Regulation of the Association of Adducin with Actin Filaments by Rho-associated Kinase (Rho-kinase) and Myosin Phosphatase*

Kazushi KimuraDagger §, Yuko Fukata§, Yoichiro Matsuoka, Vann Bennett, Yoshiharu Matsuurapar , Katsuya Okawa**, Akihiro Iwamatsu**, and Kozo Kaibuchi§Dagger Dagger

From the Dagger  Department of Anatomy and Neurobiology, Graduate School of Medicine, Kyoto University, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan, the § Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-01, Japan, the  Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, the par  Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjyuku-ku, Tokyo 162, Japan, and the ** Central Laboratories for Key Technology, Kirin Brewery Company Limited, 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated alpha -adducin in vitro and in vivo and that the phosphorylation of alpha -adducin by Rho-kinase enhanced the interaction of alpha -adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha -adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Rho is a small GTPase that exhibits both GDP/GTP binding and GTPase activities. Rho has GDP-bound inactive (GDP·Rho) and GTP-bound active (GTP·Rho) forms, which are interconvertible by GDP/GTP exchange and GTPase reactions (for reviews, see Refs. 1 and 2). When cells are stimulated with certain extracellular signals such as lysophosphatidic acid, GDP·Rho is thought to be converted to GTP·Rho, which binds to specific targets and then exerts its biological functions. Rho participates in signaling pathways that regulate actin cytoskeletons such as stress fibers and in cell-substratum adhesions such as focal adhesions in fibroblasts (3). Rho is also involved in the regulation of cell morphology (4), cell aggregation (5), cadherin-mediated cell-cell adhesion (6), cell motility (7), cytokinesis (8, 9), membrane ruffling (10), smooth muscle contraction (11, 12), c-fos gene expression (13), the synthesis of phosphatidylinositol 4,5-diphosphate via phosphatidylinositol 5-kinase (14), and endocytosis (15). In budding yeast, RHO1 (a homologue of RhoA) is implicated in the regulation of cell morphology and budding (16). We identified the following three targets of Rho: protein kinase N (17, 18), Rho-kinase1 (19), which is also known as ROKalpha (20), and the MBS of myosin phosphatase (21), which has ankyrin-like repeats in the amino-terminal domain and a poly basic region followed by a leucine zipper-like motif in the carboxyl-terminal domain (22). p160 ROCK is an isoform of Rho-kinase (23). We showed that Rho-kinase phosphorylates MBS and consequently inactivates myosin phosphatase (21). We demonstrated that Rho-kinase phosphorylates MLC and thereby activates myosin ATPase (24). Another group of investigators has identified different Rho targets: Rhophilin, Rhotekin, and Citron (18, 25). Phosphatidylinositol 5-kinase is shown to be activated by GTP·Rho (14). Among these targets, Rho-kinase appears to be involved in the formation of stress fibers and focal adhesions downstream of Rho (26-28), smooth muscle contraction through myosin phosphorylation (29), and c-fos gene expression (30).

We recently showed that MBS is accumulated at cell-cell contact sites apart from myosin fibers in polarized MDCK epithelial cells, whereas MBS is colocalized with myosin fibers in REF52 fibroblasts (31). To understand the function of MBS at cell-cell contact sites, we attempted to identify MBS-interacting molecules other than Rho and myosin. We have purified MBS-interacting proteins with molecular masses of about 85, 110, 115, 120, and 125 kDa and identified them as alpha -, beta -, and gamma -adducin.

Adducin is a membrane skeletal protein that was first purified from human erythrocytes based on calmodulin binding activity (32). Adducin associates with F-actin and spectrin-F-actin complexes to promote the association of spectrin with F-actin (33). Adducin also caps the fast growing end of actin filaments (34). Adducin is localized at cell-cell contact sites in some epithelial cells (35). It is likely that adducin participates in the assembly of the spectrin-actin network of erythrocytes and epithelial cells. Adducin is composed of alpha  and beta  or alpha  and gamma  subunits closely related in amino acid sequence and domain organization (36-38). Each adducin subunit has three distinct domains as follows: an amino-terminal head domain, connected by a neck domain to a carboxyl-terminal tail domain (36-38). alpha -Adducin and beta -adducin form heterodimers and tetramers through the head domains and tail domains (39). The tail domain of beta -adducin binds mainly to Ca2+/calmodulin (40), which inhibits both the ability of adducin to recruit additional spectrin to adducin-spectrin-F-actin complexes (33) and the ability of adducin to cap actin filaments (34). The tail domains are responsible for binding to spectrin-F-actin complexes (39).

Adducin is also a substrate for PKC and PKA (35, 41, 42). The phosphorylation of beta -adducin by PKC or PKA inhibits the calmodulin binding of beta -adducin. The phosphorylation of adducin by PKA reduces the activity of adducin to associate with F-actin and spectrin-F-actin complexes and to promote the binding of spectrin to F-actin. Phosphorylation by PKC has little effect on these activities (40).

