Inhibition of Actin Polymerization by a Synthetic Dodecapeptide Patterned on the Sequence around the Actin-binding Site of Cofilin”

Cofilin is an F-actin side-binding and -depolymeriz- ing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, l-ethyl-3-(3-di- methylamino propyl)carbodiimide, was identified as Lys”” and/or Lysi14. A synthetic dodecapeptide pat- terned on the sequence around the actin-cross-linking site of cofilin (Trp’04-Meti15) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence consti-tutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depo- lymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin- binding and -depolymerizing activity

The N and C termini of actin have been shown to interact with actin-binding proteins such as myosin (17), depactin (18,19), fragmin (20), gelsolin (21), caldesmon (22), and profilin (23). Cofilin is also cross-linked to the N-terminal region (residues 1-12) of actin by a zero length cross-linker (24). On the other hand, the identification of an actin-binding sequence in actin-binding proteins is also an important and critical step for the elucidation of interactions between actin and actin-binding proteins. Recently, a sequence essential for actin binding was identified in Dictyostelium ABP-120 (25). Cofilin has a hexapeptide sequence identical to the N-terminal portion (residues 2-7) of tropomyosin (6). This region may be part of the actin-binding domain of cofilin (26). To investigate further the actin-binding domain of cofilin, we have identified the amino acid residues of cofilin cross-linked to actin by a chemical cross-linker. Furthermore, we have shown that a synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin is a potent inhibitor of actin polymerization.

MATERIALS AND METHODS
Proteins-Recombinant cofilin (r-cofilin) was expressed in Escherichia coli and then purified to homogeneity as described previously (9). Porcine brain cofilin was purified as described previously (27). Rabbit skeletal muscle actin was prepared according to the method of Spudich and Watt (28) and further purified by gel filtration on Sephadex G-100 in 2 mM Hepes, 0.1 mM CaC12, 0.2 mM ATP, 0.05 mM dithiothreitol, and 0.01% NaN3, p H 7.9.
Polyclonal antibodies against r-cofilin were raised in rabbits. The antibody against the N-terminal extrapeptide of r-cofilin ( Fig. 1, antiextrapeptide antibody) was purified by sequential column chromatography on DEAE-cellulose and EAH-Sepharose column (Pharmacia LKB Biotechnology Inc.) conjugated with the synthetic peptide corresponding to the N-terminal extrapeptide of r-cofilin.
Synthetic Peptides-A dodecapeptide (see text) and a synthetic peptide corresponding to residues 150-166 of cofilin were synthesized by an automatic peptide synthesizer (Milligen model ~0 5 0 ) . T h e synthetic peptides were purified by preparative reverse-phase high pressure liquid chromatography on PBondasphere 5 p (C, , , 100 A) with a linear gradient of 0-40% acetonitrile in 0.1% trifluoroacetic acid. The amino acid composition of the synthetic peptides was determined by a Pico-tag work station (hydrolysis) and an amino acid analyzer JEOL JLC-300. The results were consistent with theoretical yields.

