Neurabin-II/Spinophilin
AN ACTIN FILAMENT-BINDING PROTEIN WITH ONE PDZ DOMAIN LOCALIZED AT CADHERIN-BASED CELL-CELL ADHESION SITES*

Ayako SatohDagger , Hiroyuki NakanishiDagger , Hiroshi ObaishiDagger , Manabu WadaDagger §, Kenichi TakahashiDagger , Keiko SatohDagger , Kazuyo HiraoDagger , Hideo NishiokaDagger , Yutaka HataDagger , Akira Mizoguchi, and Yoshimi TakaiDagger par **

From the Dagger  Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan, the par  Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan, and the  Department of Anatomy and Neurobiology, Graduate School, Kyoto University, Kyoto 606-01, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In a preceding paper, we reported a novel actin filament (F-actin)-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation. We purified from rat brain another F-actin-binding protein, which had a domain organization similar to that of neurabin but was ubiquitously expressed, and named it neurabin-II. The original neurabin, renamed neurabin-I, had 1095 amino acids and a calculated Mr of 122,729, whereas neurabin-II had 817 amino acids and a calculated Mr of 89,642. Both neurabin-I and -II had one F-actin-binding domain at the N-terminal region, one PDZ domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the C-terminal region. Both neurabin-I and -II bound along the sides of F-actin and showed F-actin-cross-linking activity. The subcellular distribution analysis indicated that neurabin-II was enriched at the postsynaptic density fraction in rat brain and the adherens junction fraction in rat liver. Immunofluorescence microscopic analysis revealed that neurabin-II was highly concentrated at the synapse in primary cultured rat hippocampal neurons and at the cadherin-based cell-cell adhesion sites in Madin-Darby canine kidney cells. Neurabin-II turned out to be the same as a recently reported protein phosphatase 1-binding protein named spinophilin. These results suggest that neurabin-II/spinophilin plays an important role in linking the actin cytoskeleton to the plasma membrane.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Specialized membrane domains formed with transmembrane proteins, such as cell adhesion molecules, receptors, and channels, are often associated with the actin cytoskeleton (for reviews, see Refs. 1-4). The linkage between the actin cytoskeleton and the plasma membrane plays crucial roles in various cellular events, such as cell adhesion, cell motility, and cell shape determination, and the proteins linking the actin cytoskeleton to the transmembrane proteins have been identified (1-4). However, the molecular basis of this linkage is not fully understood.

In a preceding paper, we purified from rat brain a novel F-actin-binding protein, named neurabin, which was specifically expressed in neural tissue and implicated in neurite formation (5). Neurabin had one F-actin-binding domain, one PDZ domain, and four domains predicted to form coiled-coil structures. The PDZ domain is found in many proteins, some of which are localized at cell-cell junctions (for review, see Ref. 6), such as PSD-95/SAP90 at synaptic junction, Dlg at septate junction, and ZO-1 and ZO-2 at tight junction. The PDZ domain binds to the unique C-terminal motifs of target proteins found in many transmembrane proteins, such as N-methyl-D-aspartate receptors and Shaker-type K+ channels (6). Neurabin is likely to serve as a linker between the actin cytoskeleton and a transmembrane protein(s) at synapse, although we have not yet identified its interacting transmembrane protein.

