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J Biol Chem, Vol. 274, Issue 45, 32418-32424, November 5, 1999


The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity*

Mutsuki Amano, Kazuyasu Chihara, Nao Nakamura, Takako Kaneko, Yoshiharu MatsuuraDagger , and Kozo Kaibuchi§

From the Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101 and the Dagger  Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rho-kinase is implicated in the phosphorylation of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho-kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinase beta  (MRCKbeta ). The COOH-terminal portion of Rho-kinase containing Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho-kinase and thereby inhibited the Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of protein kinase N or MRCKbeta , suggesting that the COOH-terminal region of Rho-kinase is a possible negative regulatory region of Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCKbeta or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho-kinase in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is mounting evidence that the small GTPase Rho plays crucial roles in the rearrangements of cytoskeleton and cell adhesion (1-3). Rho cycles between GDP-bound inactive and GTP-bound active forms, and the GTP-bound form binds to specific effectors and then exerts its biological functions. Numerous putative Rho effectors have been identified; PKN1 (4, 5), Rho-kinase/ROKalpha /ROCK II (6-8), myosin-binding subunit of myosin phosphatase (9), mDia1 (10), citron (11), citron kinase (12), rhophilin, rhotekin (11), Kv1.2 (13), and phospholipase D (14). ROCK I/ROKbeta is an isoform of Rho-kinase (7, 8). Rho-kinase is implicated in many processes downstream of Rho; stress fiber and focal adhesion formation (15-17), smooth muscle contraction (18), intermediate filament disassembly (19, 20), neurite retraction (21, 22), microvilli formation (23), cytokinesis (24), and cell migration (25). Rho-kinase regulates the phosphorylation of MLC by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase through the phosphorylation of myosin-binding subunit (9, 26). In addition to MLC and myosin-binding subunit, Rho-kinase phosphorylates the ezrin/radixin/moesin family proteins and adducin in vitro (27, 28). To unravel in vivo functions of Rho-kinase, it is necessary to develop specific probes for Rho-kinase. Recently, chemical compounds such as Y-27632, Y-32885, and HA1077 have been shown to inhibit the Rho-kinase activity in a manner competitive with ATP (29), and to suppress hypertension in model animals. However, the modes of action and specificity of these chemical compounds have not yet been elucidated.

Rho-kinase is composed of NH2-terminal catalytic, coiled-coil, Rho-binding, and COOH-terminal PH domains (6). When the COOH-terminal portion of Rho-kinase is deleted, the NH2-terminal portion containing the catalytic domain of Rho-kinase (CAT) becomes constitutively active and serves as the dominant active form both in vitro and in vivo (16). The Rho-binding domain (RB) of Rho-kinase inhibits Rho-dependent activation of Rho-kinase in vitro and interferes with the Rho pathway in vivo (16). The catalytic domain mutated at the ATP-binding site (kinase dead: CAT-KD) and the PH domain (PH) serve as dominant negative forms in vivo in some cases (16), while they have no or a very weak effect on the Rho-kinase activity in vitro. It has been also reported that ROCK I, an isoform of Rho-kinase, that is kinase dead and cannot bind to Rho (named as KD-IA) functions as dominant negative form in the cells (17). Recently, we found that the COOH-terminal portion of Rho-kinase containing Rho-binding and PH domains (RB/PH (TT)), in which point mutations are introduced to abolish the Rho binding activity (15, 30), inhibits the lysophosphatidic acid-induced neurite retraction in neuroblastoma cells and cytokinesis in Xenopus eggs or in mammalian cells (21, 24), although PH has minimal effects in these cases. These results suggest that RB/PH (TT) functions as the dominant negative form of Rho-kinase by the different mechanism of inhibition from those of CAT-KD, RB, and PH.

