An actin‐interacting heptapeptide in the cofilin sequence

Abstract

Cofilin, a 21-kDa actin-binding protein, has a hexapeptide sequence DAIKKK which is identical to the N-terminal portion (residues 2 -7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122 -128 of cofilin, inhibited the binding of cofilin to F-actin in a dose-dependent manner. The heptapeptide cosedimented with F-actin, decreased the fluorescence intensity of pyrene-labeled F-actin, and increased the rate of polymerization of G-actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F-actin and affected the fluorescence intensity of pyrene-labeled F-actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin-binding domain of cofilin.Cofilin is a ubiquitous actin-modulating protein that binds to both G-actin and F-actin in a 1 : 1 molar ratio of cofilin to actin molecules in vitro [l -41. It shortens the average length of F-actin and increases the steady-state concentration of G-actin to a limited extent at neutral pH [l]. Moreover, cofilin is shown to control actin polymerization in a pH-dependent manner in vitro [5].Recently, we have determined the entire amino acid sequence of cofilin derived from porcine brain by cDNA cloning and sequencing [6]. Cofilin has a hexapeptide DAIKKK (residues 122 -127) identical to the amino-terminal sequence (residues 2 -7) of tropomyosin. This is interesting because cofilin and tropomyosin compete with each other for binding to F-actin [l]. Cofilin could be cross-linked to the aminoterminal region of actin molecule with a zero-length crosslinker which links a carboxyl group to an amino group [7]. Because the amino-terminal portion of the actin molecule is rich in acidic residues, basic amino acid residues of cofilin may be involved in the binding of cofilin to actin [7]. The fact that the binding of cofilin to actin is inhibited at high ionic strength (2, 81 also suggests the importance of electrostatic interactions between cofilin and actin. Thus, the DAIKKK sequence, which contains three consecutive basic residues, is a possible candidate for an actin-binding site in the cofilin sequence.In this study, we synthesized a DAIKKKL heptapeptide corresponding to residues 122 -128 of cofilin, and examined its effect on the binding of cofilin to actin and its direct interaction with actin. The actions of other synthetic peptides, DIKKKL, DAIKKL and DIKKL, were also examined.Correspondence to E. Nishida, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo-shi, Tokyo-to, Japan 113 MATERIALS AND METHODS ProteinsCofilin was purified from porcine brain by the method described previously (81. Actin was prepared from rabbit skeletal muscle according to the method of Spudich and Watt [9], and furth...

Identification of two distinct synucleins from human brain

“…For instance, the microtubuleassociated protein MAPlA binds to tubulin through an overall negatively charged region, but with a number of clusters of acidic and basic amino acids [18]. Similarly, the microtubule-associated protein MAPlB binds to tubulin through KKE repeats [19], villin binds to actin through a KKEK motif [20] and actin depolymerisation factor, cofilin and tropomyosin carry the DATKKK motif [21]. The availability of recombinant synucleins allows investigation of possible interactions with structural proteins.…”

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

“…(22,23), the role of lower molecular weights peptides was largely overlooked. Subsequent sequence analysis has revealed homology of (4 with actobindin and cofilin, two known monomer-sequestering proteins, within the region from Asp-13 through Thr-22 (AspLys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr) (24,42,43 …”

The ADF/Cofilin Proteins: Stimulus-responsive Modulators of Actin Dynamics

“…Tropomyosin competes with cofilin for filament binding . When present at a molar concentration one thousand times that of cofilin, the DAIKKKL peptide also inhibits the binding of cofilin to F-actin (Yonezawa et al, 1989). It does not, however, affect the binding of tropomyosin to actin.…”