During the purification of neurabin, we detected another F-actin-binding protein with a Mr of about 130,000. Molecular cloning and characterization of this protein revealed that it had a domain organization similar to that of neurabin but is expressed ubiquitously. We named this protein neurabin-II and renamed the original one neurabin-I. The subcellular distribution and immunofluorescence microscopic analyses indicated that neurabin-II was enriched at synapse and cadherin-based cell-cell adhesion sites.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials and Methods-- Rabbit skeletal actin monomer (7), F-actin (5), and 125I-labeled F-actin (5, 8) were prepared as described. Various GST1 and His-6 fusion proteins of full-length and truncated forms of neurabin-II were expressed in Escherichia coli and purified (5). The fusion proteins contained the following aa residues: GST-neurabin-II-1, aa 1-154; GST-neurabin-II-2, aa 155-495; GST-neurabin-II-3, aa 400-658; GST-neurabin-II-4, aa 584-817; and His-6-neurabin-II, aa 1-817. pCMV-neurabin-II was constructed to express the full-length protein in COS7 cells as described (5). Various myc-tagged proteins of full-length and truncated forms of neurabin-II were expressed in COS7 cells as described (5). A rabbit polyclonal anti-neurabin-II antibody was raised against a 20-mer synthetic peptide corresponding to aa 290-309 and purified with the synthetic peptide covalently coupled to EAH-Sepharose (Pharmacia). A mouse monoclonal anti-neurabin-II antibody was raised against GST-neurabin-II-4 (aa 584-817) and purified with E-Z-SEP (Pharmacia). The specificities of these antibodies were confirmed by Western blot analysis on the pCMV-neurabin-II-transfected and pCMV-neurabin-I-transfected COS7 cells (5). These antibodies did not recognize neurabin-I. A rat monoclonal anti-E-cadherin antibody (Takara) and rabbit polyclonal anti-synapsin I (Chemicon) and anti-N-methyl-D-aspartate receptors 2A/B (Chemicon) antibodies were purchased from commercial sources. 125I-Labeled F-actin blot overlay was done as described (5, 8). Gel filtration and sucrose density gradient ultracentrifugation were performed as described (5). For coiled-coil prediction, the MTK and MTIDK matrices of the COILS version 2.1 algorithm were used with a 28-residue window (9). Weighting options were applied and a probability curve was generated. Electron microscopy was performed as described (5). The subcellular fractionation of rat brain (10) and liver (11) were done as described. Immunofluorescence microscopy of primary cultured rat hippocampal neurons was done as described (5). Immunofluorescence microscopy of MDCK cells was done as described (12), except that the cells were fixed in 100% methanol at -20 °C for 15 min. Protein concentrations were determined with bovine serum albumin as a reference protein (13).

Purification and Molecular Cloning of Neurabin-II-- The synaptic soluble fraction was prepared from 280 adult rat brains as described (14) and stored at -80 °C until use. One-seventh of the fraction (420 ml, 360 mg of protein) was adjusted to 0.2 M NaCl with 4 M NaCl and applied to a Q-Sepharose FF column (2.6 × 10 cm, Pharmacia) equilibrated with Buffer A (20 mM Tris-Cl at pH 7.5 and 1 mM DTT) containing 0.2 M NaCl. Elution was performed with 350 ml of Buffer A containing 0.5 M NaCl. Fractions of 10 ml each were collected. Neurabin-II appeared in Fractions 5-19. These fractions (150 ml, 152 mg of protein) were collected, and NaCl was added to give a final concentration of 2 M. The sample was applied to a phenyl-Sepharose column (2.6 × 10 cm, Pharmacia) equilibrated with Buffer A containing 2 M NaCl. Elution was performed with a 360-ml linear gradient of NaCl (2.0-0 M) in Buffer A, followed by 180 ml of Buffer A. Fractions of 6 ml each were collected. Neurabin-II appeared in Fractions 53-59. The active fractions (42 ml, 6.1 mg of protein) were collected, and CHAPS was added to give a final concentration of 0.6%. The sample was then applied to a hydroxyapatite column (0.78 × 10 cm, Koken Co. Ltd., Tokyo, Japan) equilibrated with Buffer B (20 mM potassium phosphate at pH 7.8, 1 mM DTT, 0.6% CHAPS, and 10% glycerol). Elution was performed with a 75-ml linear gradient of potassium phosphate (20-100 mM) in Buffer B and a subsequent 75-ml linear gradient (100-300 mM) in Buffer B, followed by a 50-ml linear gradient (300-500 mM) in Buffer B. Fractions of 2.5 ml each were collected. Neurabin-II appeared in Fractions 26-37. The active fractions (30 ml, 0.36 mg of protein) were collected. The active fractions were diluted with an equal volume of Buffer C (20 mM Tris-Cl at pH 7.5, 0.5 mM EDTA, 1 mM DTT, and 0.6% CHAPS) and then applied to a Mono Q HR 5/5 column (Pharmacia) equilibrated with Buffer C. Elution was performed with 5 ml of Buffer C containing 0.2 M NaCl and a subsequent 15-ml linear gradient of NaCl (0.2-0.5 M) in Buffer C, followed by 5 ml of Buffer C containing 0.5 M NaCl. Fractions of 0.6 ml each were collected. Neurabin-II appeared in Fractions 26-29. The active fractions (2.4 ml, 10 µg of protein) were collected. The rest of the synaptic soluble fraction was also subjected to the successive column chromatographies in the same manner as described above. The active fractions of the seven Mono Q column chromatographies were combined, concentrated with a Centricon 30 (Amicon) to about 1.0 ml, and stored at -80 °C.

