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J. Biol. Chem., Vol. 277, Issue 1, 725-734, January 4, 2002

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Rho-dependent Agonist-induced Spatio-temporal Change in Myosin Phosphorylation in Smooth Muscle Cells^*

Koji Miyazaki, Takeo Yano^§, David J. Schmidt, Toshiya Tokui, Masao Shibata^§, Lawrence M. Lifshitz^¶, Satoshi Kimura, Richard A. Tuft^¶, and Mitsuo Ikebe^**

From the ^¶ Department of Physiology and Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, Massachusetts 01655, ^§ Medical and Biological Laboratories, Ina, Nagano 396, Japan and the  First Department of Internal Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Received for publication, September 6, 2001, and in revised form, October 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC₂₀) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC₂₀ phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC₂₀ in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC₂₀ phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Smooth muscle contraction is primarily activated by an increase in cytosolic Ca²⁺, which activates myosin light chain kinase (MLCK),¹ a Ca²⁺-calmodulin-dependent kinase that specifically phosphorylates myosin regulatory light chain (MLC₂₀), thus activating myosin motor function and muscle contraction. However, it has been realized that the smooth muscle contractile response is modulated by factors other than Ca²⁺. One such factor is the small G-protein Rho (1, 2). Takai and co-workers (3) originally reported that GTPS enhanced contraction of saponin-permeabilized smooth muscle at submaximal Ca²⁺ concentrations, but this effect diminished in the presence of exoenzyme C3, a Rho-specific inhibitor. Subsequently it was reported that Rho can decrease myosin phosphatase activity, which anticipates the increase in the level of myosin phosphorylation (4). The target proteins of Rho have been identified recently by several laboratories. Among these Rho targets, the Rho kinases and the myosin-binding subunit of myosin phosphatase (5) have been suggested to play an important role in the regulation of smooth muscle contraction. It was found in an in vitro system that Rho kinase can phosphorylate myosin phosphatase (specifically the myosin-binding subunit), which had the effect of decreasing myosin phosphatase activity (5). It was also suggested that Rho kinase can directly phosphorylate MLC₂₀ at serine 19 in vitro, thus activating actomyosin ATPase activity (6, 7).

The question addressed in the present study is how external stimuli initiated at the plasma membrane can activate the Rho-dependent pathway to increase the phosphorylation of myosin in the contractile domain of the cells. In epithelial cells it was demonstrated by immunocytochemistry (8) and by electron microscopy (9) that the active form of RhoA in transfected cells localizes at the plasma membrane or in submembranous cortical actin networks. Consistent with these findings, a translocation of RhoA to the particulate fraction during agonist stimulation was reported using cell fractionation methods in smooth muscle (10) and fibroblasts (11). Although these previous results suggest the recruitment of Rho to the plasma membrane by external stimuli, it remains obscure whether or not the translocation of Rho is kinetically coupled with changes in myosin phosphorylation and thus contraction. This issue is directly relevant to the question of how the Ca²⁺ pathway and Rho pathway differentially contribute to changes in myosin phosphorylation, because the change in cytosolic Ca²⁺ in smooth muscle cells is achieved within a few seconds after agonist stimulation. If the activation of the Rho pathway takes place at the plasma membrane, how the signal produced at the surface membrane can be transmitted to the contractile domain remains unknown. The study of living single cells, as an experimental model, is critical to answer these questions.

In the present study, we employed smooth muscle cultured cells that retain agonist-induced signaling systems linking external stimuli to changes in myosin phosphorylation. To visualize RhoA in living cells, we expressed GFP-tagged RhoA. We succeeded in visualizing the translocation of RhoA in a living smooth muscle cell under agonist stimulation at a single cell level with a three-dimensional time course digital imaging analysis. To clarify the role of the Rho pathway in smooth muscle contraction, the effects of Rho modulators on changes in myosin phosphorylation during agonist stimulation were monitored at a single cell level. The results clearly indicated that the Rho pathway and the MLCK pathway change myosin phosphorylation at different regions of the smooth muscle cells with different kinetics after agonist stimulation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Collagenase (purified from Clostridium histolyticum) and elastase were purchased from Worthington. Protease was purchased from Sigma. Culture media, except Ham's F-12 media (Sigma), and other cell culture supplements were purchased from Life Technologies, Inc. The mammalian expression vector pEGFP-C1 was purchased from CLONTECH Laboratories (Palo Alto, CA). [-³²P]GTP was obtained from Amersham Biosciences.

