Identification of a phosphatidylinositol 4,5-bisphosphate-binding site in chicken skeletal muscle alpha-actinin.

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle α-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T.(1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on α-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T.(1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle α-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-γ1 and -δ1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed α-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of α-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-δ1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-δ1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in α-actinin and that similar sequences found in spectrin and PLC-δ1 may be involved in the interaction with PIP2.

We previously reported that phosphatidylinositol 4,5bisphosphate (PIP 2 ) dramatically increases the gelating activity of smooth muscle ␣-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150 -152) and that the hydrolysis of PIP 2 on ␣-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518 -1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168 -184 (TAPYRNVNIQNF-HLSWK) from striated muscle ␣-actinin contains a PIP 2binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-␥1 and -␦1. Furthermore, we detected an interaction between PIP 2 and a bacterially expressed ␣-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of ␣-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP 2 and the peptide (amino acids 168 -184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzymelinked immunosorvent assay. We also found this region homologous to the sequence of the PIP 2 -binding site in spectrin and the pleckstrin homology domains of PLC-␦1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-␦1 inhibited PLC activities. These results indicate that residues 168 -184 comprise a binding site for PIP 2 in ␣-actinin and that similar sequences found in spectrin and PLC-␦1 may be involved in the interaction with PIP 2 .
In addition to its role as a signal-generating lipid, PIP 2 has been shown to modulate the functions of various proteins such as PKC (5,6), -calpain (7), ADP-ribosylation factor 1 (8), and phospholipase D (9). PIP 2 also binds to actin-regulating pro-teins such as profilin (10), cofilin (11), gelsolin (12), gCap (13), and ␣-actinin (1) and regulates the functions of these proteins. When PIP 2 binds to ␣-actinin, which is an actin cross-linking protein, it further activates actin gelation by ␣-actinin (1). It is noteworthy that profilin plays crucial roles in tyrosine kinasecoupled PIP 2 hydrolysis. Under resting conditions, PLC-␥1 causes little hydrolysis of profilin-bound PIP 2 , but PLC-␥1 phosphorylated by tyrosine kinases overcomes the inhibitory effect by profilin and hydrolyzes bound PIP 2 (14). It has also been shown that the decrease in PIP 2 bound to ␣-actinin and vinculin by treatment with platelet-derived growth factor correlates with the depolymerization of actin (2). All these data suggest that the amount of PIP 2 in the actin-binding protein regulates the development of stress fibers when the cells are stimulated.
␣-Actinin was originally discovered in skeletal muscle as a protein factor promoting the superprecipitation of actomyosin and inducing the formation of actin fibers (15). The fact that ␣-actinin is found at focal contacts where actin is anchored to a variety of intercellular structures in non-muscle cells suggests that ␣-actinin plays some role in the linkage between the plasma membrane and actin. We previously reported that ␣-actinin from skeletal muscle contains large amounts of PIP 2 , whereas that from smooth muscle contains little (1). Interestingly, the addition of PIP 2 to smooth muscle ␣-actinin increases the gelation activity of actin to the level produced by skeletal muscle ␣-actinin, suggesting that PIP 2 plays important roles in the organization of the cytoskeleton.
Recently, the preckstrin homology (PH) domain has been found in a variety of functional proteins (16), including protein kinases, substrates for kinases, regulators of small G proteins, PLC isozymes, and cytoskeletal proteins. This domain has been reported to bind to PIP 2 (17), although it also associates with the ␤␥ subunit of trimeric G proteins (18,19) and PKC (20). In that case, PIP 2 is thought to act as a target for PH domaincontaining proteins in membranes.
To understand the role of PIP 2 in protein functioning or in protein-protein interactions, it is important to identify the PIP 2 -binding site in proteins. We describe here that amino acids 168 -184 in chicken skeletal muscle ␣-actinin comprise a PIP 2 -binding site and that basic amino acids, arginine 172 and lysine 184, are important for this interaction. A region homologous to the PIP 2 -binding site in ␣-actinin is also found in spectrin and the PH domains of several proteins including PLC-␦1 and Grb7.