The purified Mono Q sample (about 60 µg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel). Double protein bands corresponding to proteins with Mr values of about 130,000 and 140,000 were separately cut out from the gel and digested with a lysyl endopeptidase, and the digested peptides of each protein band were separated by reverse phase high pressure liquid column chromatography as described (15). The aa sequences of the peptides were determined with a peptide sequencer. A rat brain cDNA library in lambda ZAPII (Stratagene) was screened using the oligonucleotide probes designed from the partial aa sequences.

Assay for Cosedimentation of Neurabin-II with F-actin-- His-6-neurabin-II in an indicated amount was incubated for 30 min at room temperature with 0.3 mg/ml F-actin in a solution containing 20 mM imidazol/Cl at pH 7.0, 2 mM MgCl2, 1 mM ATP, 0.4 mM DTT, 27 mM KCl, 100 mM NaCl, and 0.1 mM EGTA, and the mixture (100 µl) was placed over a 50-µl cushion of 30% sucrose in a polymerization buffer (20 mM imidazol/Cl at pH 7.0, 2 mM MgCl2, 1 mM ATP, 0.5 mM DTT, and 90 mM KCl). After the sample was centrifuged at 130,000 × g for 20 min, the supernatant was removed from the cushion and the pellet was brought to the original volume in an SDS sample buffer. The comparable amounts of the supernatant and pellet fractions were subjected to SDS-PAGE followed by protein staining with Coomassie Brilliant Blue to quantitate His-6-neurabin-II cosedimented with F-actin using a densitometer.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

During the purification of neurabin-I from rat brain using a blot overlay method with 125I-labeled F-actin, we detected double bands of 125I-labeled F-actin-binding activity with Mr values of about 130,000 and 140,000. We highly purified them by chromatographies of Q-Sepharose, phenyl-Sepharose, hydroxyapatite, and Mono Q columns. When the purified sample was subjected to SDS-PAGE followed by protein staining and 125I-labeled F-actin blot overlay, double protein bands, which coincided the double bands of the radioactivity, were observed (Fig. 1A).


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Fig. 1.   SDS-PAGE analysis of the purified sample of neurabin-II and its amino acid sequence. A, SDS-PAGE analysis. The purified sample of neurabin-II was subjected to SDS-PAGE (8% polyacrylamide gel) followed by protein staining with Coomassie Brilliant Blue and 125I-labeled F-actin blot overlay assay. Arrowhead, p130; arrow, p140. B, deduced amino acid sequence. Underlines, aa sequences of the seven peptide peaks derived from the purified sample of neurabin-II.

