A Short Sequence Responsible for Both Phosphoinositide Binding and Actin Binding Activities of Cofilin*

Cofilin is a widely distributed actin-modulating pro- tein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trplo4-Met115 of cofilin is a po- tent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chern. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We fur-ther show that the dodecapeptide as well as cofilin binds to PIPz molecules and inhibits PIP2 hydrolysis

Previous studies using gelsolin fragments generated by the limited proteolysis or the gene truncation of plasma gelsolin cDNA revealed that the PIP,-binding site on gelsolin sequence is on residues 150-160 (12,13). It is also suggested that the sequence of residues 150-160 may be the F-actinbinding site (12,13). Cofilin does not have the sequence similar to this polyphosphoinositide binding sequence of gelsolin. The polyphosphoinositide binding sequences of other actin-binding proteins have not yet been identified. Here, we have shown that the actin binding sequence of cofilin (Trplo4-Met"')), previously identified by the chemical cross-linking study and by the use of the synthetic peptide, is a phosphoinositide binding sequence. Thus, inhibition of actin polymerization by the synthetic dodecapeptide corresponding to Trp'"4-Met"' of cofilin is canceled specifically by phosphoinositides such as PIP,, PIP, and PI.
Recent reports have revealed that profilin binds with high affinity to PIP, molecules ( 7 , 14) and that profilin inhibits the hydrolysis of PIP, by unphosphorylated phospholipase C (PLC) but not the hydrolysis by PLC phosphorylated by epidermal growth factor receptor tyrosine kinase (14,15). These results raise the interesting possibility that actin-modulating proteins function as a negative regulator of the phosphoinositide signaling pathway in quiescent cells (14,15). In this report, we show that the dodecapeptide as well as cofilin binds to PIP, molecules and inhibits PIP2 hydrolysis by PLC.

Proteins-Recombinant cofilin was expressed in Escherichia coli
and then purified to homogeneity as described previously (16). PLC-y1 and PLC-8 were purified from bovine thymus and PLC-y2 was from bovine spleen as described previously (17). PLC-8, was purified from bovine brain as described previously (23).
Synthetic Peptides-A dodecapeptide, a pentapeptide, and a peptide corresponding to residues 150-166 of cofilin were synthesized as described previously (6).
Chemicals-Phosphatidylinositol (PI), phosphatidylinositol 4monophosphate (PIP), PIP?, phosphatidylserine (PS), phosphatidylcholine (PC), inositol 1,4,5-triphosphate (IP:,), and 1-oleoyl-2-acetylglycerol were all purchased from Sigma. The lipids were dissolved in water to a final concentration of 1 mg/ml and sonicated three times for 10 s in a sonicator. IP.I was dissolved in water to a final concentration of 0.1 mg/ml. The suspensions were frozen a t -80 "C in 0.1ml aliquots. The lipid solution was quickly thawed in warm water and sonicated in a sonicator three times for 10 s just before use.

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Assay for Actin Polymerization Using Pyrene-labeled Actin-In the presence or absence of the synthetic peptide and either lipids or IP,, polymerization of G-actin (5 p~, 3.6% pyrene actin) was monitored at 25 "C by the changes in fluorescence intensity of pyrene labeled to actin in a buffer containing 90 mM KCl, 20 p M ATP, 10 pM CaCl,, and 4 mM Pipes, pH 7.0. The pyrene-labeled actin was prepared as described (1, 18). The fluorescence intensity was measured with a Hitachi 650-10s fluorescence spectrophotometer, and is shown as arbitrary units. The excitation and emission wavelengths were 365 and 407 nm, respectively.
Gel Filtration Chromatography-Gel filtration was performed on Sephadex G-25 (0.9 X 1 2 cm) at room temperature. The dodecapeptide (210 p~) was mixed with no phospholipid, PIP2 (630 FM), PIP (630 or PC (630 p~) in a solution consisting of 10 mM potassium phosphate and 100 mM NaC1, pH 7.0, and then passed through the column in the same buffer solution. The elution of the dodecapeptide was detected by measuring the absorbance a t 215 nm.
PIP, Hydrolysis by Phospholipase C-PIP, hydrolysis by PLC was performed at 37 "C for 10 min with small unilamellar vesicles consisting of ["HIPIP, (20 p~) and phosphatidylethanolamine (20 p~) in 50 mM Mes, pH 6.5, 0.1 mM CaCl,, and 1 mg/ml of bovine serum albumin. The ['HIIP, generated was separated from ["H]PIP, and then the amount of [:lH]IP:I was measured by liquid scintillation counting as described previously (17).