Actin-binding Sequence in Cofilin
Preparation of the Complex of r-Cofilin and the N-terminal Segment of Actin-A 1:l complex of actin and r-cofilin formed in 0.1 M NaCI, 1 mM Hepes, 10 mM imidazole, and 0.1 mM dithiothreitol, pH 7.0, was cross-linked with 20 mM EDC for 2 h at 25 "C. The cross-linked complex of actin and r-cofilin and the noncross-linked r-cofilin were separated on an SDS slab gel (13%) according to Laemmli (29). The gel slices were cut out, washed with methanol, and finally dried in Vacuo according to Sutoh and co-workers (19)(20)(21)30). The hydrolysis with hydroxylamine of the cross-linked complex of actin and r-cofilin was done within gel slices by incubating the gel slices in 1.5 M hydroxylamine and 6 M guanidine hydrochloride for 4 h at 45 "C. After the reaction, the gel slices were washed with methanol, dried in uacuo, soaked in 2% SDS, 10 mM Tris, 2% 2-mercaptoethanol, and 10% glycerol, pH 8.0 (presoak buffer), for 1 h at 37 "C and then loaded directly onto slab gels. After electrophoresis, the gel strips of the complex of r-cofilin and the N-terminal segment (residues 1-12) of actin (rCAI-12) were cut out, washed with methanol, and dried in uacuo.
Mapping of the Actin-Cross-linking Site of Cofilin by the End Label Fingerprinting-The partial hydrolysis with either BNPS-skatole or CNBr of the noncross-linked r-cofilin or rCA1-12 within gel slices was performed essentially according to Sutoh and co-workers (19)(20)(21)301. The gel slices were washed with methanol, dried, and finally soaked in the presoak buffer before loading onto slab gels. After electrophoresis, the polypeptides were transferred onto Durapore membrane (type GV, Millipore), and then immunoblotting was performed (31) with the antiextrapeptide antibody.
Effect of the Dodecapeptide on the Actin-Cofilin Cross-linking-r-Cofilin (final concentration 9. The open box represents the sequence of porcine cofilin. The 9-amino acid sequence shows the N-terminal extrapeptide of r-cofilin expressed in E. coli. A 1:l complex of actin and either r-cofilin or porcine brain cofilin was cross-linked with EDC, and then the reaction mixture was electrophoresed on SDS-polyacrylamide gel as described under "Materials and Methods." Noncross-linked r-cofilin (lanes l and l ' ) , cross-linked Drnduct. of r-rnfilin and act.in f Innos 2 and 2'). nnn-crnsslinked porcine brain cofilin and actin (lanes 3 and 3'), cross-linked product of porcine brain cofilin (lanes 4 and 4 ' ) , and actin (lanes 5 and 5') which were excised from the gels were reelectrophoresed on SDS-polyacrylamide gel, transferred to membrane, and stained with either Amido Black (lanes 1-5) or the antiextrapeptide antibody (lanes 1 ' 4 ' ) . amino acids cross-linked to actin in cofilin sequence are indicated by erization (see Fig. 5a) of G-actin (3 p~, 6% pyrene actin) or depolymerizatlon (see Fig. 56) ot I+'-actln (3 p~, 6% pyrefie actin) was moni-

TABLE I
The dodecapeptide has no effect on the binding of cofilin to F-actin The molar ratios of r-cofilin bound to F-actin to actin molecule in F-actin were determined by the pelleting assay as described under "Materials and Methods" in the presence of various concentrations of the dodecapeptide. and is shown in Fig. 5 in an arbitrary unit. The excitation and emission wavelengths were 365 and 407 nm, respectively.

RESULTS AND DISCUSSION
An Actin-Cross-linking Site on Cofilin Sequence-Cofilin has been expressed in E. coli and purified to homogeneity (9).
peptide containing nine amino acids (see Fig. 1) but is indistinguishable from the authentic cofilin purified from porcine brain in its interaction with actin in uitro (4,9). Because cofilin is covalently cross-linked to an N-terminal segment (24) and an Asn-Gly sequence exists only a t residues 12-13 of actin, digestion by hydroxylamine of the cross-linked product of r-cofilin and actin produced the complex of r-cofilin and the N-terminal segment of actin (we call this complex rCA1-12). rCA,-,, moved, as expected, slower than noncrosslinked r-cofilin in SDS-polyacrylamide gels as shown in Fig.  2, u and b (compare bunds A and A'). The cross-linking site of the N-terminal segment of actin on cofilin sequence was determined by means of the end label fingerprinting method, for which we made a rabbit polyclonal antibody against the N-terminal extrapeptide of r-cofilin (Fig. 1). The specificity of the antibody (antiextrapeptide antibody) is shown in Fig.  1. The antiextrapeptide antibody reacted only with r-cofilin and the cross-linked complex of actin and r-cofilin but did not react with porcine brain cofilin, actin, or the cross-linked complex of actin and porcine brain cofilin.
A fragment that comprises the amino acid residues from the N terminus to TrpIo4 of cofilin was not cross-linked to the N-terminal segment of actin (Fig. 2c) since treatment of rCAI-,, with BNPS-skatole produced a fragment containing thc cntmpcpticlc uf r-cufiliu, wlluae Illubilily UII 3DS gels was completely identical with that of a fragment generated from noncross-linked r-cofilin (Fig. 2a, bunds B and B'). CNBr mapping showed that the actin-cross-linking site is located in the segment of cofilin from Leu'5 to Met''5 (Fig. 2c) since a fragment from the N terminus to Met"5 derived from Actin-binding Sequence in Cofilin rCA1-12, moved slower than the corresponding fragment derived from noncross-linked r-cofilin (Fig. 2b, bands B and B') whereas a fragment from the N terminus to Met75, derived from both rCAI-12 and noncross-linked r-cofilin, exhibited the same electrophoretic mobility (Fig. 2b, bands C and C'). From these maps we concluded that the actin-cross-linking site of cofilin is located within Ala'05-Met115 (Fig. 2c). Because an N terminus of actin is blocked and the N-terminal segment (residues 1-12) of actin has five carboxyl groups but no amino groups, the candidates for residues cross-linked to actin in Ala'05-Met115 of cofilin are LYS"~ and/or Lys114 (Fig. 3, marked  with 0).