In the present study, we found that MBS interacts with adducin both in vitro and in vivo and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of adducin. We also found that the phosphorylation of adducin by Rho-kinase results in the interaction of adducin with F-actin.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Materials and Chemicals-- cDNA of rat Notch1 was kindly provided by Dr. M. Nakafuku (Tokyo University, Tokyo, Japan) (43). Native Rho-kinase was purified from bovine brain as described (19). GST-CAT (the catalytic domain of Rho-kinase (6-553 aa)) was produced and purified as described (24). pEF-BOS-myc-CAT was constructed as described (27). GST-RhoA was purified from Escherichia coli and loaded guanine nucleotides as described (17). F-actin was purified from an acetone powder prepared from rabbit skeletal muscle as described (44). Chicken myosin phosphatase holoenzyme was kindly provided by Dr. M. Ito (Mie University, Japan) (22). [gamma -32P]ATP and [32P]orthophosphate were purchased from Amersham Corp. (Buckinghamshire, UK). All materials used in the nucleic acid study were purchased from Takara Shuzo Corp. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources.

Production and Purification of Recombinant MBS and alpha -Adducin-- The cDNAs encoding the NH2-terminal domain (1-707 aa), the COOH-terminal domain (699-976 aa), the ankyrin repeat domain (39-295 aa) of rat MBS, human alpha -adducin (1-642 aa) (37), and the ankyrin repeat domain of rat Notch1 (1847-2099 aa) were subcloned into pGEX to produce GST-MBS-N, GST-MBS-C, GST-MBS-ANK, GST-alpha -adducin, and GST-Notch-ANK, respectively. GST-MBS-N, GST-MBS-C, GST-MBS-ANK, and GST-alpha -adducin were produced and purified from E. coli.

To obtain recombinant alpha -adducin, the cDNA encoding human alpha -adducin (1-642 aa) (37) was inserted into the KpnI site of pAcYM1-HA (hemagglutinin). HA-alpha -adducin was produced in Sf9 cells by the use of a baculovirus system (45). The cells expressing HA-alpha -adducin were suspended in homogenizing buffer (20 mM Tris/HCl at pH 8.0, 1 mM EDTA, 1 mM DTT, 10 µM A-PMSF, 10 µg/ml leupeptin). The suspension was sonicated and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was applied onto a Mono Q column (Pharmacia Biotech Inc., Uppsala, Sweden) which had been equilibrated with Buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT). After the column was washed, the proteins were eluted with a linear concentration gradient of NaCl (0-600 mM) in Buffer A. HA-alpha -adducin was eluted with about 200 mM NaCl.

GST-MBS-ANK Affinity Column Chromatography-- The membrane extract of bovine brain gray matter, 190 g, was prepared (17). The membrane extract (16 ml) was passed through a 2.5-ml glutathione-Sepharose 4B column (Pharmacia) to remove endogenous GST (17). One-tenth of the pass-through fraction was loaded onto a 0.25-ml glutathione-Sepharose 4B column containing GST-MBS-ANK, GST-Notch-ANK, or GST. After washing the columns with 0.825 ml of Buffer A containing 50 mM NaCl three times, the bound proteins were coeluted with the respective GST fusion proteins by the addition of 0.825 ml of Buffer A containing 10 mM glutathione three times. To prepare affinity purified MBS-ANK interacting proteins for peptide sequencing, the pass-through fraction (16 ml) was loaded onto a 1-ml glutathione-Sepharose 4B column containing 24 nmol of GST-MBS-ANK. The proteins were eluted by the addition of 10 ml of Buffer A containing 10 mM glutathione, and fractions of 1 ml each were collected. The same procedures were repeated three times.

Peptide Sequence Analysis-- The affinity purified p85, p110, p115, p120, and p125 were dialyzed three times against distilled water and concentrated by freeze-drying. The concentrated samples were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (46). The immobilized p85, p110, p115, p120, and p125 were digested, fractionated, and subjected to amino acid sequencing as described (46).

In Vitro Binding Assay-- GST-MBS-ANK, GST-MBS-C, and GST (1 nmol each) were separately immobilized onto 35 µl of glutathione-Sepharose 4B beads. The immobilized beads were incubated with 8 µg of HA-alpha -adducin in 300 µl of Buffer A containing 1 mg/ml bovine serum albumin for 1 h at 4 °C. The beads were washed six times with 116 µl (3.3 volumes) of Buffer A, and the bound proteins were eluted with GST-MBS-ANK, GST-MBS-C, and GST by the addition of 116 µl (3.3 volumes) of Buffer A containing 10 mM glutathione three times. The second eluates were subjected to SDS-PAGE, and the proteins were detected by silver staining.