Antibodies-- Anti-GFP polyclonal antibody was purchased from MBL International (Watertown, MA). Anti-RhoA polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-RhoGDI monoclonal antibody was from BD Transduction Laboratories (San Diego, CA). Anti-MLC₂₀ IgM monoclonal antibody was from Sigma. We raised the monoclonal antibody, which specifically recognizes phospho-Ser-19 of the regulatory light chain of myosin (12), and the polyclonal antibody against the C-terminal peptide (CGGRRVIENADGGEEEIDGRDGDFN) of smooth muscle myosin heavy chain from chicken gizzard. The latter antibody recognized a single band of about 200 kDa in the whole lysate of porcine tracheal smooth muscle cells with Western blot (Fig. 7A). All secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA), except for the horseradish peroxidase-conjugated secondary antibodies for Western blotting (Bio-Rad).

Cell Culture-- COS-7 cells (American Type Culture Collection) and NRK52E cells (gift of Dr. Y.-L. Wang, University of Massachusetts Medical School) were cultured with Dulbecco's modified Eagle's medium and Ham's F-12 media, respectively, containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.).

Isolation of Porcine Tracheal Smooth Muscle Cells-- Porcine tracheae were obtained from adult pigs, and smooth muscle cell primary culture was performed with a modification of the method as described previously (13). Briefly, myocytes were enzymatically dispersed for 40 min at 37 °C in Hanks' balanced salt solution, containing 600 units/ml collagenase, 10 units/ml elastase, and 2 units/ml protease. Isolated cells were seeded in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 50 units/ml penicillin, and 50 µg/ml streptomycin (growth media). Cells from passage 1 or 2 were used in all the present studies. To induce a differentiated phenotype, cells were cultured in differentiation medium consisting of serum-free Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) medium with insulin/transferrin/selenium supplement (final concentrations: 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium), 0.1 mM nonessential amino acids, 50 units/ml penicillin, and 50 µg/ml streptomycin.

Construction of the Expression Vectors of GFP-tagged RhoA and Its Mutants-- The expression vector pEXV/Myc-tagged RhoAVal14 was the kind gift of Dr. Alan Hall (University College of London). We replaced the valine at position 14 with glycine to get a wild type RhoA. Myc-tagged RhoA cDNA was cloned into the EcoRI site of the pEGFP-C1 mammalian expression vector (CLONTECH). The resulting frameshift was fixed by cutting with HindIII at the poly-linker region of the vector and filling in with a Klenow fragment. The constitutively active mutant (Q63L) was generated by replacing glutamine for leucine in a position corresponding to glutamine 61 of Ras (14, 15). Threonine 19 was mutated to asparagine, thus generating a dominant negative mutant (T19N) (16). Site-directed mutagenesis was done as described previously (17).

Transfection-- NRK-52E and COS-7 cells (about 10⁶ cells/ml) were mixed with 10 µg of vector DNA and electroporated at 200 kV and 950 microfarads, using the GenePulser II (Bio-Rad). Porcine tracheal smooth muscle cells, cultured with growth media, were mixed with 10 µg of vector DNA, and electroporation was performed using the GenePulser II/RF Module System (Bio-Rad) (parameters: total voltage, 180 V; 100% modulation, burst duration 3 ms; rf, 40 kHz; 10 bursts, burst interval 1 s).

Western Blot Analysis of Expressed GFP-tagged Proteins-- The expressed GFP-tagged proteins were detected by Western blot analysis. COS-7 cells, transfected with pEGFP-C1/RhoA(wild type), pEGFP-C1/RhoA(Q63L), pEGFP-C1/RhoA(T19N), or pEGFP-C1 vector (control), were treated with ice-cold 5% trichloroacetic acid, followed by sonication. The trichloroacetic acid precipitates were dissolved in 5% SDS, 0.5 M NaHCO₃ buffer. These samples were then subjected to 7.5-20% gradient SDS-PAGE, and the proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was incubated with anti-GFP polyclonal antibody (MBL International, Watertown, MA), followed by horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. The immunoreactive bands were detected with enhanced chemiluminescence (Amersham Biosciences).

