Interaction of thymosin .beta.4 with muscle and platelet actin: implications for actin sequestration in resting platelets

Weber, Annemarie; Nachmias, Vivianne T.; Pennise, Cynthia R.; Pring, Martin; Safer, Daniel

doi:10.1021/bi00142a002

Cited by 157 publications

(172 citation statements)

References 55 publications

(64 reference statements)

Supporting

Mentioning

152

Contrasting

Unclassified

Order By: Relevance

“…Apparently, actin bound to thymosin ␤ 4 is participating in the polymerization process, either during nucleation or elongation. Previous investigators have suggested that this might also be the case in the absence of jasplakinolide (7), but others have not found evidence for anything other than monomer sequestration by thymosin ␤ 4 (20). A previous report (21) that thymosin ␤ 4 -actin complexes could directly associate with phalloidin-stabilized actin neglected the fact that phalloidin, like jasplakinolide, lowers the critical concentration and therefore decreases the amount of actin sequestered by thymosin ␤ 4 (17).…”

Section: The Effects Of Jasplakinolide On the Time Course Of Actin Pomentioning

confidence: 99%

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Bubb

Spector

Beyer

et al. 2000

Journal of Biological Chemistry

485

399

View full text Add to dashboard Cite

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin ␤ 4 . Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.Jasplakinolide is a cyclic peptide isolated from the marine sponge, Jaspis johnstoni, that we have previously shown to bind to and stabilize filamentous actin in vitro (1). In vivo data suggests that jasplakinolide-treated prostate cancer cells have both decreased labeling of F-actin and decreased amounts of rhodamine-phalloidin bound to cell extracts (2), results that could be explained by the observation that jasplakinolide and phalloidin bind competitively to actin (1). In addition, however, in vivo data also convincingly show that jasplakinolide disrupts actin filaments with alterations in cellular architecture (2, 3), an effect that cannot be explained simply by competitive binding. We now present kinetic data characterizing the steady state and time-dependent in vitro interactions between jasplakinolide and actin that provide a plausible explanation for the effects of jasplakinolide on actin distribution in cultured cells. EXPERIMENTAL PROCEDURESMaterials-Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in buffer G (5.0 mM Tris, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl 2 , and 0.01% sodium azide, pH 7.8) (4). Non-muscle actin from bovine brain was prepared by the method of Ruscha and Himes (5). Muscle and non-muscle pyrenyl-labeled actins 1 were prepared with 0.67-0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (6). Labeled and unlabeled actins were further purified by gel filtration on Superose 12 (Amersham Pharmacia Biotech). Thymosin ␤ 4 cDNA was a gift from Dr. Vivian Nachmias and was inserted in a pET-11a vector, expressed in BL21(DE3) Escherichia coli, and purified as described previously (7). Jasplakinolide was a gift from ...

show abstract

Section: The Effects Of Jasplakinolide On the Time Course Of Actin Pomentioning

confidence: 99%

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Bubb

Spector

Beyer

et al. 2000

Journal of Biological Chemistry

485

399

View full text Add to dashboard Cite

show abstract

“…The relatively low affinity of T~4 for actin (ca. 1 ~tM [4]) may be functionally significant in allowing the instant release of polymerizable actin into cytoplasm, if required.…”

Section: In~oductionmentioning

confidence: 99%

The ternary complex of DNase I, actin and thymosin β4

et al. 1996

View full text Add to dashboard Cite

Key words: Actin; Thymosin 134; DNase I; Ternary complex; Thiol-specific crosslinking the differently positioned thiol groups in TIM come close enough to distinct thiol groups in actin to allow the formation of crosslinks. In this way two contact sites between the two proteins could be identified, one between the C-terminus of actin (Cys 374) and position 6 of TIM and a second between the )'-phosphate of the actin-bound nucleotide in actin and the sequence 17 28 in TIM.X-ray analysis of monomeric actin took advantage of a facilitated crystallization of actin when complexed with DNase I. Provided DNase I exerted a similar effect on TIMactin as well, co-crystallization with DNase I would allow the interface of actin and TIM in the ternary complex to be studied. Corresponding complexes have been reported for DNase I, actin and profilin, or ADF/cofilin, respectively [14,15]. However, the ternary complex can be expected to form only if the binding of DNase I and that of TIM to actin do not strongly interfere. We therefore examined whether DNase I affects the affinity of TIM for actin by studying one of the crosslinking reactions mentioned above in the presence of DNase I. Materials and methods In~oductionThymosin 134 (TIM) is one of the small proteins in nonmuscle cells that bind to monomeric actin and thus prevent unregulated polymerization [14]. From the TiM-complexed monomers, polymerization can be started in vitro either by decapping barbed ends of filaments [5,6], or by adding nucleus stabilizers such as myosin S1 or phalloidin [7][8][9][10][11]. The relatively low affinity of T~4 for actin (ca. 1 ~tM [4]) may be functionally significant in allowing the instant release of polymerizable actin into cytoplasm, if required.Since an X-ray analysis of the actin-TiM complex has so far not been achieved we have tried to identify contact sites between the two proteins by a chemical approach [12]. For this, five TIM analogs were synthesized, each of them with one cysteine residue substituted for a hydrophobic amino acid in the TIM chain. Using a set of thiol-specific crosslinkers of varying length, we assayed whether in the complex with actin

show abstract

“…2.5 nM [8]), a much higher concentration was found inside cells (ca. 600 ,uM in the cytoplasm of platelets [7,9]). This led to the suggestion that the main physiological role of j&thymosins may be other than in modulating immune activities.…”

Section: Introductionmentioning

confidence: 99%

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

et al. 1993

View full text Add to dashboard Cite

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosinp4 (T/94), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation, Based on this effect, we determined the Kn value of the thymosin @l complex as 0.8 PM, a value that is in agreement with previous determinations. In addition to the main compound thymosin/?4, bovine tissue contains a related peptide, thymosin p (Tp9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T/$?, similar to Tp4, forms a 1:l complex with monomeric actin, and hereby inhibits actin polymerization. With a K, value of 1.1 FM the affinity of TjT9 is in the same range as that of Tp4, suggesting that T@9, like T/?4, contributes to maintaining the pool of monomeric. actin in bovine non-muscle cells. Further proof of the interaction of T/I9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslink~g experiments. Using different crosslinking reagents, like water-soluble ~~iimide (EDC), ~-maleimido~~oyl-~-hydroxys~nimi~te (MBS), and disu~inimidylsu~rate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tfi9 could be identified by imm~oblot~ng. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T/39 must be located close to the carboxy-terminus.

show abstract

Interaction of thymosin .beta.4 with muscle and platelet actin: implications for actin sequestration in resting platelets

Cited by 157 publications

References 55 publications

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

Effects of Jasplakinolide on the Kinetics of Actin Polymerization

The ternary complex of DNase I, actin and thymosin β4

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

Contact Info

Product

Resources

About