Cell Culture-- MDCK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum, streptomycin, and penicillin. COS7 cells were maintained in DMEM containing 10% fetal bovine serum, streptomycin, and penicillin. For the transfection of DNA, COS7 cells were seeded at the density of 1.7 × 105 cells in 35-mm tissue culture dishes and cultured overnight.

Immunoprecipitation Assay-- Rabbit anti-rat MBS pAb and rabbit anti-human alpha -adducin pAb were generated by use of GST-MBS-N and GST-alpha -adducin. MDCK cells were grown in 100-mm tissue culture dishes. After being washed with PBS, the cells were lysed with 1 ml of extraction Buffer A (20 mM Tris/HCl at pH 8.0, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Nonidet P-40, 10 µM A-PMSF, 10 µg/ml leupeptin). The lysate was removed from the dishes with a rubber policeman, incubated in a 1.5-ml tube for 15 min, and then clarified by centrifugation at 12,000 × g for 10 min. The soluble supernatant was incubated with 2 µg of anti-alpha -adducin Ab or 2 µg of control rabbit IgG. The immunocomplex was then precipitated with protein A-Sepharose CL 4B (Pharmacia). The immunocomplex was washed five times with the extraction Buffer A containing 0.5% Nonidet P-40, then eluted by boiling in sample buffer for SDS-PAGE and subjected to immunoblot analysis using the anti-MBS Ab as described (47).

Immunofluorescence Analysis-- The coiled-coil domain of Rho-kinase (421-701 aa) was produced and purified as GST fusion protein (GST-COIL). Rabbit anti-Rho-kinase pAb was generated by use of GST-COIL. For anti-MBS Ab, MDCK cells were fixed with 3.7% formaldehyde in PBS for 10 min and treated with ice-cold methanol for 10 min. For anti-alpha -adducin Ab and anti-Rho-kinase Ab, MDCK cells were fixed with ice-cold methanol for 10 min. After being washed with PBS three times, the cells were incubated with anti-MBS Ab, anti-Rho-kinase Ab, or anti-alpha -adducin Ab overnight at room temperature. Then MDCK cells were incubated with fluorescein isothiocyanate-conjugated anti-rabbit Ig Ab. After being washed with PBS three times, the cells were examined using a Zeiss axiophoto microscope or a confocal microscope (Carl Zeiss, Oberkochen, Germany).

Phosphorylation Assay-- The kinase reaction for Rho-kinase was carried out in 50 µl of the kinase buffer (50 mM Tris/HCl at pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing 100 µM [gamma -32P]ATP (1-20 GBq/mmol), purified enzyme, and 2.4 µg of HA-alpha -adducin with 1 µM GTPgamma S·GST-RhoA, GDP·GST-RhoA, or GST. After an incubation for 10 min at 30 °C, the reaction mixtures were boiled in SDS sample buffer and subjected to SDS-PAGE. The radiolabeled bands were visualized and estimated by an image analyzer (BAS-2000, Fuji, Tokyo).

Cosedimentation Assay-- HA-alpha -adducin (7 µg of protein) was phosphorylated with GST-CAT (3 µg of protein) in 200 µl of kinase buffer containing 0.1 µM calyculin A with or without ATP for 1 h at 30 °C. F-actin was mixed with HA-alpha -adducin phosphorylated as above in Buffer B (30 mM Hepes at pH 7.4, 0.5 mM DTT, 2 mM MgCl2, 50 mM KCl, 1 mM EGTA, 10% (w/v) sucrose, 0.5 mM ATP) for 2 h at 4 °C. After the incubation, 50 µl of each reaction mixture was layered onto a 100-µl sucrose barrier (20% (w/v) sucrose in Buffer B) and centrifuged at 200,000 × g for 1 h at 4 °C. The supernatants and pellets were separated and subjected to immunoblot analysis using anti-alpha -adducin Ab.

Protein Phosphatase Assay-- HA-alpha -adducin (300 ng of protein) was phosphorylated with GST-CAT (120 ng of protein) in 20 µl of kinase buffer containing 100 µM [gamma -32P]ATP for 1 h at 30 °C, and the reaction was stopped by the addition of 200 nM staurosporine. Native myosin phosphatase (5-75 ng) was preincubated in 30 µl of reaction mixture (30 mM Tris/HCl at pH 7.5, 3 mM MgCl2, 0.4 mM EDTA, 0.55 mM EGTA, 0.1 mg/ml bovine serum albumin, 0.3 mM CoCl2, 5-75 ng of myosin phosphatase) with or without 100 µM ATPgamma S and GST-CAT (80 ng of protein) for 15 min at 30 °C, and the reaction was stopped by the addition of 200 nM staurosporine. The phosphatase reaction was then performed in 50 µl of the reaction mixture containing 300 ng of 32P-labeled HA-alpha -adducin for 15 min at 30 °C. The reaction mixture was then boiled in sample buffer for SDS-PAGE and resolved by SDS-PAGE. The 32P-labeled band corresponding to HA-alpha -adducin was visualized and estimated with an image analyzer. As a control experiment, 32P-labeled MLC was used as the substrate of myosin phosphatase.

