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

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 adducinin 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 α-adducin in vitro and in vivo and that the phosphorylation of α-adducin by Rho-kinase enhanced the interaction of α-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward α-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.

Rho is a small GTPase that exhibits both GDP/GTP binding and GTPase activities. Rho has GDP-bound inactive (GDP⅐Rho) and GTP-bound active (GTP⅐Rho) 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, GDP⅐Rho is thought to be converted to GTP⅐Rho, 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-kinase 1 (19), which is also known as ROK␣ (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 GTP⅐Rho (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 ␣-, ␤-, and ␥-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 ␣ and ␤ or ␣ and ␥ subunits closely related in amino acid sequence and domain organization (36 -38). Each adducin subunit has three distinct domains as follows: an aminoterminal head domain, connected by a neck domain to a carboxyl-terminal tail domain (36 -38). ␣-Adducin and ␤-adducin form heterodimers and tetramers through the head domains and tail domains (39). The tail domain of ␤-adducin binds mainly to Ca 2ϩ /calmodulin (40), which inhibits both the ability of adducin to recruit additional spectrin to adducinspectrin-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 ␤-adducin by PKC or PKA inhibits the calmodulin binding of ␤-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 Rhokinase results in the interaction of adducin with F-actin.

EXPERIMENTAL PROCEDURES
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). [␥-32 P]ATP and [ 32 P]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.
To obtain recombinant ␣-adducin, the cDNA encoding human ␣-adducin (1-642 aa) (37) was inserted into the KpnI site of pAcYM1-HA (hemagglutinin). HA-␣-adducin was produced in Sf9 cells by the use of a baculovirus system (45). The cells expressing HA-␣-adducin were suspended in homogenizing buffer (20 mM Tris/HCl at pH 8.0, 1 mM EDTA, 1 mM DTT, 10 M A-PMSF, 10 g/ml leupeptin). The suspension was sonicated and centrifuged at 100,000 ϫ 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-␣-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 l of glutathione-Sepharose 4B beads. The immobilized beads were incubated with 8 g of HA-␣-adducin in 300 l of Buffer A containing 1 mg/ml bovine serum albumin for 1 h at 4°C. The beads were washed six times with 116 l (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 l (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 ϫ 10 5 cells in 35-mm tissue culture dishes and cultured overnight.
Immunoprecipitation Assay-Rabbit anti-rat MBS pAb and rabbit anti-human ␣-adducin pAb were generated by use of GST-MBS-N and GST-␣-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 MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Nonidet P-40, 10 M A-PMSF, 10 g/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 ϫ g for 10 min. The soluble supernatant was incubated with 2 g of anti-␣-adducin Ab or 2 g 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-␣-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-␣-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 l of the kinase buffer (50 mM Tris/HCl at pH 7.5, 5 mM Cosedimentation Assay-HA-␣-adducin (7 g of protein) was phosphorylated with GST-CAT (3 g of protein) in 200 l of kinase buffer containing 0.1 M calyculin A with or without ATP for 1 h at 30°C. F-actin was mixed with HA-␣-adducin phosphorylated as above in Buffer B (30 mM Hepes at pH 7.4, 0.5 mM DTT, 2 mM MgCl 2 , 50 mM KCl, 1 mM EGTA, 10% (w/v) sucrose, 0.5 mM ATP) for 2 h at 4°C. After the incubation, 50 l of each reaction mixture was layered onto a 100-l sucrose barrier (20% (w/v) sucrose in Buffer B) and centrifuged at 200,000 ϫ g for 1 h at 4°C. The supernatants and pellets were separated and subjected to immunoblot analysis using anti-␣adducin Ab.
Protein Phosphatase Assay-HA-␣-adducin (300 ng of protein) was phosphorylated with GST-CAT (120 ng of protein) in 20 l of kinase buffer containing 100 M [␥-32 P]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 l of reaction mixture (30 mM Tris/HCl at pH 7.5, 3 mM MgCl 2 , 0.4 mM EDTA, 0.55 mM EGTA, 0.1 mg/ml bovine serum albumin, 0.3 mM CoCl 2 , 5-75 ng of myosin phosphatase) with or without 100 M ATP␥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 l of the reaction mixture containing 300 ng of 32 Plabeled HA-␣-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 32 P-labeled band corresponding to HA-␣-adducin was visualized and estimated with an image analyzer. As a control experiment, 32 P-labeled MLC was used as the substrate of myosin phosphatase.
In Vivo Phosphorylation of ␣-Adducin by Rho-kinase-To express HA epitope-tagged ␣-adducin, the cDNA encoding human ␣-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 DEAEdextran method (17,21). The plasmid pEF-BOS-HA-␣-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 [ 32 P]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 MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Nonidet P-40, 2 mM Na 3 VO 4 , 50 mM NaF, 0.1 M calyculin A, 10 M A-PMSF, 10 g/ml leupeptin). The lysates were clarified by centrifugation at 12,000 ϫ g for 15 min. HA-␣-adducin was then immunoprecipitated from the soluble supernatant. The washed immunocomplexes were subjected to SDS-PAGE for peptide mapping.
Peptide Mapping-HA-␣-adducin was phosphorylated by Rho-kinase in vitro as described above. HA-␣-adducins phosphorylated in vitro and in vivo were isolated by SDS-PAGE and digested with trypsin. Twodimensional 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).