GTPase Activity of the Expressed GFP-tagged RhoA-- The GTPase activity of the GFP-tagged RhoA was determined as follows. COS-7 cells transfected with GFP-tagged RhoA were harvested and lysed with buffer A, consisting of 0.1 mg/ml tyrosine inhibitor type II (Sigma), 120 µg/ml N-p-tosyl-L-lysine chloromethyl ketone, 120 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10% glycerol, 1% Nonidet P-40, 30 mM Tris-HCl, pH 8.8, 1.25 mM EGTA, and 10 mM dithiothreitol. GFP-tagged RhoA was immunoprecipitated with anti-GFP antibody using Affi-Prep® protein A support (Bio-Rad), as described below. The immunoprecipitates were subjected to a GTPase assay as described previously (18).

Subcellular Fractionation of Porcine Tracheal Smooth Muscle Cells-- Subcellular fractionation was performed as described previously (11). Briefly, porcine tracheal smooth muscle cells under the conditions shown in each figure were prepared for the cell fractionation. After washing with cold PBS, the cells were scraped in 1 ml/dish of lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM MgCl₂, 2 mM EDTA, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 500 µM sodium orthovanadate, 10 mM sodium fluoride, and 1 mM dithiothreitol). The cells were lysed by 10 passes through a 26-gauge needle on ice. Trypan blue staining of lysate indicated more than 95% disruption of the plasma membrane. The lysate was centrifuged at 120,000 × g for 45 min to pellet the remainder of the particulate fraction. Each fraction was washed twice with the lysis buffer to remove cytosolic proteins. Each fraction was dissolved in the same volume of sampling buffer for SDS-PAGE, followed by immunoblotting.

Immunoprecipitation-- Samples were prepared in the same way as described above in subcellular fractionation. The samples were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants were preincubated with Affi-Prep® Protein A support (Bio-Rad) for 1 h on ice to prevent nonspecific binding of proteins in the immunoprecipitated complex. The precleared homogenates were incubated with anti-GFP polyclonal antibody (MBL International), conjugated to Affi-Prep® Protein A support overnight at 4 °C, and rotated. The beads were centrifuged and the supernatants collected and saved. The precipitates were washed five times in ice-cold washing buffer (50 mM Tris-HCl, pH 8.8, and 0.1 M KCl). The supernatants and the precipitates were subjected to the further analysis.

Immunofluorescence-- Cells were fixed with 4% formaldehyde for 10 min, followed by permeabilization with 0.1% Triton X-100 in PBS. After blocking with 3% bovine serum albumin/PBS at room temperature for 1 h, the preparations were incubated with primary antibody at 37 °C for 1 h. After washing with PBS for 3 times, they were incubated with fluorescence-labeled secondary antibodies at 37 °C for 1 h. After washing out excess antibodies, the samples were mounted in 3% 1,4-diazabicyclo[2.2.2]octane (Sigma), 90% glycerol in PBS.

Three-dimensional Digital Imaging Microscopy and Image Restoration-- For the imaging experiments with living cells, we used DIM-1 system as described below to minimize the expected photo damage to the living cells. Images of labeled living cells were acquired with a Nikon Diaphot 200 microscope equipped with a 100-watt Hg arc lamp for epifluorescence microscopy. Cells were viewed with 60 (NA 1.4) or 100× Nikon (NA 1.3) Planapo objectives with a 2.5 or 5× camera eyepiece, and images were projected onto the face of a Photometrics thermoelectrically cooled CCD camera. Digitized optical sections of labeled cells were generally obtained at 0.25-µm z axis intervals spanning the cell volume. This through-focus image series was transferred to Silicon Graphics workstations (Mountain View, CA) for analysis. 190 nm diameter fluorescent beads on separate slides were imaged under identical optical conditions to record fluorescent beads through-focus images, thus providing an empirical measure of the microscope point spread function.

For restorations, three-dimensional images were dark current-subtracted, flat field-corrected, background-subtracted, and normalized to constant integrated optical density to correct for non-uniformity in illumination intensity and camera sensitivity across the field of view. Prepared images were then processed with an iterative deconvolution algorithm with non-negativity constraints, by using the empirically determined point spread function for the microscope to at least partially reverse the blurring introduced by the optics, thus increasing the quantitative reliability of the data (19).