We separately determined the partial aa sequences of the upper (p140) and lower (p130) protein bands. The aa sequences of p140 were identical to those of neurabin-I (GenBankTM accession number U72994), and p140 appeared to be a proteolytic product of neurabin-I (5). The aa sequences of p130 (neurabin-II) were not found in the current protein data base. On the basis of this information, we isolated the neurabin-II cDNA from a rat brain cDNA library and determined its nucleotide sequence. The encoded protein consisted of 817 aa and a calculated Mr of 89,642 (GenBankTM accession number AF027181) (Fig. 1B). The Mr value calculated from the predicted aa sequence was less than that estimated by SDS-PAGE. To confirm whether this clone contained a full-length cDNA of p130, we constructed the eukaryotic expression vector with this cDNA and expressed the protein in COS7 cells. The expressed protein showed the mobility similar to that of native p130 on SDS-PAGE and the 125I-labeled F-actin-binding activity (Fig. 2). Although the reason for the remarkable difference between the Mr value calculated from the predicted aa sequence and that estimated by SDS-PAGE is not known, we concluded that this clone encoded the full-length cDNA of neurabin-II.


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Fig. 2.   Comparison of mobility on SDS-PAGE and 125I-labeled F-actin-binding activity between native and recombinant samples of neurabin-II. A, mobility on SDS-PAGE. The purified native sample of neurabin-II (0.01 µg of protein) and the cell extracts of control and pCMV-neurabin-II-transfected COS7 cells (0.2 µg of protein each) were subjected to SDS-PAGE (8% polyacrylamide gel) followed by immunoblot with the polyclonal or monoclonal anti-neurabin-II antibody. Aa, with polyclonal anti-neurabin-II antibody; Ab, with monoclonal anti-neurabin-II antibody. B, 125I-labeled F-actin-binding activity. The purified native sample of neurabin-II (0.1 µg of protein) and the cell extracts of control and pCMV-neurabin-II-transfected COS7 cells (1 µg of protein each) were subjected to SDS-PAGE (8% polyacrylamide gel) followed by 125I-labeled F-actin blot overlay.

By use of fusion proteins of several truncated forms of neurabin-II with GST, we determined the minimum 125I-labeled F-actin-binding domain to be aa 1-154 (Fig. 3A). The aa sequence of this domain showed 43% identity to that of neurabin-I (Fig. 3B). We expressed myc-tagged full-length neurabin-II or a myc-tagged mutant, which lacked the F-actin-binding domain, in COS7 cells and compared the localization of each expressed protein with that of endogenous F-actin. Neurabin-II having the F-actin-binding domain was colocalized with F-actin, but neurabin-II lacking the domain was not (data not shown). In addition to the F-actin-binding domain, neurabin-II had one PDZ domain (aa 496-586) and three domains predicted to form coiled-coil structures (aa 665-725, aa 729-772, aa 783-817). This domain organization was similar to that of neurabin-I (Fig. 3B).


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Fig. 3.   Domain organization of neurabin-II. A, 125I-labeled F-actin-binding activity of various truncated forms of neurabin-II. The purified proteins (0.2 µg of protein each) were subjected to SDS-PAGE (8-15% polyacrylamide gradient gel) followed by 125I-labeled F-actin blot overlay. GST-Neurabin-II-1, aa 1-154; GST-Neurabin-II-2, aa 155-495; GST-Neurabin-II-3, aa 400-658; GST-Neurabin-II-4, aa 584-817. B, comparison of domain organizations of neurabin-I and -II.

When His-6-full-length neurabin-II was incubated with F-actin followed by ultracentrifugation the fusion protein was recovered with F-actin in the pellet (Fig. 4Aa). The stoichiometry of the binding of His-6-neurabin-II to actin was one His-6-neurabin-II molecule per approximately 10 actin molecules (Fig. 4Ab). The Kd value was about 5 × 10-7 M. The binding of His-6-neurabin-II to 125I-labeled F-actin was completely inhibited by an excessive amount of myosin S1, a protein which binds along the sides of F-actin (16, 17) (Fig. 4B). This inhibition was reversed by the addition of MgATP because MgATP dissociates the actin-myosin complex (18), indicating that neurabin-II binds along the sides of F-actin. The F-actin-cross-linking activity of His-6-neurabin-II was examined by transmission electron microscopy of negatively stained specimens. Neurabin-II caused F-actin to associate into bundles (Fig. 4C). These biochemical properties of neurabin-II were similar to those of neurabin-I (5).