In Vivo Phosphorylation of alpha -Adducin by Rho-kinase-- To express HA epitope-tagged alpha -adducin, the cDNA encoding human alpha -adducin (1-642 aa) was inserted into the KpnI site of pEF-BOS-HA. The transfection of plasmids into COS7 cells was carried out by the standard DEAE-dextran method (17, 21). The plasmid pEF-BOS-HA-alpha -adducin was transfected with or without pEF-BOS-myc-CAT. The transfected cells were cultured in DMEM containing 10% fetal bovine serum for 2 days. The cells were then labeled with 18.5 MBq of [32P]orthophosphate for 3 h and lysed with 0.3 ml of extraction Buffer B (20 mM Tris/HCl at pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Nonidet P-40, 2 mM Na3VO4, 50 mM NaF, 0.1 µM calyculin A, 10 µM A-PMSF, 10 µg/ml leupeptin). The lysates were clarified by centrifugation at 12,000 × g for 15 min. HA-alpha -adducin was then immunoprecipitated from the soluble supernatant. The washed immunocomplexes were subjected to SDS-PAGE for peptide mapping.

Peptide Mapping-- HA-alpha -adducin was phosphorylated by Rho-kinase in vitro as described above. HA-alpha -adducins phosphorylated in vitro and in vivo were isolated by SDS-PAGE and digested with trypsin. Two-dimensional peptide mappings were performed with silica gel thin layer plates as described (48). Phosphorylated peptides were visualized by autoradiography.

Other Procedures-- SDS-PAGE was performed as described (49). Protein concentrations were determined with bovine serum albumin as the reference protein as described (50).

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Identification of MBS-interacting Molecule-- To detect MBS- interacting molecules, bovine brain membrane extract was loaded onto a glutathione-Sepharose affinity column on which GST, GST-MBS-ANK, or GST-Notch-ANK was immobilized. The proteins bound to the affinity columns were then coeluted with GST or GST fusion proteins by the addition of glutathione. Proteins with molecular masses of about 85 kDa (p85), 110 kDa (p110), 115 kDa (p115), 120 kDa (p120), and 125 kDa (p125) were detected in the glutathione eluate from the GST-MBS-ANK affinity column but not detected from the GST or the GST-Notch-ANK affinity column (Fig. 1A).


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Fig. 1.   A, purification of MBS-ANK-interacting molecules. The bovine brain membrane extract was loaded onto a glutathione-Sepharose column containing the indicated GST fusion proteins. The bound proteins were coeluted with the respective GST fusion proteins by the addition of glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GST; lane 2, GST-MBS-ANK; lane 3, GST-Notch-ANK. Arrowheads from the top denote the positions of p125, p120, p115, p110, and p85, respectively. The results shown are representative of three independent experiments. B, immunoblot analysis of p85, p110, p115, p120, and p125. Aliquots of the eluates from the affinity columns were subjected to immunoblot analysis with control rabbit IgG (lanes 1-3) or anti-alpha -adducin Ab (lanes 4-6). Lanes 1 and 4, GST; lanes 2 and 5, GST-MBS-ANK; lanes 3 and 6, GST-Notch-ANK. Arrowheads from the top denote the positions of p125, p120, p115, p110, and p85, respectively. The results shown are representative of three independent experiments.

To identify the p85, p110, p115, p120, and p125, they were subjected to amino acid sequencing as described (46). The peptide sequences derived from p85, p110, p115, p120, and p125 were determined. The amino acid sequences are KIDHAGFSPHAA (derived from p85), KGLSQMTTSADTDVDT, KGVSCSEVTASSL, QRPHEVGSVXWAG, KIFHLQAACEIQVSALSSAGG (derived from p115), KIREQNLQDIK, KHSDVEAPA, and KEDGHRTSTSAVPNL (derived from p110, p120, and p125). The peptide sequence of p85 was almost identical to that of human and rat gamma -adducin. The peptide sequences of p115 were almost identical to that of human beta -adducin. The peptide sequences of p110, p120, and p125 were almost identical to that of human alpha -adducin. p110 and p120 were probably degradation products of p125. p110, p120, and p125 were recognized by anti-alpha -adducin antibody, whereas p85 and p115 were weakly recognized by this antibody (Fig. 1B). Adducin is a membrane-cytoskeletal protein localized at the spectrin-actin junction that was first purified from human erythrocytes based on calmodulin binding activity (32). Since the molecular masses of alpha -, beta -, and gamma -adducins are estimated to be about 110, 104, and 80 kDa (38), respectively, by SDS-PAGE, we concluded that p85, p115, and p125 were bovine counterparts of rat and human adducin and hereafter refer to them as adducin.