Here, we examined the mode of action and the specificity of inhibitors of Rho-kinase. We found that RB/PH (TT) directly and specifically inhibited the kinase activity of catalytic fragment of Rho-kinase in vitro. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MLC.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Chemicals-- GST-Rho-kinase-CAT (6-553 amino acids), GST-MRCKbeta -CAT (1-550 amino acids), and GST-PKN-CAT (581-942 amino acids) were produced in Sf9 cells with a baculovirus system and purified on a glutathione-Sepharose column (6,16,26). MBP-RB/PH (TT) (941-1388 amino acids with mutations; N1036T,K1037T), MBP-RB (941-1075 amino acids), MBP-PH (1125-1388 amino acids), GST-RB, GST-PH, and GST-coil gamma  (941-1131 amino acids) were expressed in Escherichia coli and purified with amylose resin (New England Biotech) for MBP fusion proteins or with glutathione-Sepharose (Amersham Pharmacia Biotech) for GST fusion proteins, respectively. MLC was kindly provided by Dr. Ito (Mie University, Mie, Japan). Rsk kinase S6 substrate peptide (RRRLSSLRA) and PKC substrate peptide (RFARKGSLRQKNVHEVK) were synthesized. [gamma -32P]ATP was purchased from Amersham Pharmacia Biotech. Staurosporine was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). HA1077 was kindly provided by Asahi Chemical Industry (Shizuoka, Japan). Y-32885 was synthesized (29). Tetramethyl rhodamine isothiocyanate-labeled phalloidin and anti-vinculin monoclonal antibody were purchased from Sigma. Anti-Myc polyclonal antibody was purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA). All materials used in the nucleic acid study were purchased from Takara Shuzo Co. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources.

Kinase Assay-- The kinase reaction for GST-Rho-kinase-CAT, GST-MRCKbeta -CAT, or GST-PKN-CAT was carried out in 50 µl of kinase buffer (50 mM Tris/HCl at pH 7.5, 2 mM EGTA, 1 mM EDTA, 5 mM MgCl2) containing 100 µM [gamma -32P]ATP (1-20 GBq/mmol), recombinant kinase (30 ng of Rho-kinase-CAT, 250 ng of MRCKbeta -CAT, or 60 ng of PKN-CAT), and 40 µM substrate peptide. Rsk kinase S6 substrate peptide and PKC substrate peptide were used for Rho-kinase and MRCKbeta , and for PKN as substrates, respectively. After incubation for 10 min at 30 °C, the reaction mixtures were spotted onto a Whatman P81 paper and washed with 75 mM phosphoric acid three times. Incorporation of 32P into the substrates was assessed by scintillation counting.

The kinase reaction for GST-Rho-kinase-CAT was carried out in 50 µl of kinase buffer (50 mM Tris/HCl at pH 7.5, 2 mM EGTA, 1 mM EDTA, 5 mM MgCl2, 3 mM KCl) containing 100 µM [gamma -32P]ATP (1-20 GBq/mmol), recombinant kinase (6 ng of Rho-kinase-CAT or 50 ng of MRCKbeta -CAT), and purified MLC. After 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 by an image analyzer (Fuji).

Affinity Chromatography-- The cytosol fraction and membrane extract of bovine brain gray matter (50 g) were prepared (4). The cytosol fraction (4 ml; 200 mg of protein) or membrane extract (4 ml; 20 mg of protein) was preincubated with 2 ml of amylose resin. Half of the bovine cytosol or membrane extract was loaded onto a 0.25-ml amylose resin containing MBP or MBP-RB/PH (TT). After washing the columns three times with 0.825 ml of buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT) containing 50 mM NaCl, the bound proteins were eluted by addition of 0.825 ml of buffer A containing 200 mM NaCl. The eluates were subjected to immunoblot analysis using antibody against Rho-kinase.

Cell Culture and Microinjection-- NIH 3T3 cells were maintained in DMEM containing 10% calf serum. For expression of RhoVal-14, Rho-kinase-CAT, or MRCKbeta -CAT, cells were seeded at a density of 2 × 103 onto 13-mm glass coverslips. After 5 days, the cells were deprived of serum for 24 h in DMEM. Plasmid DNA was microinjected into nuclei and the cells were incubated for 24 h in DMEM. For expression of MLC or MLCT18D,S19D, cells were seeded at a density of 2 × 104 onto 13-mm glass coverslips, and cultured in DMEM containing 10% calf serum. After 24 h, plasmid DNA was microinjected into nuclei, and the cells were incubated in DMEM containing 10% calf serum for 24 h, then deprived of serum for 24 h in DMEM. If necessary, the cells were treated with HA1077 or vehicle for 20 min after incubation.

Immunofluorescent Staining-- The cells were fixed with 3.7% formaldehyde in PBS for 10 min, washed with PBS, and permeabilized with PBS containing 0.2% Triton X-100 for 10 min. After being washed with PBS three times, the cells were double stained with tetramethyl rhodamine isothiocyanate-labeled phalloidin and anti-Myc polyclonal antibody with fluorescein isothiocyanate-conjugated anti-rabbit antibody, or with anti-vinculin monoclonal antibody with fluorescein isothiocyanate-conjugated anti-mouse antibody and anti-Myc polyclonal antibody with Texas red-conjugated anti-rabbit antibody. After being washed three times with PBS, the cells were examined using a Zeiss axiophoto microscope.

Other Procedures-- SDS-PAGE was performed as described previously (31).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemical Compounds Inhibit the Rho-kinase Activity in Vitro-- We examined the effect of chemical compounds, which are thought to interact with the kinase domain of Rho-kinase and compete with ATP, on the activity of the catalytic domain of Rho-kinase (Rho-kinase-CAT). Staurosporine, HA1077, and Y-32885 inhibited the activity of Rho-kinase-CAT in a dose-dependent manner (Fig. 1), and the IC50 values were approximately 7 nM, 1.7 µM, and 0.4 µM respectively, under the conditions. The Ki values for staurosporine, HA1077, and Y-32885 were calculated to be approximately 1.6 nM, 0.4 µM, and 0.1 µM, respectively. The Ki values for HA1077 and Y-32885 are roughly the same as reported previously (29). PKN is one of the Rho targets. PKN has sequence similarity to protein kinase C within the kinase domain and substrate specificity similar to that of protein kinase C (32). PKN is activated by the active form of Rho like Rho-kinase, but shows substrate specificity distinct from that of Rho-kinase. MRCKbeta was identified as a Cdc42 target molecule, and a member of the myotonic dystrophy kinase family like Rho-kinase. MRCKbeta shares sequence similarity with Rho-kinase within the kinase domain and shows substrate specificity similar to that of Rho-kinase (33).2 To test the specificity of the above compounds, we examined their effects on the activity of the catalytic domain of PKN (PKN-CAT) and the catalytic domain of MRCKbeta (MRCKbeta -CAT). These compounds inhibited the activity of PKN-CAT with similar doses as for Rho-kinase, whereas they had less inhibitory effect on that of MRCKbeta -CAT despite their similarity in primary structure (Fig. 1). Thus, it is likely that these compounds inhibit both Rho-kinase and PKN activities with a similar efficiency and cannot distinguish Rho-kinase and PKN under the conditions.


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  		Fig. 1.   Effect of protein kinase inhibitors on the activity of Rho-kinase. The activities of recombinant GST-Rho-kinase-CAT (hatched square), GST-MRCKbeta -CAT (open circle), and GST-PKN-CAT (closed circle) were assayed in the presence of the indicated concentrations of inhibitors as described under "Experimental Procedures." S6 peptide or PKC peptide was used as substrate for Rho-kinase and MRCKbeta , or for PKN, respectively. All data are mean ± S.E. of triplicate determinations.

The COOH-terminal Portion of Rho-kinase Inhibits the Rho-kinase Activity in Vitro-- We then examined whether the various fragments of Rho-kinase affect the Rho-kinase activity in vitro. We previously reported that RB or PH fragment expressed in E. coli as a GST fusion protein had no or a very weak effect on the activity of Rho-kinase-CAT in vitro (16).3 Here, we found that the COOH-terminal portion of Rho-kinase containing the RB and PH domains (RB/PH (TT)), in which point mutations are introduced to abolish the Rho binding activity, inhibited the Rho-kinase activity. The RB/PH (TT) region expressed in E. coli as an MBP fusion protein (MBP-RB/PH (TT)) inhibited the kinase activity of Rho-kinase-CAT toward both Rsk kinase S6 peptide (RRRLSSLRA) and MLC in a dose-dependent manner (Fig. 2, A and C). IC50 values of MBP-RB/PH (TT) were approximately 0.1 and 0.02 µM, respectively, under the conditions. MBP-RB/PH (TT) had no effect on the activity of MRCKbeta -CAT or PKN-CAT (Fig. 2, A and C). Kinetic analyses revealed that MBP-RB/PH (TT) inhibited the activity of Rho-kinase-CAT toward S6 peptide in a competitive manner (Fig. 2B) and toward MLC in a noncompetitive-competitive mixed manner (Fig. 2D). We confirmed that MBP-RB/PH (TT) inhibited the activity of native Rho-kinase purified from bovine brain (data not shown). Since MBP-RB/PH (TT) inhibited the activity of Rho-kinase-CAT in a manner competitive with S6 peptide, it is possible that MBP-RB/PH (TT) interacts with the catalytic domain of Rho-kinase and thereby competes with S6 peptide. To prove this rationale, we examined whether MBP-RB/PH (TT) interacts with Rho-kinase. Bovine cytosol or membrane extract fraction containing Rho-kinase was loaded onto affinity beads coated with MBP or MBP-RB/PH (TT). The proteins bound to the affinity beads were eluted by the addition of 0.2 M NaCl and the eluates were subjected to immunoblot analysis with the anti-Rho-kinase antibody. The band corresponding to Rho-kinase was detected in the eluate from the beads coated with MBP-RB/PH (TT), but not in that from the beads coated with MBP (Fig. 3), MBP-RB, or MBP-PH (data not shown). We also confirmed that Rho-kinase-CAT interacted with MBP-RB/PH (TT) under similar conditions (data not shown). Taken together, these results indicate that MBP-RB/PH (TT) interacts with the catalytic domain of Rho-kinase and thereby inhibits the kinase activity more specifically than do the chemical compounds.


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  		Fig. 2.   Effect of RB/PH (TT) on the activity of Rho-kinase. A, dose-effect of MBP-RB/PH (TT) on phosphorylation of S6 peptide (40 µM) by GST-Rho-kinase-CAT (closed circle) or GST-MRCKbeta -CAT (open circle), or on phosphorylation of PKC peptide (40 µM) by GST-PKN-CAT (hatched circle). B, effect of MBP-RB/PH (TT) on phosphorylation of S6 peptide by Rho-kinase. Phosphorylation of S6 peptide at the indicated concentrations by GST-Rho-kinase-CAT was examined with 0.1 µM MBP (open circle) or with 0.1 µM MBP-RB/PH (TT) (closed circle). The maximal activity assayed with MBP is regarded as 100% activity. C, dose-effect of MBP-RB/PH (TT) on phosphorylation of MLC (2 µM) by GST-Rho-kinase-CAT (closed circle) or GST-MRCKbeta -CAT (open circle). D, effect of MBP-RB/PH (TT) on phosphorylation of MLC by Rho-kinase. Phosphorylation of MLC at the indicated concentrations by GST-Rho-kinase-CAT was examined with 20 nM MBP (open circle) or with 20 nM MBP-RB/PH (TT) (closed circle). The maximal activity assayed with MBP is regarded as 100% activity. All data are mean ± S.E. of triplicate determinations.


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  		Fig. 3.   Interaction of RB/PH (TT) with Rho-kinase. The bovine brain cytosol (100 mg of protein) or membrane extract fraction (10 mg of protein) was loaded onto an amylose resin column containing MBP or MBP-RB/PH (TT). The bound proteins were eluted by the addition of 0.2 M NaCl. Aliquots of the eluates were resolved by SDS-PAGE and subjected to immunoblot analysis using anti-Rho-kinase antibody. Lane 1, cytosol fraction (1 µl); lane 2, eluate from MBP column applied cytosol fraction (40 µl); lane 3, eluate from MBP-RB/PH (TT) column applied cytosol fraction (40 µl); lane 4, membrane extract fraction (1 µl); lane 5, eluate from MBP column applied membrane extract fraction (40 µl); lane 6, eluate from MBP-RB/PH (TT) column applied membrane extract fraction (40 µl).

To identify the minimal region in RB/PH fragment responsible for the inhibition, we examined whether various fragments within the RB/PH region inhibit the activity of Rho-kinase-CAT. Except RB/PH (TT), all fragments or combinations of fragments tested had no or very weak inhibitory effects on the activity of Rho-kinase-CAT as summarized in Fig. 4. These results suggest that the entire structure of RB/PH (TT) is necessary for the inhibition of the activity of Rho-kinase-CAT.


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  		Fig. 4.   Diagrams of various fragments within the RB/PH region. MBP-RB/PH (TT) (941-1388 amino acids with mutations; N1036T, K1037T), MBP-RB (941-1075 amino acids), MBP-PH (1125-1388 amino acids), GST-RB, GST-PH, and/or GST-coil gamma  (941-1131 amino acids) were added into the kinase reactions of Rho-kinase-CAT. Numbers indicate the amino acid residues. Functional and structural domains of Rho-kinase are shown schematically. +, indicates the fragment with inhibitory effect.