EXPERIMENTAL PROCEDURES
Materials-Striated muscle ␣-actinin was purified from chicken pectoralis muscle according to the methods described by Feramisco and Burridge (21). Mouse monoclonal antibody to PIP 2 was developed as described previously (22). Transformer site-directed mutagenesis kit and QIA express vector system were obtained from Clontech (Palo Alto, CA) and QIAGEN. PIP 2 was prepared from bovine spinal cords by the method of Schacht (23). [ 3 H]PIP 2 (7.6 Ci/mmol) was from DuPont NEN. DEAE-cellulose (Whatman), cellulose phosphate (Whatman), the Hi-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Sequencing of the Proteolytic Digestion Fragments of ␣-Actinin-Purified striated ␣-actinin was digested with ␣-chymotrypsin at an enzyme to substrate ratio of 1:200 (mol/mol) in 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 1 mM 2-mercaptoethanol at 37°C for the indicated times. The proteolytic fragments of ␣-actinin were electrophoresed on 8.5% SDS-polyacrylamide gels and the gels were subjected to Western blot analysis with anti-PIP 2 antibody as described previously (2).
Cleavage with lysylendopeptidase was carried out as follows. ␣-Actinin was digested overnight with 1:200 (mol/mol) lysylendopeptidase in 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM 2-mercaptoethanol, and 1 M urea at 37°C. The digests were separated by high performance liquid chromatography on a C18 reverse-phase column. For the detection of PIP 2 -bound peptide, every fraction, including the front, was lyophilized, dot-blotted on nitrocellulose, and stained with anti-PIP 2 antibody. The amino acid sequence of the PIP 2 -containing fragment was determined with a protein sequencer (ABI 477A/120A).
PLC Assay-PLC activity was assayed by the methods described previously (25). In brief, a reaction mixture containing 50 mM Mes buffer, pH 6.5, 400 M Ca 2ϩ , 1 mg/ml bovine serum albumin, 20 M PIP 2 , 20,000 dpm of [ 3 H]PIP 2 , and PLC-␥1 or -␦1 was incubated at 37°C for 10 min in the presence or absence of various proteins or peptides. The reaction was terminated by the addition of 2 ml of chloroform/ methanol (2:1) and radioactive inositol trisphosphate was extracted with 1 N HCl. The radioactivity was measured by a scintillation counter.
Construction of ␣-Actinin Mutants-Four point mutants, with mutations in the PIP 2 -binding site of chicken skeletal muscle ␣-actinin, were produced using a transformer site-directed mutagenesis kit. The cDNA, EP␣An1 (26), encoding chicken muscle ␣-actinin was subcloned into pUC19. Four mutagenic primers, TAAGGAGCAGTTTTTATTTGAC, TGAATGTTCACATTTATGTAAG, AATCCAAGGCCATCTATCCAGC, and AGGTCAGGTCGGTGTATGTGGA, were used to generate pointmutated cDNAs. These mutations result in substitutions of Ile for Arg 166 , Arg 172 , Lys 184 , and Arg 195 (designated as ␣An.166.1, ␣An.172.2, ␣An.184.2, and ␣An.195., respectively). The mutated nucleotide sequences were confirmed by sequence analysis. DNA fragments corresponding to amino acids 137-259 were amplified by polymerase chain reaction, using these point-mutated cDNAs as templates, and subcloned into a six histidine (6 ϫ His)-tagged expression vector. We also constructed a non-mutated DNA fragment and subcloned it into the same vector to obtain ␣An.0.1.
Expression and Purification of Recombinant Protein-Escherichia coli JM109 cells containing ␣An fusion constructs were grown in LB containing 100 g/ml ampicillin, and induced with 1 mM isopropyl-1thio-␤-D-galactopyranoside. Cells were harvested by centrifugation and sonicated on ice in buffer (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0). After centrifugation, the supernatant was applied to Ni-nitrilotriacetic acid resin, which has a high affinity for the 6 ϫ His tag, and incubated for 1 h with rotation. The expressed proteins were eluted from the resin with 8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 6.3, and 100 mM EDTA and dialyzed against 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 6.3. These proteins migrated with the expected relative molecular mass of 13 kDa (data not shown).