Interaction of MBS with alpha -Adducin in Vitro and in Vivo-- We examined whether recombinant adducin interacts with MBS in a cell-free system. The purified recombinant HA-alpha -adducin was incubated with GST-MBS-ANK, GST-MBS-C, or GST immobilized beads. After washing the beads, the GST fusion proteins were eluted by the addition of glutathione. HA-alpha -adducin was coeluted with GST-MBS-ANK but not with GST-MBS-C or GST (Fig. 2A). This result indicates that recombinant alpha -adducin binds directly to the ankyrin repeat domain of MBS.


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Fig. 2.   Interaction of MBS with alpha -adducin in vitro and in vivo. A, purified HA-alpha -adducin was incubated with GST-MBS-ANK, GST-MBS-C, or GST immobilized beads. After washing, the bound proteins were eluted with GST, GST-MBS-ANK, or GST-MBS-C by the addition of glutathione. The eluates were subjected to SDS-PAGE and detected by silver staining. GST, lane 1; GST-MBS-ANK, lane 2; GST-MBS-C, lane 3. The arrow denotes the position of the purified HA-alpha -adducin. B, MDCK cells were lysed and solubilized with extraction Buffer A. This sample was incubated without antibody (lane 1), with control rabbit IgG (lane 2), or with anti-alpha -adducin Ab (lane 3). The immunocomplexes were then precipitated with protein A-Sepharose 4B. The immunocomplexes were subjected to immunoblot analysis using anti-MBS Ab. The arrowhead denotes the position of MBS. The results shown are representative of three independent experiments.

We examined whether MBS forms a complex with adducin in vivo. When alpha -adducin was immunoprecipitated with anti-alpha -adducin antibody from MDCK cells, some MBS was co-immunoprecipitated with alpha -adducin (Fig. 2B). Little MBS was immunoprecipitated with control rabbit IgG or without antibody. The catalytic subunit of myosin phosphatase was also detected in the immunoprecipitates (data not shown), whereas other Rho targets such as Rho-kinase and protein kinase N were not detected. Taken together, these findings indicate that MBS binds to alpha -adducin directly in vivo and in vitro.

Similar Localization of MBS and alpha -Adducin at Cell-Cell Contact Sites-- We recently showed that MBS is accumulated at cell-cell contact sites apart from myosin fibers in polarized MDCK epithelial cells, whereas MBS is colocalized with myosin fibers in REF52 fibroblasts (31). Adducin is also localized at the cell-cell contact sites of MDCK epithelial cells (35). We then compared the localization of MBS with that of alpha -adducin in confluent MDCK cells, which show characteristics of polarized epithelial cells and form the junctional complexes (including the tight junctions, adherens junctions, and desmosomes) at cell-cell contact sites. Immunofluorescence analysis revealed that MBS showed a distribution similar to that of alpha -adducin at the cell-cell contact sites (Fig. 3, A and B). Because Rho-kinase phosphorylates MBS (21), we then examined the localization of Rho-kinase in confluent MDCK cells. Rho-kinase was partly accumulated at the cell-cell contact sites (Fig. 3C).


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Fig. 3.   Localization of MBS, Rho-kinase, and alpha -adducin at cell-cell contact sites in MDCK cells. MDCK cells were seeded at 1 × 104 cells per 13-mm cover glass. After culturing for 4 days, confluent MDCK cells were fixed and stained with anti-MBS Ab, anti-Rho-kinase Ab, or anti-alpha -adducin Ab. A, with anti-MBS Ab; B, with anti-alpha -adducin Ab; C, with anti-Rho-kinase Ab. Fluorescein isothiocyanate-conjugated anti-rabbit Ig Ab was used as the secondary antibody. The results shown are representative of three independent experiments. Bar, 25 µm. All photographs were taken with the same magnification.

In Vitro Phosphorylation of alpha -Adducin by Rho-kinase-- We next examined whether Rho-kinase phosphorylates adducin in a cell-free system. Native Rho-kinase purified from bovine brain phosphorylated HA-alpha -adducin, and this phosphorylation was markedly enhanced by the addition of GTPgamma S·GST-RhoA but not of GDP·GST-RhoA (Fig. 4A). We found that GST-CAT (the catalytic domain of Rho-kinase) phosphorylated recombinant HA-alpha -adducin (Fig. 4A). We previously showed that CAT serves as a constitutively active form in vitro and in vivo (27). About 0.8 mol of phosphate could be maximally incorporated into 1 mol of HA-alpha -adducin by GST-CAT (Fig. 4B). It is reported that PKC and PKA phosphorylate alpha -adducin (35, 41, 42). alpha -Adducin is primarily phosphorylated at Ser-408, Ser-436, Ser-481, and Ser-726 by PKA and at Ser-726 by PKC (40). We performed a phosphoamino acid analysis and found that phosphorylation by Rho-kinase occurred mainly on the threonine residue. It is thus likely that the phosphorylation sites by Rho-kinase are different from those by PKC and PKA. (The identification of phosphorylation sites by Rho-kinase is currently under investigation and will be described elsewhere.)