Leica-- The fixed cells (Figs. 7 and 8) were viewed using the Leica DM IRBE inverted microscope equipped with TCS SP2 confocal system, a 65-milliwatt argon laser, two HeNe lasers (1.2 and 10 milliwatts), and the interference contrast accessories (Leica Microsystems Inc., Heidelberg, Germany). TIFF images were acquired and analyzed with LCS software and Adobe® Photoshop® 5.5 software (Adobe Systems Inc., San Jose, CA).

Determination of the Relative Change in Myosin Phosphorylation Level in Single Smooth Muscle Cells-- To quantify phosphorylated MLC₂₀ levels during agonist stimulation (see Fig. 7), the cultured differentiated smooth muscle cells were stimulated by carbachol (20 µM) for an indicated time, and the reaction was stopped by washing once with ice-cold PBS with 1 mM CaCl₂, followed by addition of 4% formaldehyde for fixation. Cells were permeabilized and double-stained with anti-phospho-MLC₂₀ monoclonal IgG antibody (12), detected by indocarbocyanine (Cy3)-conjugated anti-mouse IgG secondary antibody, and anti-myosin heavy chain polyclonal antibody, detected by indodicarbocyanine (Cy5)-conjugated anti-rabbit IgG secondary antibody. Three-dimensional digital image series of transfected cells were captured with Leica TCS SP2 spectral confocal microscope as described above. The step size between the each confocal plane section was 0.5 µm. The plasma membrane of the cells was outlined manually on each plane section. The total fluorescence of both Cy3 (corresponds to phosphorylated MLC₂₀) and Cy5 (myosin heavy chain) in each cell was calculated as the sum of the fluorescence within the outline on each plane section. The relative level of myosin phosphorylation in each smooth muscle cell was determined as the ratio of the total fluorescence between Cy3 and Cy5. Five to ten transfected cells at each indicated time were analyzed and their values determined. Similar analysis was performed in three separate experiments.

Determination of MLC₂₀ Phosphorylation Level of Porcine Tracheal Smooth Muscle Cells by Western Blotting-- The extent of MLC₂₀ phosphorylation of smooth muscle cells during agonist stimulation was determined by SDS-PAGE, followed by Western blotting using anti-phospho-MLC₂₀ antibody. The cultured differentiated smooth muscle cells were stimulated by carbachol (20 µM), and the reaction was terminated by washing once with ice-cold PBS with 1 mM CaCl₂, followed by soaking in 1 ml of ice-cold 5% trichloroacetic acid. The samples were subjected to Western blots using either anti-phospho-MLC₂₀ IgG or anti-MLC₂₀ IgM monoclonal antibody.

Measurement of Cytosolic [Ca²⁺]-- To monitor [Ca²⁺] in the cytoplasm, tracheal smooth muscle cells were loaded with 1.7 µM fura-2 AM (Molecular Probes, Eugene, OR) for 40 min, and fluorescence was measured using a custom-built multiwavelength microfluorimeter as described previously (20). The time required for [Ca²⁺] to resume the resting level after carbachol stimulation was measured on each tracheal smooth muscle cell.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of GFP-tagged RhoA and Its Variants-- RhoA cDNA was linked to the 3'-end of GFP cDNA to produce C-terminal fusion protein. It is known that the C-terminal end of Rho is subjected to post-translational modification that is critical for Rho activity (21); therefore, GFP (enhanced GFP) was tagged at the N-terminal end of RhoA to avoid any possible functional disruption to RhoA (Fig. 1). To confirm the expression of GFP-tagged RhoA in cells, COS-7 cells were transfected with the GFP-RhoA expression vectors described in Fig. 1. After the transfection was confirmed with fluorescence microscopy, the cells were harvested, and the lysates were applied to SDS-PAGE followed by Western blotting. As shown in Fig. 2, the expressed GFP-tagged RhoA was detected as a single band showing a molecular mass of 48 kDa, the expected molecular mass of a chimeric protein composed of GFP (27 kDa) and RhoA (21 kDa). To investigate the effects of RhoA activation on myosin phosphorylation in smooth muscle cells, two mutant RhoAs tagged with GFP were also produced as follows: Q63L, expected to decrease the GTP hydrolysis rate (22); and T19N, expected to bind to guanine nucleotide exchange factors (GEFs) and inactivate endogenous Rho family homologues (16), thus producing active and dominant negative RhoA, respectively. As shown in Fig. 2, these mutants also showed the expected molecular mass of the chimeric proteins. No degradation products were observed with SDS-PAGE analysis indicating that all the GFP fluorescence signals in the cells reflect the localization of the intact GFP-RhoA molecules.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   The construction of GFP-tagged RhoA and its mutants. The figure shows the construction of GFP-tagged RhoA and its variants vectors (see "Experimental Procedures"). RhoA cDNA flanked with EcoRI sites was subcloned into the multicloning site of pEGFP-C1 vector, and the frameshift was fixed as described under "Experimental Procedures." Thr-19 and Gln-63 were substituted by Asn and Leu, respectively, by using site-directed mutagenesis strategy. MCS, multicloning site. CMV, cytomegalovirus.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Western blotting analysis of expressed GFP-tagged RhoA. The whole lysate of COS-7 cells, transfected with either pEGFP-C1/RhoA (wild type) (lane 1), pEGFP-C1/RhoA(Q63L mutant) (lane 2), pEGFP-C1/RhoA(T19N mutant) (lane 3), or pEGFP-C1 vector (lane 4), was subjected to immunoblotting as described under "Experimental Procedures" using anti-GFP polyclonal antibody.