Effects of HA1077 and RB/PH (TT) on the Cytoskeletal Organization Induced by Rho-kinase-- We then investigated the specificity of RB/PH (TT) and HA1077 in vivo by the nuclear injection into NIH 3T3 cells of the cDNA encoding RB/PH (TT) with the cDNA encoding activated Rho (RhoVal-14), Rho-kinase-CAT, or MRCKbeta -CAT. As previously reported, the expression of RhoVal-14 (34), Rho-kinase-CAT (16), or MRCKbeta -CAT (33) resulted in the formation of stress fibers and focal adhesions in serum-starved NIH 3T3 cells (Fig. 5). In contrast, coexpression of RB/PH (TT) with RhoVal-14 or Rho-kinase-CAT inhibited the formation of stress fibers in more than 50% of cells expressing RhoVal-14 or Rho-kinase-CAT (Fig. 5). We examined the various ratios between RB/PH (TT) and Rho-kinase-CAT and confirmed that an RB/PH (TT) excess was required for inhibition of the Rho-kinase-CAT-induced stress fiber formation (data not shown). Most cells expressing both MRCKbeta -CAT and RB/PH (TT) showed stress fibers (Fig. 5), although the number or thickness of stress fibers decreased in a few cases. Coexpression of COIL (amino acids 421 to 701) of Rho-kinase did not affect the RhoVal-14, Rho-kinase-CAT-, or MRCKbeta -CAT-induced stress fiber formation (data not shown). Twenty-four h after the injection, treatment of the cells expressing RhoVal-14 with HA1077 for 20 min disrupted the actin stress fibers. Treatment of the cells expressing Rho-kinase-CAT with HA1077 also resulted in disruption of stress fibers, although the inhibitory effect by HA1077 on stress fibers induced by Rho-kinase-CAT was weaker than on those induced by RhoVal-14 at the same concentration. This may be explained by the notion that higher concentrations of HA1077 are required to inhibit overexpressed Rho-kinase-CAT efficiently. HA1077 did not affect the stress fibers induced by MRCKbeta -CAT.


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  		Fig. 5.   Effect of RB/PH (TT) and HA1077 on the stress fiber formation. A, pEF-BOS-HA-RhoVal-14 (a, d, and g), pEF-BOS-myc-Rho-kinase-CAT (b, e, and h), or pEF-BOS-myc-MRCKbeta -CAT (c, f, and i) (0.4 mg/ml) was injected into nuclei of serum-starved NIH 3T3 cells with pEF-BOS-myc vector (a-c, g-h) or with pEF-BOS-myc-Rho-kinase-RB/PH (TT) (d-f) (0.8 mg/ml). The cells were incubated for 24 h after injection in the serum-depleted medium, and the cells were fixed and doubly stained by phalloidin and anti-Myc or anti-HA polyclonal antibody. For g-i, the cells were treated with 100 µM HA1077 for 20 min before fixation. The arrows show the injected cells. Scale bar, 20 µm. B, the percentages of cells with stress fibers in Myc- or HA-positive cells are indicated. The cells which had more stress fibers than the adjacent uninjected cells were regarded as stress fiber positive cells. Data are mean ± S.E. of at least triplicate determinations.

The coexpression of RB/PH (TT) with RhoVal-14 or Rho-kinase-CAT also inhibited the formation of focal adhesions, which were visualized as vinculin accumulation at the tip of stress fibers with an arrowhead-like shape, but had no effect on focal adhesion formation induced by MRCKbeta -CAT (Fig. 6). The vinculin accumulation at the tips occasionally remained after the treatment of the cells expressing RhoVal-14 with HA1077 (Fig. 6), although the stress fiber structure was mostly disrupted under the same conditions. The vinculin accumulation at the tips in the cells expressing RhoVal-14 or Rho-kinase-CAT became less prominent after treatment with HA1077, while the staining pattern of vinculin in the cells expressing MRCKbeta -CAT did not change after treatment. These results indicate that RB/PH (TT) and HA1077 inhibit the activity of Rho-kinase in vivo but not that of MRCKbeta , as expected from the results of the in vitro experiments.