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Fig. 4.   Biochemical properties of neurabin-II. Aa, cosedimentation of His-6-neurabin-II with F-actin. His-6-full-length neurabin-II (3 µg of protein) was mixed with F-actin followed by ultracentrifugation. S, supernatant; P, pellet; arrow, actin; arrowhead, His-6-neurabin-II; Ab, binding of His-6-neurabin-II to F-actin. Various amounts of His-6-neurabin-II were mixed with F-actin followed by ultracentrifugation. Amounts of free and bound His-6-neurabin-II were calculated by determining the protein amounts from the supernatant and pellet fractions with a densitometer. B, inhibition by myosin S1 of the binding of neurabin-II to 125I-labeled F-actin. His-6-neurabin-II (0.1 µg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel) followed by blot overlay with 125I-labeled F-actin pretreated with myosin S1 in the presence or absence of ATP. C, F-actin-cross-linking activity of His-6-neurabin-II estimated by electron microscopy. Scale bars, 200 nm.

When His-6-neurabin-II was subjected to Superdex 200 column chromatography, it appeared at a position corresponding to a Mr of about 440,000 (Stokes radius, approximately 66 Å). On sucrose density gradient ultracentrifugation, His-6-neurabin-II appeared at a position corresponding to a Mr of about 370,000 (S value, approximately 15.1). The Mr value of neurabin-II was calculated to be about 420,000 from both the Stokes radius and S value (19). The frictional ratio of neurabin-II was calculated to be about 1.3. Because the Mr values of neurabin-II estimated by SDS-PAGE and calculated from its predicted aa sequence were about 130,000 and 90,000, respectively, and neurabin-II had predicted coiled-coil structures at the C-terminal region, these results suggest that neurabin-II forms a trimer or a tetramer.

Northern and Western blot analyses indicated that neurabin-II was expressed in various rat tissues examined (Fig. 5, Aa and Ab). The subcellular fractionation analysis of rat brain indicated that both neurabin-I and -II were enriched at the postsynaptic density fraction (Fig. 5Ba). The immunofluorescence microscopic analysis of primary cultured rat hippocampal neurons using the monoclonal anti-neurabin-II antibody indicated that the staining pattern of neurabin-II was similar to that of synapsin I (Fig. 6A). Neurabin-II was highly concentrated in the synapse. A similar result was obtained with the polyclonal anti-neurabin-II antibody (data not shown). In rat liver, immunofluorescence microscopic analysis of neurabin-II was not performed because of a low level of expression (Fig. 5, Aa and Ab), but the subcellular fractionation analysis indicated that it was enriched at the AJ fraction in which E-cadherin was enriched (Fig. 5Bb), suggesting that neurabin-II is localized at the cadherin-based cell-cell adhesion sites. Consistently, in MDCK cells, neurabin-II was colocalized with E-cadherin at cell-cell adhesion sites (Fig. 6, Ba1 and Ba2). We have recently reported that cadherin and F-actin are densely accumulated at adhesion sites in a MDCK cell line stably expressing a dominant active mutant of Rac1 small GTP-binding protein (V12Rac1) and that the levels of their accumulation are markedly reduced in a MDCK cell line stably expressing a dominant negative mutant of Rac1 (N17Rac1) compared with those in wild-type cells (20). The staining patterns of neurabin-II in these cell lines were similar to those of E-cadherin (Fig. 6, Bb and Bc). Similar results were obtained with the polyclonal anti-neurabin-II antibody (data not shown).