RESULTS AND DISCUSSION
The synthetic dodecapeptide, WAPECAPLKSKM, corresponding to T~p '~~-M e t '~' of cofilin inhibited actin polymerization as previously reported (  (O), PS (A), 1-oleoyl-3-acetylglycerol (0), or IP:, (+) did not affect the activity of the dodecapeptide at all. Thus, the inhibitory effect of the dodecapeptide on actin polymerization was inhibited specifically by phosphoinositides. Because phosphoinositides do not affect the actin polymerization and do not bind to actin ( l l ) , these results suggest that the phosphoinositides block the interaction of the dodecapeptide with actin by binding to the dodecapeptide.
To show the binding of the dodecapeptide to the phosphoinositides directly, we carried out the gel filtration chromatography. In gel filtration on the Sephadex G-25 column, PIP, or P I P eluted at void volume while the dodecapeptide alone eluted at the position corresponding to an apparent molecular weight of -1000 (Fig. 2,O). When the dodecapeptide mixed with PIP, or PIP was passed through the column, however, the dodecapeptide co-eluted with PIP, or PIP at void volume (Fig. 2, 0 ) . This indicated that the dodecapeptide binds to PIP, or PIP. In contrast, neither PC nor PS affected the elution position of the dodecapeptide (Fig. 2, X). A synthetic peptide corresponding to residues 150-166 of cofilin, that does not inhibit actin polymerization at all (6), eluted by itself at the position corresponding to an apparent molecular weight of -2000. Neither PIP, nor P I P affected the elution position of this 17-amino acid synthetic peptide (data not shown). These results suggest that phosphoinositides such as PIP, and PIP specifically bind to the dodecapeptide, that is, the actin-binding sequence of cofilin. Thus, the inhibition of interaction of cofilin with actin by phosphoinositides may be accounted for by assuming that phosphoinositides and actin compete for binding to the same site, the dodecapeptide sequence, in cofilin.
As the dodecapeptide as well as cofilin was found to bind to PIP, tightly, we tested the effect of cofilin and the dodecapeptide on PIP, hydrolysis by PLC. Cofilin inhibited the hydrolysis of PIP, by PLC-7. in a dose-dependent manner (Fig. 3a). The rate of hydrolysis was 0 at molar ratios of 1 cofilin per 2.5-3 PIP, molecules. The stoichiometry of cofilin

TABLE I Effects of cofilin and the dodecapeptide on PIP, hydrolysis
by PLC isoforms PIP, hydrolysis by PLC isoforms, PLC-p,, PLC-yl, PLC-72, or PLC-6 was performed in the presence of cofilin (final concentration was 4.5 p~) or the dodecapeptide (final concentration was 120 pM) or in their absence as described under "Materials and Methods." The activities of PLC isoforms in the absence of cofilin or the dodecapeptide were regarded as 100%. required for the complete inhibition was nearly the same as that of profilin (14). The dodecapeptide also inhibited the hydrolysis of PIP, by PLC--y, in a dose-dependent manner (Fig. 3b). At a molar ratio of 10 dodecapeptides per 1 PIPz molecule, the rate of hydrolysis was almost 0. Neither actin nor the other synthetic peptide, corresponding to residues 150-166 of cofilin, affected the rate of PIPz hydrolysis by PLC--y2 (data not shown). Both cofilin and the dodecapeptide also inhibited PIPz hydrolysis by other isoforms of PLC, PLC-Dl, PLC--yl, and PLC-6 (

Comparison of activities of the dodecapeptide and the pentapeptide
The concentrations of the synthetic peptides required for the halfmaximal inhibition of the initial rate of actin polymerization (assay conditions were the same as in Fig. 1 except for 3 p~ actin containing 6% pyrene actin) and for the half-maximal inhibition of PIP, hydrolysis by PLC-72 (as in Fig. 3) were determined. As both abilities of the synthetic pentapeptide, LKSKM, were weak, the data for the pentapeptide cannot be accurately determined and are rough estimates. These results suggest the possibility that not only profilin but also cofilin may bind to PIP2 on the membrane and maintain the very low rate of PIP, hydrolysis in resting cells.
Because the basic residues (Lys112 and Lys114) in the dodecapeptide sequence are thought to be involved in the interactions of cofilin with the acidic residues of the N-terminal segment of actin and the acidic polar head of phosphoinositides, we assume that the latter portion of the dodecapeptide, LKSKM, might be important for cofilin binding to actin and phosphoinositides. Then, we examined whether the synthetic pentapeptide, LKSKM (residues 111-115 of cofilin), interacts with actin and phosphoinositides, and found that the pentapeptide has abilities to inhibit actin polymerization in a phosphoinositide-sensitive manner and to inhibit PIP2 hydrolysis by PLC--y2, although both abilities are weak. The concentrations of the pentapeptide required for 50% decrease in the initial rate of actin polymerization and 50% inhibition of PIPz hydrolysis by PLC-y2 were roughly 50 and 20 times higher than those of the dodecapeptide, respectively (Table  11). These results suggest that although this pentapeptide segment containing 2 lysine residues contributes to the actin and PIPp binding of cofilin, the N-terminal portion (WAPE-CAP) of the dodecapeptide is also necessary for full activities of the dodecapeptide.
In summary, the dodecapeptide sequence (Trp104-Met115) of cofilin has the ability to interact with both actin and phosphoinositides, and thus may be responsible for cofilin's abilities to interact with actin and to inhibit PIPz hydrolysis by PLC by binding to PIP2. Thus, the dodecapeptide sequence may constitute a multifunctional region of cofilin. As the sequence very similar to the dodecapeptide exists in a family of actin-depolymerizing proteins, destrin (19, ZO), depactin (21), and actin depolymerizing factor (22) as reported previously (6), this sequence region may constitute a domain responsible for not only actin depolymerizing activity but also PIPz binding activity of these proteins. These actin-depolymerizing proteins in concert with profilin may function as negative regulators of the phosphoinositide signaling pathway in addition to functioning as regulators of actin cytoskeleton.

Phosphoinositide Binding
Sequence of Cofilin