©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Phosphatidylinositol 4,5-Bisphosphate-binding Site in Chicken Skeletal Muscle -Actinin (*)

(Received for publication, August 24, 1995; and in revised form, November 17, 1995)

Kiyoko Fukami (1) Norio Sawada (2) Takeshi Endo (2) Tadaomi Takenawa (1)(§)

From the  (1)Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan and the (2)Department of Biology, Faculty of Science, Chiba University, Inage-ku, Chiba 263, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP(2)) dramatically increases the gelating activity of smooth muscle alpha-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 alpha-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 alpha-actinin contains a PIP(2)-binding 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 alpha-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-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 enzyme-linked 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 alpha-actinin and that similar sequences found in spectrin and PLC-1 may be involved in the interaction with PIP(2).


INTRODUCTION

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) (^1)is a trace phospholipid, which generates two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol that respond to phospholipase C (PLC) activation by a variety of physiological stimuli. Inositol 1,4,5-trisphosphate and diacylglycerol are known to mobilize Ca from the endoplasmic reticulum and to activate protein kinase C (PKC), respectively(3, 4) .

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) , µ-calpain(7) , ADP-ribosylation factor 1(8) , and phospholipase D(9) . PIP(2) also binds to actin-regulating proteins such as profilin(10) , cofilin(11) , gelsolin(12) , gCap(13) , and alpha-actinin (1) and regulates the functions of these proteins. When PIP(2) binds to alpha-actinin, which is an actin cross-linking protein, it further activates actin gelation by alpha-actinin(1) . It is noteworthy that profilin plays crucial roles in tyrosine kinase-coupled 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 alpha-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.

alpha-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 alpha-actinin is found at focal contacts where actin is anchored to a variety of intercellular structures in non-muscle cells suggests that alpha-actinin plays some role in the linkage between the plasma membrane and actin. We previously reported that alpha-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 alpha-actinin increases the gelation activity of actin to the level produced by skeletal muscle alpha-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 beta subunit of trimeric G proteins (18, 19) and PKC(20) . In that case, PIP(2) is thought to act as a target for PH domain-containing 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 alpha-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 alpha-actinin is also found in spectrin and the PH domains of several proteins including PLC-1 and Grb7.


EXPERIMENTAL PROCEDURES

Materials

Striated muscle alpha-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) . [^3H]PIP(2) (7.6 Ci/mmol) was from DuPont NEN. DEAE-cellulose (Whatman), cellulose phosphate (Whatman), the HiTrap-heparin column (Pharmacia), Sephadex 26/40 (Pharmacia), and MonoQ 5/5 column (Pharmacia) were purchased as indicated. alpha-Chymotrypsin and lysylendopeptidase were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Sequencing of the Proteolytic Digestion Fragments of alpha-Actinin

Purified striated alpha-actinin was digested with alpha-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 alpha-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. alpha-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).

Peptide Synthesis

Four peptides with the following sequences were synthesized. Peptide I, WEKQQRKTE; peptide II, GERLPKPDRGKMRFHK; peptide III, HRHRPDLIDYSKLNKDD; and peptide IV, TAPYRNVNIQNFHLSWK.

Three more peptides corresponding to the homologous sites of several proteins were synthesized. Human spectrin beta-chain (PEP-spectrin), TAGYPHVNVTNFTSSWK; PLC-1 (PEP-PLC-1), LKGSQLLKVKSSSWR; and Grb7 (PEP-Grb7), QLRGSGRGSGRKLWK. We also synthesized a peptide which is known as PIP(2)-binding site in gelsolin (24) (PEP-gelsolin), KLFQVKGRR.

Preparation of PLC Enzymes

PLC-1 and -1 were partially purified by the methods described previously(25) . Bovine thymus (900 g) was homogenized in 1.8 liters of buffer containing 50 mM Tris-HCl, pH 7.6, 0.25 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 mM diisopropyl fluorophosphate, 10 µg/ml leupeptin, 2 µg/ml bestatin, and 2 µg/ml pepstatin, and centrifuged at 105,000 times g for 1 h at 4 °C. The resulting supernatants were used for PLC-1 purification by five chromatographic steps including DEAE-cellulose (8 times 40 cm), cellulose phosphate (4 times 25 cm), HiTrap-heparin (10 ml), Sephadex 26/60, and MonoQ 5/5. PLC-1 was purified by DEAE-cellulose and HiTrap-heparin.

