation.