Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin.

Thymosin beta(4) possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin beta(4) serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin beta(4) may serve as a useful tool for further investigations in cell biology. Thymosin beta(4) could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin beta(4) concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin beta(4) may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.

Plant profilin induces actin polymerization from actin : beta-thymosin complexes and competes directly with beta-thymosins and with negative co-operativity with DNase I for binding to actin.

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.

Mapping the binding site of thymosin beta4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin.

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta4 was determined by analysing the binding of thymosin beta4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin beta4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin beta4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin beta4 was obtained only when using the chemically cross-linked actin-thymosin beta4 complex, indicating that thymosin beta4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin.

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.