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 µM Ca, 1 mg/ml bovine serum albumin, 20 µM PIP(2), 20,000 dpm of [^3H]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 alpha-Actinin Mutants

Four point mutants, with mutations in the PIP(2)-binding site of chicken skeletal muscle alpha-actinin, were produced using a transformer site-directed mutagenesis kit. The cDNA, EPalphaAn1(26) , encoding chicken muscle alpha-actinin was subcloned into pUC19. Four mutagenic primers, TAAGGAGCAGTTTTTATTTGAC, TGAATGTTCACATTTATGTAAG, AATCCAAGGCCATCTATCCAGC, and AGGTCAGGTCGGTGTATGTGGA, were used to generate point-mutated cDNAs. These mutations result in substitutions of Ile for Arg, Arg, Lys, and Arg (designated as alphaAn.166.1, alphaAn.172.2, alphaAn.184.2, and alphaAn.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 times His)-tagged expression vector. We also constructed a non-mutated DNA fragment and subcloned it into the same vector to obtain alphaAn.0.1.

Expression and Purification of Recombinant Protein

Escherichia coli JM109 cells containing alphaAn fusion constructs were grown in LB containing 100 µg/ml ampicillin, and induced with 1 mM isopropyl-1-thio-beta-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 times 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 alpha-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.


RESULTS

Determination of a PIP(2)-binding Site in Chicken Skeletal alpha-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 alpha-actinin, where two homodimers bind to each other. Cleavage of alpha-actinin with alpha-chymotrypsin for the indicated times revealed that the 102-kDa alpha-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(2)-binding site exists within the N-terminal actin-binding domain.


Figure 1: alpha-Chymotryptic cleavage pattern of chicken skeletal alpha-actinin. 50 µg of skeletal muscle alpha-actinin was digested with alpha-chymotrypsin (molar ratio, 1:200) at 37 °C for the indicated times as described under ``Experimental Procedures.'' The digests were subjected to 8.5% acrylamide gels and stained with Coomassie Brilliant Blue (A) or transferred to nitrocellulose and immunostained with anti-PIP(2) antibody (B). The sizes of the alpha-chymotryptic cleavage fragments as reported previously (29) are shown in C.



To more closely locate the PIP(2)-binding site, limited proteolysis with lysylendopeptidase was carried out. The digests of alpha-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 SDS-polyacrylamide 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 corresponds to the sequence of amino acid residues 168-184 in chicken skeletal alpha-actinin. We conclude therefore that this region in the actin-binding domain contains a binding site for PIP(2).


Figure 2: Determination of the peptide sequence in alpha-actinin involved in PIP(2) binding by digestion with lysylendopeptidase. 100 µg of alpha-actinin was treated overnight with 0.38 µM lysylendopeptidase at 37 °C. The digests were applied to a C18 reverse-phase column and eluted with 0-60% gradient of acetonitrile (A). The separated peptides were lyophillized and dot-blotted on nitrocellulose membranes. The membrane was stained with anti-PIP(2) antibody (B). The peptide containing PIP(2) was sequenced by a protein sequencer.



A computer-assisted sequence homology search revealed that this sequence is homologous to chicken smooth muscle alpha-actinin (88.8%), human spectrin beta-chain (58.8%), yeast mRNA capping protein (37.5%), and mouse integrin beta-7 subunit precursor (35.7%). Moreover we found the homologous regions in the PH domains of several proteins including PLC-1, Grb 7, pleckstrin, Ras-GAP, and racKbeta (Fig. 3).


Figure 3: Comparison of the PIP(2)-binding domain of chicken striated alpha-actinin with various protein sequences. Highly conserved (&cjs2110;) and similar (&cjs2106;) amino acids residues are boxed and shaded, respectively. SK.alpha-actinin, chicken skeletal muscle alpha-actinin; SM.alpha-actinin, chicken smooth muscle alpha-actinin; spectrin, human spectrin beta-chain; integrin, mouse integrin beta7 subunit precursor; mRNA.CP, yeast mRNA capping protein; PLC1.PH, Grb7.PH, plec(N).PH, plec(C).PH, Ras-GAP.PH, and racKbeta.PH are the PH domains of various proteins. Amino acids alignment was done on the basis of RXXXXXXX(H/R/K)XX(X)W(K/R).



Inhibition of Phospholipase C Activity by Synthetic Peptides

To examine whether a peptide that includes a PIP(2)-binding site inhibits the activity of PLC, we synthesized the peptide corresponding to amino acids 168-184 (TAPYRNVNIQNFHLSWK, peptide IV) of alpha-actinin. We also synthesized peptides rich in basic amino acids corresponding to amino acids 39-47 (WEKQQRKTE, peptide I), 84-99 (GERLPKPDRGKMRFHK, 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 µM 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 alpha-actinin contains a PIP(2)-binding site. We also examined the effect of alpha-actinin purified from chicken smooth muscle on PLC activities (Fig. 4C). At maximum soluble concentration, 19.2 µM, alpha-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 alpha-actinin in the spectrin beta-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 µM 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 µM 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).