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Fig. 4.   In vitro phosphorylation of alpha -adducin by Rho-kinase. A, purified HA-alpha -adducin was phosphorylated by native Rho-kinase in the presence of GST (lane 1), GDP·GST-RhoA (lane 2), or GTPgamma S·GST-RhoA (lane 3), or by GST-CAT (lane 4). The phosphorylated HA-alpha -adducin was resolved by SDS-PAGE and visualized by an image analyzer. The arrow denotes the position of HA-alpha -adducin. The results shown are representative of three independent experiments. B, purified HA-alpha -adducin was phosphorylated by GST-CAT for various periods. The values shown are means ± S.E. of triplicate experiments.

The Effect of the Phosphorylation of alpha -Adducin by Rho-kinase on Its F-Actin Binding Activity-- Adducin binds to F-actin and spectrin-F-actin complex to promote the binding of spectrin to F-actin (33). We speculated that phosphorylation by Rho-kinase might regulate the F-actin binding activity of adducin. To examine whether the phosphorylation of adducin by Rho-kinase modulates its F-actin binding activity, a cosedimentation assay of recombinant alpha -adducin with F-actin was performed. HA-alpha -adducin phosphorylated by GST-CAT was cosedimentated with F-actin more efficiently than non-phosphorylated HA-alpha -adducin (Fig. 5). A similar result was obtained in the presence of spectrin (data not shown). These findings suggest that the phosphorylation of adducin by Rho-kinase enhances the F-actin binding activity of adducin.


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Fig. 5.   The effect of phosphorylation of alpha -adducin by Rho-kinase on its F-actin binding activity. Purified HA-alpha -adducin was phosphorylated by GST-CAT with or without ATP. Various doses of HA-alpha -adducin were mixed with or without F-actin (3 µM) and incubated at room temperature for 1 h as indicated. After the incubation, 50 µl of each reaction mixture was layered onto 100 µl of 20% (w/v) sucrose barrier and was centrifuged at 200,000 × g for 1 h at room temperature. Pellets samples were subjected to immunoblot analysis using anti-alpha -adducin Ab. The arrow denotes the position of HA-alpha -adducin. The results shown are representative of three independent experiments.

Phosphatase Activity of Myosin Phosphatase toward alpha -Adducin Phosphorylated with Rho-kinase-- Rho-kinase phosphorylated adducin stoichiometrically, as described above. We speculated that myosin phosphatase might regulate the phosphorylation states of adducin downstream of Rho as described for MLC (21). We next examined whether myosin phosphatase dephosphorylates adducin which was phosphorylated by Rho-kinase. The myosin phosphatase showed phosphatase activity toward HA-alpha -adducin phosphorylated by GST-CAT (Fig. 6). We previously observed that the MBS of the native myosin phosphatase was thiophosphorylated with Rho-kinase in the presence of ATPgamma S and that this thiophosphorylation of MBS was associated with a decrease of phosphatase activity toward MLC (21). Here, we examined whether Rho-kinase also modulates the phosphatase activity of myosin phosphatase toward adducin through the thiophosphorylation of MBS. We found that the thiophosphorylation of MBS was associated with a decrease of phosphatase activity toward HA-alpha -adducin (Fig. 6).


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Fig. 6.   Phosphatase activity of myosin phosphatase toward HA-alpha -adducin. Purified HA-alpha -adducin was phosphorylated with GST-CAT as described under "Experimental Procedures." The phosphatase activity of myosin phosphatase toward phosphorylated HA-alpha -adducin was assayed for 10 min at 30 °C after the MBS of myosin phosphatase was thiophosphorylated with (closed diamonds) or without (open triangles) ATPgamma S and GST-CAT. The reaction mixture was then boiled in sample buffer for SDS-PAGE and resolved by SDS-PAGE. The 32P-labeled bands corresponding to HA-alpha -adducin were visualized, and the amounts of [32P]-HA-alpha -adducin were estimated with an image analyzer. The values shown are means ± S.E. of triplicate experiments.