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Fig. 5.   Tissue and subcellular distributions of neurabin-II. Aa, Northern blot analysis. A RNA blot membrane (CLONTECH) was hybridized with the 32P-labeled 1.1-kilobase pair ApaI fragment of the neurabin-II cDNA according to the manufacturer's protocol. Ab, Western blot analysis. The homogenates of various rat tissues (10 µg of protein each) were subjected to SDS-PAGE (8% polyacrylamide gel) followed by immunoblot with the polyclonal anti-neurabin-II antibody. Ba, subcellular distribution of neurabin-II in rat brain. Each subcellular fraction (4 µg of protein each) was subjected to Western blot analysis with the polyclonal anti-neurabin-II, anti-neurabin-I, and anti-N-methyl-D-aspartate receptors 2A/B antibodies. Ho, homogenate; P1, nuclear pellet fraction; S1, crude synaptosomal fraction; P2, crude synaptosomal pellet fraction; S2, cytosolic synaptosomal fraction; S3, crude synaptic vesicle fraction; P3, lysed synaptosomal membrane fraction; SPM, synaptosomal membrane fraction. Synaptosomal membrane fractions were extracted with 0.5% Triton X-100, twice (One Triton and Two Triton). Bb, subcellular distribution of neurabin-II in rat liver. Each subcellular fraction (20 µg of protein each) was subjected to Western blot analysis with the polyclonal anti-neurabin-II and monoclonal anti-E-cadherin antibodies. Ho, homogenate, S1, soluble fraction; P1, pellet fraction; BC, fraction rich in bile canaliculi; AJ, fraction rich in AJ.


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Fig. 6.   Immunofluorescence microscopic analysis of neurabin-II. A, localization of neurabin-II in primary cultured rat hippocampal neurons. The neurons were doubly stained with the mouse anti-neurabin-II and rabbit anti-synapsin I antibodies and visualized with rhodamine-conjugated anti-mouse and fluorescein isothiocyanate-conjugated anti-rabbit antibodies, respectively. Scale bars, 20 µm. Ba, localization of neurabin-II in wild-type MDCK cells. Ba1, junctional levels; Ba2, vertical sections; Bb, localization of neurabin-II in MDCK cells stably expressing the dominant active mutant of Rac1 (V12Rac1); Bc, localization of neurabin-II in MDCK cells stably expressing the dominant negative mutant of Rac1 (V12Rac1). The samples were doubly stained with the mouse anti-neurabin-II and rat anti-E-cadherin antibodies and visualized with rhodamine-conjugated anti-mouse and fluorescein isothiocyanate-conjugated anti-rat antibodies, respectively. Scale bars, 20 µm.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have isolated and characterized a novel F-actin-binding protein named neurabin-II. Neurabin-II showed physical and biochemical properties similar to those of neurabin-I, but neurabin-II was ubiquitously expressed, whereas neurabin-I was specifically expressed in neural tissue. In rat brain, subcellular distributions of neurabin-I and -II were apparently similar. We have attempted to isolate an ubiquitously expressed isoform of neurabin-I by the degenerated polymerase chain reaction and low stringency hybridization methods, but all the clones isolated were those encoding neurabin-II. Neurabin-II is likely an ubiquitous isoform of neurabin-I.