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  		Fig. 6.   Effect of RB/PH (TT) and HA1077 on the focal adhesion formation. A, pEF-BOS-HA-RhoVal-14 (a, d, and g), pEF-BOS-myc-Rho-kinase-CAT (b, e, and h), or pEF-BOS-myc-MRCKbeta -CAT (c, f, and i) (0.4 mg/ml) were injected into nuclei of serum-starved NIH 3T3 cells with pEF-BOS-myc vector (a-c, g-h) or with pEF-BOS-myc-Rho-kinase-RB/PH (TT) (d-f) (0.8 mg/ml). The cells were incubated for 24 h after injection in the serum-depleted medium, and the cells were fixed and doubly stained by anti-vinculin antibody and anti-Myc or anti-HA polyclonal antibody. For g-i, the cells were treated with 100 µM HA1077 for 20 min before fixation. The arrows show the injected cells. Scale bar, 20 µm. B, the percentage of cells with vinculin accumulation in Myc- or HA-positive cells is indicated. Data are mean ± S.E. of at least triplicate determinations.

Effects of HA1077 and RB/PH (TT) on the Cytoskeletal Organization Induced by Activated MLC-- The replacement of MLC by recombinant MLCT18D,S19D in myosin II partially activates the ATPase activity and forms myosin filaments in vitro (35-37). We have recently found that the expression of MLCT18D,S19D induces neurite retraction in N1E-115 neuroblastoma cells and stress fiber formation in NIH 3T3 cells (21). Since the contractility driven by myosin II activation is presumed to play a critical role in focal adhesion formation (38), we examined whether the expression of MLCT18D,S19D is sufficient for the formation of focal adhesion. The expression of MLCT18D,S19D, but not MLC, conferred not only stress fibers but also vinculin accumulation at the tip of stress fibers in NIH 3T3 cells (Fig. 7). We further examined the effect of RB/PH (TT) or HA1077 on the MLCT18D,S19D-induced stress fibers and vinculin accumulation at the tip. Stress fibers and vinculin accumulation at the tip remained in the cells coexpressing RB/PH (TT) with MLCT18D,S19D, although levels were slightly weakened (Fig. 7). HA1077 had a minimal effect on both stress fibers and vinculin accumulation at the tip. Thus, it is likely that RB/PH (TT) and HA1077 do not inhibit stress fiber and focal adhesion formation induced by active form of MLC.


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  		Fig. 7.   Effect of RB/PH (TT) and HA1077 on the stress fiber and focal adhesion formation in the cells expressing MLC or MLC mutant. A, pEF-BOS-myc-MLC (a and e) or pEF-BOS-myc-MLCT18D,S19D (b-d, f-h) (0.4 mg/ml) was injected into nuclei of NIH 3T3 cells with pEF-BOS-myc vector (a, b, d-f, and h) or with pEF-BOS-myc-Rho-kinase-RB/PH (TT) (c and g) (0.8 mg/ml). After injection, the cells were incubated for 24 h in the presence of 10% calf serum, and for another 24 h in serum-depleted medium. Then the cells were fixed and doubly stained by phalloidin (a-d) or anti-vinculin antibody (e-h) and anti-Myc polyclonal antibody. For d and h, the cells were treated with 100 µM HA1077 for 20 min before fixation. The arrows show the injected cells. Scale bar, 20 µm. B, percentage of cells with stress fibers (open bar) or vinculin accumulation (closed bar) in Myc-positive cells is indicated. Data are mean ± S.E. of at least triplicate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibitory Mechanism and Specificity of Chemical Compounds-- HA1077 and Y-32885 have been shown to inhibit the Rho-kinase activity (29). We here found that these compounds inhibited the PKN activity at similar doses as Rho-kinase, whereas they had a less inhibitory effect on the MRCKbeta activity. The IC50 values of HA1077 and Y-32885 for PKN-CAT were almost the same as those for Rho-kinase-CAT under the above conditions, although Ki values for PKN-CAT were higher than those for Rho-kinase-CAT depending on the Km values for ATP.3 These compounds affected the activity of not only Rho-kinase but also PKN at the same concentrations. Thus, it is unlikely that these compounds can distinguish Rho-kinase and PKN under the conditions used. HA1077 is an isoquinolinesulfonic acid derivative. A similar isoquinolinesulfonic acid derivative known as H-89 interacts with the ATP-binding site of protein kinase A through its isoquinoline ring and thereby inhibits the kinase activity (39). Y-32885 is a compound containing pyridine moiety. Since both HA1077 and Y-32885 inhibit the Rho-kinase activity in a manner competitive with ATP, they may interact with the ATP-binding site of the catalytic domain of Rho-kinase. The ATP-binding sites are well conserved among protein kinases (40). Thus, it is possible that these compounds interact with protein kinases other than Rho-kinase and PKN, and inhibit their kinase activities. One has to be careful when using these compounds as specific probes for Rho-kinase, although they are convenient and useful.