Phosphorylation of alpha -Adducin by Rho-kinase in COS7 Cells-- We performed a two-dimensional peptide map analysis of the phosphorylated HA-alpha -adducin. HA-alpha -adducin phosphorylated by GST-CAT in vitro was digested with trypsin and was subjected to thin layer chromatography using a silica gel plate, followed by autoradiography. Two major radioactive spots (named spots a and b) and several minor radioactive spots were detected (Fig. 7A). To examine whether Rho-kinase can induce the phosphorylation of adducin in vivo, the plasmid pEF-BOS-HA-alpha -adducin was transfected with or without pEF-BOS-CAT into COS7 cells, and the cells were labeled with [32P]orthophosphate. HA-alpha -adducin was then immunoprecipitated from the cell lysates and subjected to two-dimensional peptide mapping. When HA-alpha -adducin was expressed alone, several radioactive spots were observed, whereas spots corresponding to spots a or b were not detected (Fig. 7B). When HA-alpha -adducin was coexpressed with CAT, the spot corresponding to spot a was detected, and the spot corresponding to spot b was weakly detected (Fig. 7, C and D). Similar results were obtained when HA-alpha -adducin was coexpressed with the dominant active RhoA (data not shown). These findings suggest that the site of adducin corresponding to spot a was phosphorylated by Rho-kinase both in vitro and in vivo.


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Fig. 7.   Peptide map analysis of alpha -adducin phosphorylated by Rho-kinase in vitro and in vivo. Purified HA-alpha -adducin was phosphorylated by GST-CAT with [gamma -32P]ATP in vitro (A). COS7 cells were cotransfected with pEF-BOS-HA-alpha -adducin and pEF-BOS-myc (B) or pEF-BOS-myc-CAT (C). The cells were labeled with 18.5 MBq of [32P]orthophosphate and lysed with the extraction Buffer B. HA-alpha -adducin was then immunoprecipitated. The in vitro phosphorylated HA-alpha -adducin and the immunocomplexes were subjected to SDS-PAGE. Phosphorylated HA-alpha -adducin was isolated and digested with trypsin, and each sample was loaded onto a silica gel plate. Phosphopeptides were separated by electrophoresis (horizontal dimension) and chromatography (vertical dimension), followed by autoradiography. Phosphopeptides derived from HA-alpha -adducin phosphorylated in COS7 cells expressed with CAT were mixed with those from HA-alpha -adducin phosphorylated in vitro (D). Asterisks denote origins. The arrowheads denote major radioactive spots derived from HA-alpha -adducin phosphorylated by Rho-kinase. The results shown are representative of three independent experiments.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Complex Formation between MBS and Adducin-- We purified MBS-interacting proteins by GST-MBS-ANK affinity column chromatography and identified them as adducin. Adducin is a membrane skeletal protein that associates with F-actin and spectrin-F-actin complexes and promotes the association of spectrin with F-actin (33). Adducin is localized at cell-cell contact sites in some epithelial cells (35). Adducin is thought to participate in the assembly of the spectrin-actin network. We showed that alpha -adducin interacts with the ankyrin-repeat domain of MBS in vitro and that some population of MBS interacts with alpha -adducin in vivo (Fig. 2). We also showed that the localization of MBS is similar to that of alpha -adducin at cell-cell contact sites in confluent MDCK epithelial cells (Fig. 3) and that myosin phosphatase dephosphorylates the alpha -adducin phosphorylated by Rho-kinase (Fig. 6). We confirmed that the interaction of alpha -adducin with MBS is not modulated by the activated RhoA and that MBS is co-immunoprecipitated with alpha -adducin from the bovine brain cytosol where the activated RhoA is absent (data not shown). Thus, it is likely that MBS constitutively binds to adducin and that myosin phosphatase efficiently regulates the state of phosphorylation of adducin.

In contrast, in non-confluent MDCK cells, a high level of MBS and alpha -adducin immunoreactivities were not observed at cell-cell contact sites (data not shown). Activated RhoA was detected at the cell-cell contact sites as described (51). We previously demonstrated that activated RhoA translocates MBS from the cytosol to the plasma membrane in COS7 cells (21). We showed here that MBS binds to alpha -adducin both in vitro and in vivo as described above. The microinjection of C3 into keratinocytes is known to inhibit the cadherin-mediated cell-cell adhesion (6). We also confirmed that the microinjection of C3 transferase into MDCK cells resulted in the perturbation of the cell-cell contacts, followed by a decrease in the accumulation of MBS and alpha -adducin at the cell-cell contact sites (data not shown). Taken together, these findings suggest that MBS may be translocated with adducin to the cell-cell contact sites under the control of Rho.

Dual Regulation of the Phosphorylation State of Adducin-- Rho regulates MLC phosphorylation via two pathways through its targets, Rho-kinase and MBS, as follows (21, 24). Activated Rho interacts with Rho-kinase and the MBS of myosin phosphatase and activates Rho-kinase. The activated Rho-kinase subsequently phosphorylates MBS, thereby inactivating myosin phosphatase (21). Rho-kinase by itself phosphorylates MLC at the same site that is phosphorylated by MLC kinase and activates myosin ATPase (24). Both pathways appear to be important for an increase of the phosphorylation of MLC (29).