The subcellular distribution analysis of neurabin-II in rat liver and immunofluorescence microscopic analysis in MDCK cells indicated that neurabin-II was localized at cadherin-based cell-cell adhesion sites. These results, together with the fact that neurabin-II has a PDZ domain, suggest that neurabin-II serves as a linker between the actin cytoskeleton and the plasma membrane at cadherin-based cell-cell adhesion sites. The cytoplasmic domain of cadherin is associated with cytoplasmic proteins such as alpha -, beta -, and gamma -catenins (for reviews, see Refs. 4 and 21). alpha -Catenin directly interacts with F-actin (22). alpha -Catenin also indirectly interacts with F-actin through alpha -actinin and/or ZO-1 (23, 24). Vinculin, another F-actin-binding protein, is concentrated at cadherin-based cell-cell AJ, although its interacting molecule at the junction has not yet been identified (1, 4). Many F-actin-binding proteins, including alpha -actinin, talin, and vinculin, serve as linkers between the actin cytoskeleton and integrin at cell-matrix AJ (for review, see Ref. 25). The cytoplasmic domain of the beta -subunit of integrin interacts directly with alpha -actinin and talin and indirectly with vinculin through alpha -actinin and talin (25). Therefore, the molecular linkage mechanism between the actin cytoskeleton and cadherin is apparently similar to that between the actin cytoskeleton and integrin, in the sense that F-actin is linked to each transmembrane protein through multiple F-actin-binding proteins.

In contrast to cadherin-based cell-cell adhesion sites, the molecular mechanism of the synaptic junction is not fully understood. Recent studies have revealed that cadherin is concentrated at the synaptic junction (26, 27). Because both neurabin-I and -II are concentrated at the synapse, they may also serve as linkers between the actin cytoskeleton and the plasma membrane at the synaptic junction where cadherin functions as a cell adhesion molecule. Further studies are necessary for our understanding of the role of neurabin-I and -II at the synapse.

During the preparation of this manuscript, Allen et al. (28) reported a novel PP1-binding protein and named it spinophilin. Spinophilin formed a complex with the catalytic subunit of PP1 and modulated its enzymatic activity. This protein was enriched at the dendritic spines. Neurabin-II turned out to be the same as this PP1-binding protein. We found that neurabin-I was copurified with a protein with a Mr of approximately 36,000.2 The aa sequence analysis of this protein showed that it was PP1. Moreover, we found that PP1 was coimmunoprecipitated with neurabin-I and that they interacted with each other by the yeast two-hybrid method. Neurabin-I had an Arg/Lys-Val/ILe-Xaa-Phe motif (aa 457-460), which is present in almost all the PP1-binding proteins (29). This motif is conserved in neurabin-II/spinophilin (aa 448-451). These results and those of tissue and subcellular distributions of neurabin-II are consistent with those reported by Allen et al. (28). The phosphorylation and dephosphorylation events at cadherin-based cell-cell adhesion sites have been shown to regulate the cadherin-catenin system and affect the assembly and stability of the adhesion sites (30). Neurabin-I and neurabin-II/spinophilin have potential phosphorylation sites and may serve as the substrates for PP1. The phosphorylation and dephosphorylation of neurabin-I and neurabin-II/spinophilin may modulate their F-actin-binding activity and affect the adhesion sites. Alternatively, there may be a molecule(s) interacting with neurabin-I and neurabin-II/spinophilin, which regulates the assembly and stability of the adhesion sites by its phosphorylation and dephosphorylation. Further studies are necessary for our understanding of the physiological function of the binding of PP1 to these F-actin-binding proteins.

    ACKNOWLEDGEMENTS

We thank Drs. M. Takeichi, S. Tsukita, and M. Itoh (Kyoto University, Kyoto, Japan) for helpful discussions and Dr. K. Takaishi (Osaka University, Osaka, Japan) for providing the MDCK cell lines expressing the Rac1 mutants. We also thank Dr. J. Koga (JCR Pharmaceuticals Co., Ltd., Kobe, Japan) for advice in producing the monoclonal anti-neurabin-II antibody.

    FOOTNOTES

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

§ Present address: JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan.

** To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.

1 The abbreviations used are: GST, glutathione S-transferase; AJ, adherens junction; aa, amino acid(s); MDCK cells, Madin-Darby canine kidney cells; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; S1, subfragment 1; PP1, protein phosphatase 1.

2 H. Obaishi, H. Nakanishi, and Y. Takai, unpublished observations.

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Abstract
Introduction
Procedures
Results
Discussion
References

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