Formation and Implications of a Ternary Complex of Profilin, Thymosin β4, and Actin

Abstract

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin ␤ 4 , and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin ␤ 4 (residues 6 -22) confirm the presence of an extensive binding surface between actin and thymosin ␤ 4 , and explain why thymosin ␤ 4 and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin ␤ 4 peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin ␤ 4 . Intracellular concentrations of thymosin ␤ 4 and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin ␤ 4 -actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin ␤ 4 can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.The amount of unpolymerized actin in many cells is large. Several actin-monomer sequestering proteins have been identified that are responsible for maintaining this pool, and attempts have been made to account for the quantity of unpolymerized actin by calculation of the sum of sequestered actin in cells (1-3). These calculations depend not only on the concentration of each sequestering protein and its equilibrium dissociation constant for actin, but also on several other parameters, including an estimate of the critical concentration of actin (i.e. the amount of unpolymerized, unsequestered actin), the stoichiometry with which the sequestering proteins bind to actin, the potential qualitative and quantitative effects should different sequestering proteins interact simultaneously with a single actin subunit, and on assumptions regarding the effects of cytoplasmic molecular crowding on equilibrium association constants. Not surprisingly, with so many parameters to evaluate, even small quantitative errors in measurement or qualitative errors in mechanism result in a wide range of plausible estimates of total sequestered actin. In this report we investigate assumptions that have very significant effects on predictions related to the amount of sequestered actin, finding significant discrepancies with previously reported results, and discuss the expected consequences of these observations. Based on assays measuring steady-state F-actin levels and the failure to obtain a covalently cross-linked ternary complex, thymosin ␤ 4 and profilin have been reported to bind ...

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

“…We assume here, as supported by our previous data (26), that any ternary complex of actin, thymosin ␤ 4 , and another actin-binding protein, for example profilin (PAT), has the same value of r b as r b for the actin-thymosin ␤ 4 complex (AT). Then in the presence of actin and profilin…”

“…Proteins and Peptides-Pyrenyl-labeled and unlabeled rabbit skeletal muscle Ca 2ϩ -actin, recombinant human profilin, and recombinant rat thymosin ␤ 4 (identical in sequence to human) were purified as previously described (26). For labeling with tetramethylrhodamine-5-maleimide (T-6027; Molecular Probes Inc., Eugene, OR), the thymosin ␤ 4 cDNA was modified by the addition of a C-terminal cysteine.…”

“…Because the assay is most accurate when the binding constant of the labeled thymosin ␤ 4 peptide is similar to the free actin concentration (or for A c , when measuring at steady state), the use of 1-25 rhod-t␤ 4 peptide provides a standard curve with utility at higher free actin concentrations than does intact thymosin ␤ 4 . We previously showed that thymosin ␤ 4 competes with rhodamine-labeled thymosin ␤ 4 in this anisotropy assay; with a dose response as expected if both have similar affinities for actin (26). Moreover, because labeled thymosin ␤ 4 also inhibits binding by profilin, either competitively (9) or noncompetitively (26), the use of thymosin ␤ 4 uniquely determines the free actin concentration in the presence of either or both of these actin monomer-sequestering proteins.…”

“…We previously showed that thymosin ␤ 4 competes with rhodamine-labeled thymosin ␤ 4 in this anisotropy assay; with a dose response as expected if both have similar affinities for actin (26). Moreover, because labeled thymosin ␤ 4 also inhibits binding by profilin, either competitively (9) or noncompetitively (26), the use of thymosin ␤ 4 uniquely determines the free actin concentration in the presence of either or both of these actin monomer-sequestering proteins. Binding of 1-25 rhod-t␤ 4 peptide to actin is inhibited by profilin in a manner consistent with either competitive or noncompetitive inhibition, with the equilibrium dissociation constant for the ternary complex formation K dPT Ն 80 M (Fig.…”

“…Specifically, we have previously used this technique to assess binding of thymosin ␤ 4 to actin (26). The binding isotherm for this interaction, when plotted as anisotropy versus total actin for trace levels of rhodamine-labeled thymosin ␤ 4 (or synthetic 1-25 rhod-t␤ 4 peptide), provides a standard curve for determination of the free actin concentration in any sample in which the anisotropy is determined under the same standard conditions (Fig.…”