Detection of Direct Interaction of PIP 2 with Peptides or Bacterially Expressed ␣-Actinin Fragments by Enzyme-linked Immunosorvent Assay-Peptides (I-IV) or proteins were coated on the 96-well multiplates overnight at room temperature. After the plates were blocked with 2% bovine serum albumin in phosphate-buffered saline, various amounts of PIP 2 were added to each well and incubated at room temperature for 30 min. After washing the plates with phosphate-buffered saline containing 0.05% Tween 20, antibody against PIP 2 was added to wells, followed by the treatment with peroxidase-conjugated anti-mouse immunoglobulins. The interactions were visualized with 0.4 mg/ml orthophenilenediamine in 100 mM citrate buffer, pH 5.0.

Determination of a PIP 2 -binding Site in Chicken
Skeletal ␣-Actinin-First, we examined whether the PIP 2 -binding site was located in the N-terminal actin-binding domain or the C-terminal tails of ␣-actinin, where two homodimers bind to each other. Cleavage of ␣-actinin with ␣-chymotrypsin for the indicated times revealed that the 102-kDa ␣-actinin was converted to 88-, 68-, 55-, and 34-kDa fragments as previously reported (27) (Fig. 1, A and C). Among them, PIP 2 was strongly detected in the 34-kDa fragment by Western blot analysis with anti-PIP 2 antibody (Fig. 1B). These results show that the PIP 2binding site exists within the N-terminal actin-binding domain.
To more closely locate the PIP 2 -binding site, limited proteolysis with lysylendopeptidase was carried out. The digests of ␣-actinin were separated on a C18 reverse-phase column ( Fig. 2A) and binding ability to PIP 2 was assayed by dot-blot analysis with anti-PIP 2 antibody (Fig. 2B). Two positive peaks (shown by the arrowhead and * in Fig. 2A) were obtained. The later peak (*), which was very broad and weakly positive for PIP 2 , was found to be the 34-kDa N-terminal domain by SDSpolyacrylamide gel electrophoresis (data not shown). On the other hand, the earlier peak was very sharp and gave a very strong positive signal for PIP 2 binding. We found the sequence of this peptide to be TAPYRNVNIQNFHLSWK, which corre- sponds to the sequence of amino acid residues 168 -184 in chicken skeletal ␣-actinin. We conclude therefore that this region in the actin-binding domain contains a binding site for PIP 2 .
Inhibition of Phospholipase C Activity by Synthetic Peptides-To examine whether a peptide that includes a PIP 2binding site inhibits the activity of PLC, we synthesized the peptide corresponding to amino acids 168 -184 (TAPYRN-VNIQNFHLSWK, peptide IV) of ␣-actinin. We also synthesized peptides rich in basic amino acids corresponding to amino acids 39 -47 (WEKQQRKTE, peptide I), 84 -99 (GERLPKPDRGKM-RFHK, peptide II), and 194 -210 (HRHRPDLIDYSKLNKDD, peptide III) as controls (Fig. 4A). As shown in Fig. 4B, peptide IV inhibited the activities of both PLC-␥1 and -␦1 in a dose-dependent manner, with almost complete inhibition produced by 125 M peptide IV. Although there was no remarkable difference between the inhibitions of PLC-␥1 and PLC-␦1, the presence of 0.5% octyl glucoside reduced the inhibition. This may be due to the formation of smaller micelles of PIP 2 leading to increased utility of PIP 2 as a substrate. On the other hand, peptides I, II, and III had no effect on the activities of PLC. These results suggest that the amino acid sequence 168 -184 of ␣-actinin contains a PIP 2 -binding site. We also examined the effect of ␣-actinin purified from chicken smooth muscle on PLC activities (Fig. 4C). At maximum soluble concentration, 19.2 M, ␣-actinin caused a decrease in activity down to about 70% of control. Since we found that there are regions homologous to the PIP 2 -binding site of ␣-actinin in the spectrin ␤-chain and the PH domains of several proteins, we also synthesized the corresponding peptides (Fig. 4A) and examined the effects of these peptides on PLC activities. We chose PLC-␦1 and Grab7 on the basis of alignment, RXXXXXXX(H/R/K)XX(X)W(K/R). A peptide from gelsolin, which is a known PIP 2 -binding site (24), was also synthesized. PEP-PLC-␦1 strongly inhibited the activity of PLC-␥1 in a dose-dependent manner and 200 M PEP-PLC-␦1 caused a decrease in activity down to 23% of control. PEP-spectrin also caused a decrease in the activity of PLC-␥1 to 53%. On the other hand, 100 M PEP-Grb7 and PEP-gelsolin had no significant effect on PLC-␥1 activity. Similar effects were observed when PLC-␦1 was used, but the degrees of inhibition or stimulation of PLC-␦1 activity were weaker than those of PLC-␥1 (Fig. 4D).