Inhibitory Mechanism and Specificity of RB/PH (TT)-- We here found that RB/PH (TT) inhibited the Rho-kinase activity in vitro, but not the activities of PKN and MRCKbeta . RB/PH (TT) inhibited the Rho-kinase activity in a manner competitive with S6 peptide. We also found that Rho-kinase bound to MBP-RB/PH (TT). Taken together, these results indicate that MBP-RB/PH (TT) interacts with the catalytic domain of Rho-kinase and competes with S6 peptide, and thereby inhibits the kinase activity. It should be noted that RB/PH (TT) inhibited the activity of Rho-kinase-CAT toward MLC in a noncompetitive-competitive mixed manner. Both RB/PH (TT) and MLC are thought to interact with the active center of Rho-kinase-CAT. Nevertheless, MLC does not efficiently compete with MBP-RB/PH (TT) under the conditions in which S6 peptide competes with MBP-RB/PH (TT). These results raise the possibility that MLC interacts with the catalytic domain of Rho-kinase with at least two sites, including the active center. MBP-RB/PH (TT) may mask the MLC-binding sites of the catalytic domain. Further studies are necessary for understanding how the catalytic domain recognizes its substrates.

Protein kinases such as protein kinase C are usually composed of catalytic and regulatory domains (41). The regulatory domain interacts with and masks the catalytic domain to inactivate the kinase activity in the resting state. Upon stimulation, the regulatory domain dissociates from the catalytic domain and subsequently induces the activation of the kinase. The active form of Rho interacts with the Rho-binding domain of Rho-kinase, and thereby activates it (6). Rho-kinase becomes constitutively active when the COOH portion containing the RB and PH domains is deleted (15-17). We here found that RB/PH (TT) interacts with Rho-kinase and inhibits the kinase activity of Rho-kinase-CAT. Thus, it is likely that the RB and PH domains interact with the catalytic domain and inactivate the activity in the resting state, and that the active form of Rho interacts with the RB domain, alters the conformation of Rho-kinase, and thereby cancels the inhibition by the RB and PH domains in response to extracellular signals such as lysophosphatidic acid.

We also found that both RB and PH domains are required to inhibit the activity of Rho-kinase-CAT. The regulatory domains of protein kinase such as protein kinase C usually contain a pseudosubstrate region that is thought to mimic substrates and interact with the active center of the catalytic domain. Consensus amino acid sequences for phosphorylation sites by Rho-kinase are (R/K)X0-2(S/T). There are several potential pseudosubstrate regions in RB and PH domains. We cannot explain at present why both RB and PH domains are required for the inhibition of the kinase activity. The regulatory subunit of protein kinase A has been shown to inhibit the protein kinase A activity by its interaction with the catalytic subunit through the pseudosubstrate region and an additional binding site (42, 43). Thus, it is possible that RB/PH (TT) interacts with the catalytic domain of Rho-kinase through at least two sites including the putative pseudosubstrate region.

RB/PH (TT) Inhibits the Rho-kinase Activity in Vivo-- We found that RB/PH (TT) inhibited both the RhoVal-14- and Rho-kinase-CAT-induced formation of stress fibers and focal adhesions in NIH 3T3 cells, but not those induced by MRCKbeta -CAT. We recently showed that RB/PH (TT) inhibits the serum-induced MLC phosphorylation in NIH 3T3 cells (21), the RhoVal-14-induced moesin phosphorylation as well as microvilli formation in COS7 cells (23), and the 12-O-tetradecanoylphorbol-13-acetate-induced adducin phosphorylation as well as membrane ruffling in Madin-Darby canine kidney cells (25). We also found that alpha -adducinT445A,T480A (phosphorylation sites of Thr by Rho-kinase are replaced by Ala), which is not phosphorylated by Rho-kinase, inhibits the 12-O-tetradecanoylphorbol-13-acetate-induced membrane ruffling in Madin-Darby canine kidney cells, and that alpha -adducinT445D,T480D (phosphorylation sites of Thr by Rho-kinase are replaced by Asp), which might mimic the phosphorylated alpha -adducin, counteracts the inhibitory effect of RB/PH (TT) on the 12-O-tetradecanoylphorbol-13-acetate-induced membrane ruffling. Taken together, these results indicate that RB/PH (TT) specifically inhibits the Rho-kinase activity in vivo, and that RB/PH (TT) is a useful probe to analyze the functions of Rho-kinase.