Homology of the Sequence of the Dodecapeptide with Other
Actin-binding Proteins-A dodecapeptide corresponding to the sequence of Trplo4-Met115 of cofilin was synthesized to examine the function of this region (Fig. 3, top row). Interestingly, prediction of the secondary structure according to the method of Chou and Fasman (33) showed that only this region and the C-terminal short segment displayed random coil potential. As shown in Fig. 3, a sequence very similar to the dodecapeptide is found in destrin ( 9 ) , ADF (14,15), and depactin (16), mammalian, chicken, and echinodermatous actin-depolymerizing proteins, respectively. It should be noted that only this region in the depactin sequence exhibits close similarity with the corresponding sequence of cofilin, destrin, or ADF. Moreover, other actin-binding proteins, severin (residues 257-268) (34), dystrophin (residues 2978-2989) (35), villin (residues 485-496) (36), and a-spectrin (residues 218-229) (37), have a portion weakly homologous to the dodecapeptide sequence.

Inhibition of the Actin-Cofilin Cross-linking by the Dodeca-
peptide-The synthetic dodecapeptide inhibited the crosslinking of cofilin to actin by EDC in a dose-dependent manner ( Fig. 4, a and b), and instead of cofilin the dodecapeptide itself was cross-linked to actin (Fig. 4b, lane 2). On the other hand, a synthetic peptide corresponding to the C-terminal portion (residues 150-166) of cofilin did not inhibit the cross-linking of cofilin to actin (Fig. 4a, 0; and Fig. 4b, lane 3 ) . These results suggest that the dodecapeptide specifically competes with cofilin for binding to actin.
Inhibition of Actin Polymerization by the Dodecapeptide-The pelleting assay revealed that the dodecapeptide did not inhibit the binding of cofilin to F-actin at all (Table I). The amount of F-actin was, however, decreased in the presence of the dodecapeptide irrespective of the presence or absence of cofilin (data not shown). We examined the effect of the dodecapeptide on the time course of actin polymerization by using pyrene-labeled actin. Fig. 5a shows that the dodecapeptide increased the lag time for actin polymerization, slowed down the speed of polymerization, and decreased the final extent of polymerization. Actin polymerization was almost completely inhibited in the presence of 100 dodecapeptidesl actin (Fig. 5a). The pelleting assay confirmed that the decrease in the fluorescence intensity of pyrene actually reflects the decrease in the amount of F-actin (data not shown). The inhibitory activity of the peptide for actin polymerization was not dependent on changes in pH of the medium between pH 7.0 and 8.3 (data not shown). The dodecapeptide had the ability to depolymerize F-actin (Fig. 5b). Almost complete depolymerization occurred in the presence of 100 dodecapeptides/actin. The other synthetic peptide, corresponding to the C-terminal portion (residues 150-166) of cofilin, had no effect on actin polymerization and did not depolymerize F-actin at all (Fig. 5, a and b, broken lines). The F-actin depolymerization induced by the dodecapeptide was much slower than that induced by cofilin at pH 8.3 (3). This suggests that the dodecapeptide depolymerizes F-actin by sequestering G-actin equilibrated with F-actin. However, the possibility cannot be excluded that the dodecapeptide might also have the ability to attack F-actin directly because the initial rate of F-actin depolymerization increased slightly with increasing amounts of dodecapeptide (Fig. 5b). The apparent dissociation constant for the interaction of the dodecapeptide with actin was roughly estimated to be 20-60 PM, based on the dodecapeptide concentration required for complete inhibition of actin polymerization (Fig. 5a) and the critical concentration of actin under the ionic conditions used. Thus, the affinity of the dodecapeptide for actin is 2-3 orders of magnitude lower than that of cofilin (0.1-0.2 PM). It should be noted that such a short peptide with a potent inhibitory activity for actin polymerization has not been reported previously.
Thus, determination of an actin-cross-linking site on cofilin led us to identify a dodecapeptide capable of inhibiting actin polymerization strongly. As a sequence very similar to the dodecapeptide exists in a family of actin-depolymerizing proteins, this sequence region may constitute the domain responsible for actin-depolymerizing activity.