Identification of MBS-interacting
Molecule-To detect MBSinteracting 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).
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 KIDHAGF-SPHAA (derived from p85), KGLSQMTTSADTDVDT, KGVSC-SEVTASSL, QRPHEVGSVXWAG, KIFHLQAACEIQVSALS-SAGG (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 ␥-adducin. The peptide sequences of p115 were almost identical to that of human ␤-adducin. The peptide sequences of p110, p120, and p125 were almost identical to that of human ␣-adducin. p110 and p120 were probably degradation products of p125. p110, p120, and p125 were recognized by anti-␣-adducin antibody, whereas p85 and p115 were weakly recognized by this antibody (Fig. 1B). Adducin is a membranecytoskeletal 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 ␣-, ␤-, and ␥-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 ␣-Adducin in Vitro and in Vivo-We examined whether recombinant adducin interacts with MBS in a cell-free system. The purified recombinant HA-␣-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-␣-adducin was coeluted with GST-MBS-ANK but not with GST-MBS-C or GST ( Fig. 2A). This result indicates that recombinant ␣-adducin binds directly to the ankyrin repeat domain of MBS.
We examined whether MBS forms a complex with adducin in vivo. When ␣-adducin was immunoprecipitated with anti-␣adducin antibody from MDCK cells, some MBS was co-immunoprecipitated with ␣-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 ␣-adducin directly in vivo and in vitro.
Similar Localization of MBS and ␣-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 ␣-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 ␣-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).
In Vitro Phosphorylation of ␣-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-␣-adducin, and this phosphorylation was markedly enhanced by the addition of GTP␥S⅐GST-RhoA but not of GDP⅐GST-RhoA (Fig. 4A). We found that GST-CAT (the catalytic domain of Rho-kinase) phosphorylated recombinant HA-␣-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-␣-adducin by GST-CAT (Fig. 4B). It is reported that PKC and PKA phosphorylate ␣-adducin (35,41,42). ␣-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.) The Effect of the Phosphorylation of ␣-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 ␣-adducin with F-actin was performed. HA-␣-adducin phosphorylated by GST-CAT was cosedimentated with F-actin more efficiently than non-phosphorylated HA-␣-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.
Phosphatase Activity of Myosin Phosphatase toward ␣-Addu- cin 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 Rhokinase. The myosin phosphatase showed phosphatase activity toward HA-␣-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 ATP␥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-␣-adducin (Fig. 6).
Phosphorylation of ␣-Adducin by Rho-kinase in COS7 Cells-We performed a two-dimensional peptide map analysis of the phosphorylated HA-␣-adducin. HA-␣-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-␣-adducin was transfected with or without pEF-BOS-CAT into COS7 cells, and the cells were labeled with [ 32 P]orthophosphate. HA-␣-adducin was then immunoprecipitated from the cell lysates and subjected to two-dimensional peptide mapping. When HA-␣-adducin was expressed alone, several radioactive spots were observed, whereas spots corresponding to spots a or b were not detected (Fig. 7B). When HA-␣-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-␣-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.

DISCUSSION
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 ␣-adducin interacts with the ankyrin-repeat domain of MBS in vitro and that some population of MBS interacts with ␣-adducin in vivo (Fig. 2). We also showed that the localization of MBS is similar to that of ␣-adducin at cell-cell contact sites in confluent MDCK epithelial cells (Fig. 3) and that myosin phosphatase dephosphorylates the ␣-adducin phosphorylated by Rho-kinase (Fig. 6). We confirmed that the interaction of ␣-adducin with MBS is not modulated by the activated RhoA and that MBS is co-immunoprecipitated with ␣-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 ␣-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 ␣-adducin both in vitro and in vivo as described above. The microinjection of C3 into keratinocytes is known to inhibit the cadherin-mediated cellcell 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 ␣-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 ␣-adducin in the presence of the activated RhoA (Fig. 4). We also showed that myosin phosphatase dephosphorylates ␣-adducin, which is phosphorylated by Rho-kinase. The phosphatase activity of myosin phosphatase toward ␣-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 ␣-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). ␣-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 ␣-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 ␣-adducin at the same sites as those phosphorylated in vitro (Fig.  7). We demonstrated that the ␣-adducin phosphorylated by Rho-kinase is cosedimentated with F-actin more efficiently than the non-phosphorylated ␣-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 Factin 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 cadherinmediated 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 ␣-adducin, and this phosphorylation is dually regulated by the Rho targets Rho-kinase and MBS and that the phosphorylation of ␣-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.