Here we found that Rho-kinase phosphorylates alpha -adducin in the presence of the activated RhoA (Fig. 4). We also showed that myosin phosphatase dephosphorylates alpha -adducin, which is phosphorylated by Rho-kinase. The phosphatase activity of myosin phosphatase toward alpha -adducin is inhibited by the thiophosphorylation of MBS with Rho-kinase (Fig. 6). The expression of the dominant active form of Rho-kinase induces the phosphorylation of alpha -adducin in COS7 cells (Fig. 7). These results suggest that the phosphorylation state of adducin is regulated through MBS and Rho-kinase downstream of Rho in a manner similar to MLC.

Phosphorylation of Adducin by Rho-kinase-- Adducin is a substrate for PKC and PKA (35, 41, 42). The phosphorylation of adducin by PKA reduces the activity of adducin to associate with F-actin and spectrin-F-actin complexes and to promote the binding of spectrin to F-actin. As mentioned earlier, phosphorylation by PKC has little effect on this activity (40). alpha -Adducin is primarily phosphorylated at Ser-408, Ser-436, Ser-481, and Ser-726 by PKA and at Ser-726 by PKC (40).

Here we found that the phosphorylation of alpha -adducin by Rho-kinase occurred mainly on the threonine residue. We also showed that in COS7 cells, the expression of the constitutively active form of Rho-kinase induced the phosphorylation of alpha -adducin at the same sites as those phosphorylated in vitro (Fig. 7). We demonstrated that the alpha -adducin phosphorylated by Rho-kinase is cosedimentated with F-actin more efficiently than the non-phosphorylated alpha -adducin (Fig. 5). These results suggest that the phosphorylation sites of adducin by Rhokinase are different from those by PKC and PKA and that the phosphorylation of adducin by Rho-kinase stimulates the F-actin binding activity of adducin. This action of adducin may promote the spectrin-actin complex formation at the cell-cell contact sites in epithelial cells.

Roles of Rho in the Regulation of Adducin Activity-- Accumulating evidence indicates that Rho participates in signaling pathways that regulate actin cytoskeletons such as stress fibers and in cell substratum adhesions such as focal adhesions in fibroblasts (3). Rho is also involved in the regulation of cell morphology (4) and cell aggregation (5). It has recently been shown that activated Rho is required for maintaining cadherin-mediated cell-cell adhesion (6). Rho appears to be involved in the assembly of adhesion molecules such as cadherin and peripheral proteins including cortical actin filaments, ERM (ezrin, radixin, and moesin), and vinculin at cell-cell contact sites (6, 52). Cortical actin filaments consist of a number of proteins including spectrin-F-actin-adducin complexes. Adducin is thought to promote the formation of this complex (33). We showed herein that Rho-kinase phosphorylates alpha -adducin, and this phosphorylation is dually regulated by the Rho targets Rho-kinase and MBS and that the phosphorylation of alpha -adducin by Rho-kinase enhances its binding activity to F-actin. Taken together, these findings suggest that Rho-kinase and MBS can regulate the F-actin binding activity of adducin through its phosphorylation downstream of Rho, thereby resulting in the assembly of the spectrin-F-actin network.

    ACKNOWLEDGEMENTS

We thank Dr. Masato Nakafuku for providing cDNA of rat Notch1, Dr. Masaaki Ito for providing chicken gizzard myosin phosphatase, and Dr. Masaki Inagaki for valuable discussions. We are also grateful to Akemi Takemura for secretarial assistance and Nagatoki Kinoshita for technical assistance.

    FOOTNOTES

* This investigation was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture of Japan (1997) and by grants from the Mitsubishi Foundation and Kirin Brewery Co. Ltd.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.

Dagger Dagger To whom correspondence should be addressed. Tel.: 81-743-72-5440; Fax: 81-743-72-5449; E-mail: kaibuchi{at}bs.aist-nara.ac.jp.

1 The abbreviations used are: Rho-kinase, Rho-associated kinase; MBS, myosin-binding subunit; MLC, myosin light chain; PKC, protein kinase C; PKA, protein kinase A; pAb, polyclonal antibody; GST, glutathione S-transferase; A-PMSF, (p-amidinophenyl)-methanesulfonyl fluoride; GTPgamma S, guanosine 5'-(3-O-thio)-triphosphate; ATPgamma S, adenosine 5'-O-(3-thiophosphate); PAGE, polyacrylamide gel electrophoresis; Ab, antibody; DTT, dithiothreitol; PBS, phosphate-buffered saline; HA, hemagglutinin; MDCK, Madin-Darby canine kidney; aa, amino acid(s); DMEM, Dulbecco's modified Eagle's medium.

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