Figure 4: Inhibition of PLC-1 and PLC-1 activities by synthetic peptides and alpha-actinin. Synthetic peptide sequences are shown in A. The inhibition of PLC-1 and PLC-1 activities by peptide I (circle), peptide II (box), peptide III (up triangle), and peptide IV () at various doses is shown in B. PLC activities were measured as described under ``Experimental Procedures'' using 20 µM PIP(2) as a substrate. The total activity of PLC-1 is 5400 dpm and that of PLC-1 is 8600 dpm. Inhibition by peptide IV (bullet) in the presence of 0.5% octyl glucoside was also examined (B). C shows the inhibition of PLC-1 and PLC-1 activities by smooth muscle alpha-actinin at concentrations of 0, 3.8, and 19.1 µM. Inhibition of PLC-1 and PLC-1 by peptides corresponding to PEP-spectrin (circle), PLC-1 (bullet), PEP-Grb7 (box), and PEP-gelsolin (Delta) is shown in D.



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 alpha-actinin, we produced various bacterial histidine tag proteins. We examined whether these proteins inhibited PLC activities as strongly as peptide IV or alpha-actinin. We used 20 µM recombinant peptides as maximal soluble concentration. As shown in Fig. 5, alphaAn.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 alpha-actinin and more effective than that for peptide IV. Two point-mutated proteins, alphaAn.166.1 and alphaAn.195.1, also inhibited the activities almost as identically to alphaAn.0.1, while alphaAn.172.2 and alphaAn.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 alpha-actinin PIP(2)-binding site.


Figure 5: Inhibition of PLC-1 and PLC-1 activities by bacterial expression of PIP(2)-binding sites on alpha-actinin. Activities of PLC-1 (A) and PLC-1 (B) were measured in the presence of 20 µM various point mutants or a non-mutant as described under ``Experimental Procedures.''



Direct Interactions of PIP(2)with Peptides and Bacterially Expressed alpha-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 alpha-An.0.1, alpha-An.161.1, and alpha-An.195.1 also bound to PIP(2) tightly, while two mutated proteins, alpha-An.172.2 and alpha-An.184.2, caused the decrease in this binding down to about 68 and 51% of control, respectively (Fig. 6B).


Figure 6: Direct interactions of PIP(2) with peptides and bacterial expression alpha-actinin fragments. 96-well multiplates were coated with 50 µl of 10 µM peptide I (box), peptide II (circle), peptide III (Delta), peptide IV (bullet), or no peptide (&cjs0800;) (A), and 50 µl of 20 µg/ml alphaAn.0.1 (bullet), alphaAn.166.1 (&cjs0800;), alphaAn.172.2 (box), alphaAn.184.2 (circo]), or alphaAn.195.1 () (B) overnight at room temperature. Various amounts of PIP(2) were added to each well and incubated at room temperature for 30 min. Procedures were the same as described under ``Experimental Procedures.''




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) , µ-calpain(7) , and many actin-regulating proteins(1, 10, 11, 12, 13, 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 µ-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 alpha-actinin from chicken striated muscle contains large amounts of PIP(2) while alpha-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 alpha-actinin(1) . Furthermore, it has been shown that the amount of PIP(2) bound to alpha-actinin and vinculin decrease in response to platelet-derived growth factor stimulation in vivo(2) . These facts suggest that alpha-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 alpha-actinin.

Amino acid sequences which contain PIP(2)-binding site in skeletal muscle alpha-actinin are homologous to that in chicken smooth muscle alpha-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 alpha-actinin also exist in some cytoskeletal-related proteins such as spectrin beta-chain or integrin beta-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 alpha-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 beta subunit of trimeric G protein (18, 19) and PKC(20) . An arginine to cysteine substitution in the N-terminal PH domain (beta(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 alpha-actinin also exist in the beta(1)- and beta(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-trisphosphate binding site on PLC-1 is thought to comprise amino acids 30-43, which overlaps with the site which we aligned (amino acids 23-37). 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 alpha-actinin, a bacterially-expressed protein, and a synthetic peptide corresponding to amino acids 168-184 of alpha-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 alpha-actinin may be hydrolyzed by activated PLC-1 when cells are activated. This problem remains to be solved in future.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-5449-5510; Fax: 81-3-5449-5417.

(^1)
The abbreviations used are: PIP(2), phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PKC, protein kinase C; PH domain, pleckstrin homology domain; Mes, 4-morpholineethanesulfonic acid; PEP, P-enolpyruvate.


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