HA1077 disrupted the organized actin stress fibers in the cells expressing RhoVal-14 or Rho-kinase-CAT under the conditions in which HA1077 had little effects on the MRCKbeta -CAT-induced stress fibers. Disorganized actin filaments remained in most of the cells expressing RhoVal-14 but not in the cell expressing Rho-kinase-CAT after treatment with HA1077. Disorganized actin filaments were also observed in the cells expressing both RhoVal-14 and RB/PH (TT). These results suggest that HA1077 inhibits the Rho-kinase activity more efficiently than the MRCKbeta activity in vivo, and that actin polymerization is induced by activated Rho in a fashion independent of Rho-kinase. Although HA1077 disrupted most actin stress fibers, a small size of vinculin accumulation sometimes remained in the cells expressing RhoVal-14 or Rho-kinase-CAT. Because HA1077 was applied 24 h after the injection of plasmids to the cells, in which stress fibers and focal adhesions were formed before treatment with HA1077, inhibition of the Rho-kinase may result in the breakdown of vinculin accumulation at the tip.

Activated MLC Maintains Stress Fibers and Focal Adhesions Independent of Rho/Rho-kinase-- Burridge and co-worker (38) have proposed that the contractility driven by Rho plays a critical role in focal adhesion formation based on the observation that inhibition of contractility leads to inhibition of the Rho-induced stress fibers and focal adhesions. We here found that the expression of MLCT18D,S19D, but not of MLC, induced not only stress fiber formation but also focal adhesion formation in NIH 3T3 cells, and that the MLCT18D,S19D-induced focal adhesion was not inhibited by RB/PH (TT) or HA1077. MLCT18D,S19D shows ATPase activity and forms myosin filaments when reconstituted with myosin heavy chain in vitro (35-37). MLCT18D,S19D as well as phosphorylated MLC is preferentially incorporated into myosin fibers associated with actin stress fibers in fibroblasts (21, 44), suggesting that MLCT18D,S19D mimics the phosphorylated MLC. MLCT18D,S19D appears to exert the specific functions of Rho-kinase. Indeed, the expression of MLCT18D,S19D in COS7 cells or NIH 3T3 cells did not induce microvilli formation, which was induced by the expression of RhoVal-14 or Rho-kinase-CAT through the phosphorylation of moesin (23). On the other hand, the expression of moesinT558D induced microvilli formation but not stress fiber formation. Taken together, these results indicate that myosin II activation plays a critical role in focal adhesion formation downstream of Rho-kinase.

    ACKNOWLEDGEMENTS

We are grateful to A. Takemura for secretarial assistance. We also thank Dr. M. Ito (Mie University School of Medicine) for providing MLC, and Dr. M. Inagaki (Aichi Cancer Center Research Institute) for helpful discussions.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research and cancer research from the Ministry of Education, Science, and Culture of Japan, by the Research for the Future of Japan Society for the Promotion of Science, and by a grant from Kirin Brewery Company Limited.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.

§ To whom correspondence should be addressed: Div. of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan. Tel.: 81-743-72-5440; Fax: 81-743-72-5449; E-mail: kaibuchi@bs.aist-nara.ac.jp.

2 N. Nakamura, M. Fukata, Y. Fukata, N. Oshiro, M. Amano, S. Kuroda, T. Yano, M. Shibata, M. Ikebe, Y. Matsuura, K. Ookawa, A. Iwamatsu, and K. Kaibuchi, manuscript in preparation.

3 M. Amano, K. Chihara, N. Nakamura, T. Kaneko, Y. Matsuura, and K. Kaibuchi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PKN, protein kinase N; MLC, myosin light chain; PH, pleckstrin homology; CAT, catalytic domain; RB, Rho-binding domain; CAT-KD, kinase-deficient catalytic domain; GST, glutathione S-transferase; MRCKbeta , myotonic dystrophy kinase-related Cdc42-binding kinase beta ; MBP, maltose-binding protein; PAGE, polyacrylamide gel electophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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