Inhibition of PLC Activity by Bacterial Expression Proteins-To clarify the precise mechanism of the interaction between PIP 2 and the PIP 2 -binding site in ␣-actinin, we produced various bacterial histidine tag proteins. We examined whether these proteins inhibited PLC activities as strongly as peptide IV or ␣-actinin. We used 20 M recombinant peptides as maximal soluble concentration. As shown in Fig. 5, ␣An.0.1 partially inhibited the activities of PLC-␥1 (Fig. 5A) and PLC-␦1 (Fig. 5B) to about 69 and 63% of the control level, respectively. These values are comparative to those obtained for ␣-actinin and more effective than that for peptide IV. Two point-mutated proteins, ␣An.166.1 and ␣An.195.1, also inhibited the activities almost as identically to ␣An.0.1, while ␣An.172.2 and ␣An.184.2 inhibited the activity of PLC-␥1 to about 80 -85% and PLC-␦1 to about 82-87% of control, respectively. These results show that the basic amino acids arginine 173 and lysine 184 play important roles in the interaction between PIP 2 and the ␣-actinin PIP 2 -binding site.
Direct Interactions of PIP 2 with Peptides and Bacterially Expressed ␣-Actinin Fragments-To confirm whether the residue 168 -184 comprise a binding site for PIP 2 , direct interactions between PIP 2 and peptides or bacterially expressed fragments were examined by enzyme-linked immunosorvent assay. As shown in Fig. 6A, peptide IV bound very tightly to PIP 2 and this binding reached plateau at 166 ng/well PIP 2 , while peptides I, II, and III did not bind to PIP 2 . Moreover, bacterial expression proteins ␣-An.0.1, ␣-An.161.1, and ␣-An.195.1 also bound to PIP 2 tightly, while two mutated proteins, ␣-An.172.2 and ␣-An.184.2, caused the decrease in this binding down to about 68 and 51% of control, respectively (Fig. 6B). DISCUSSION There are many reports of specific interactions between phospholipids and proteins. The C2 domains of PKC (5, 6), phospholipase A 2 (28), PLC, Ras-GTPase activating protein (29), rabphilin (30), and synaptotagmin I (31,32) have been proposed to contain phospholipid binding domains. ADP-ribosylation factor I (8), dynamin (33), myristoylated alanine-rich protein kinase C substrate (34), -calpain (7), and many actinregulating proteins (1, 10 -14) have also been shown to interact with acidic phospholipids including PIP 2 . These interactions induce the translocation of PKC, synaptotagmin I, and dynamin to the plasma membrane, or activate phospholipase D, ADP-ribosylation factor I, and -calpain. Synaptotagmin I is thought to be involved in the docking and fusion steps in calcium-dependent exocytosis. Interestingly, it has become clear that PIP 2 synthesis by phosphatidylinositol 4-phosphate 5-kinase is also concerned in exocytosis (35). Additional evidence for a role of PIP 2 in vesicular trafficking was provided by Cantley et al. (36). They reported that PIP 2 stimulates in vitro the activity of partially purified membrane phospholipase D, in which PIP 2 functions as a phospholipase D cofactor (9). These results suggest that phospholipids by themselves play important roles in modulating enzyme activities and targeting for translocation.
We have shown that ␣-actinin from chicken striated muscle contains large amounts of PIP 2 while ␣-actinin from chicken smooth muscle has little PIP 2 , but that the latter can bind to exogenous PIP 2 . In vitro, the addition of PIP 2 dramatically stimulates the gelating activity of actin by smooth muscle ␣-actinin (1). Furthermore, it has been shown that the amount of PIP 2 bound to ␣-actinin and vinculin decrease in response to platelet-derived growth factor stimulation in vivo (2). These facts suggest that ␣-actinin-bound PIP 2 is dynamically metabolized under physiological conditions and that PIP 2 by itself regulates the organization of stress fibers. Thus, we tried to clarify the binding site of PIP 2 in ␣-actinin.
Amino acid sequences which contain PIP 2 -binding site in skeletal muscle ␣-actinin are homologous to that in chicken smooth muscle ␣-actinin (Fig. 3), except for the substitution of a basic amino acid, arginine, to another basic amino acid, lysine. This substitution may have no effect on PIP 2 -binding, but these basic amino acids seem to be very important for binding, because mutants in which either arginine 172 or lysine 184 is replaced by isoleucine partially lose their inhibitory effect on PLC activities (Fig. 5) and their direct binding with PIP 2 (Fig. 6B). Sequences homologous to the PIP 2 -binding domain in ␣-actinin also exist in some cytoskeletal-related proteins such as spectrin ␤-chain or integrin ␤-7 subunit precursor, although these are not yet reported as PIP 2 -binding proteins. On the other hand, we found no homologous sequence in gelsolin or cofilin, which have been reported previously to be  PIP 2 -binding proteins (11,12). For gelsolin, cofilin, and profilin, we could detect no PIP 2 binding by Western blot analysis. This may be due to the low affinity of PIP 2 for these proteins compared to ␣-actinin.
We also found homologous regions in the PH domains of several proteins. The PH domain is suggested to be involved in protein-protein or lipid-protein interactions, because the PH domain is reported to associate not only with PIP 2 (17), but also with the ␤␥ subunit of trimeric G protein (18,19) and PKC (20). An arginine to cysteine substitution in the N-terminal PH domain (␤ 2 -sheet) of Bruton's tyrosine kinase is thought to be the cause of X-linked immunodeficiency in mice (37). This result suggests that the basic amino acid arginine in the PH domain may play a critical role in the signaling of Bruton's tyrosine kinase. Regions homologous to the PIP 2 -binding site in ␣-actinin also exist in the ␤ 1 -and ␤ 2 -sheets of the PH domains of PLC-␦1 and Grb7. There has been another report that inositol 1,4,5-trisphosphate binds to PLC-␦1 and that this interaction is inhibited by PIP 2 (38). The inositol 1,4,5-trisphos-phate binding site on PLC-␦1 is thought to comprise amino acids 30 -43, which overlaps with the site which we aligned (amino acids [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. In fact, the peptide from PLC-␦1 strongly inhibited the activity of PLC-␥1, but PEP-Grb7 did not inhibit. Although we do not know the precise reason, three-dimensional conformation may be important for the interaction of the peptide and PIP 2 . We have shown that ␣-actinin, a bacterially-expressed protein, and a synthetic peptide corresponding to amino acids 168 -184 of ␣-actinin inhibit the activities of PLC-␥1 and PLC-␦1. From these results, it appears likely that PLC inhibition is induced by PIP 2 competition. Goldschmidt-Clermont et al. (14) have reported that PLC-␥1 causes little hydrolysis of PIP 2 bound to profilin, but that PLC-␥1 phosphorylated by tyrosine kinase overcomes the inhibitory effect of profilin. Additionally, PIP 2 bound to ␣-actinin may be hydrolyzed by activated PLC-␥1 when cells are activated. This problem remains to be solved in future.