Functional Authenticity of GFP-tagged RhoA and Its Variants-- To confirm that the GFP tagging does not disturb RhoA activity, the function of GFP-tagged RhoA was examined by two means. First, the GTPase activity of GFP tagged RhoA was tested. GFP-RhoA was expressed in COS-7 cells, and the expressed protein was extracted and isolated by immunoprecipitation using anti-GFP antibodies as described under "Experimental Procedures." The specific activity was calculated to be 1.01 nmol of P_i/min/mg, which is comparable with the GTPase activity of naturally isolated RhoA (23, 24), indicating the expressed GFP-tagged RhoA is a functional G-protein. The GTPase activities of the GFP-tagged RhoA mutants were 0.18 nmol of P_i/min/mg (for Q63L) and 10.7 nmol of P_i/min/mg (for T19N). The activities of T19N and Q63L GFP-RhoAs were an order of magnitude higher and lower, respectively, than that of wild type GFP-RhoA. The inhibition of the GTPase activity by Q63L mutation is consistent with the previous report (22) of non-GFP-tagged RhoA and other small GTP-binding proteins. The high GTPase activity of T19N, which has not been accurately studied, could imply a shorter life of the GTP bound form, thus the inactivated form dominates. This is consistent with the previous finding (16) that showed a decrease in the affinity of untagged T19N mutant for GTP but not for GDP. This mutant is also expected to show dominant negative activity, because it strongly binds to GEF, thus competing with wild type Rho for GEF binding (16). The results are thus consistent with earlier reports of non-GFP-tagged small G-proteins and indicate that GFP-RhoA(T19N) and GFP-RhoA(Q63L) biochemically function as the dominant negative and constitutively active forms of RhoA, respectively.

It has been known that activation of the Rho pathway induces stress fiber formation in cultured cells (25, 26). GFP-tagged RhoA and its constitutively active form, GFP-tagged RhoA(Q63L), both induced actin stress fiber formation in NRK52E cells, whereas the GFP-tagged dominant negative form of RhoA(T19N) did not induce but rather decreased stress fibers (not shown). The results demonstrate that GFP-tagged RhoA and its derivatives properly function in vivo with regard to stress fiber induction.

It has been shown that the GDP-bound form of wild type RhoA binds to RhoGDI that stabilizes its GDP-bound form, and this property constitutes an important part of RhoA function. Furthermore, the intracellular localization of RhoA is likely to be influenced by the binding to RhoGDI that is present in cytosol. Therefore, we examined the binding of GFP-tagged RhoA and its variants to RhoGDI. GFP-RhoA variants were expressed in COS-7 cells, and the binding of GFP-RhoAs with the endogenous RhoGDI was determined by co-immunoprecipitation. As shown in Fig. 3, RhoGDI co-immunoprecipitated with wild type GFP-RhoA indicating that GFP-tagged RhoA retains RhoGDI binding activity. On the other hand, the binding of GFP-RhoA(Q63L) to RhoGDI was significantly lower than that of the wild type. This is consistent with the notion that Q63L mutation stabilizes the GTP form and that RhoGDI binds preferentially the GDP form of RhoA (27). GFP-RhoA(T19N), while the GDP form is stabilized, failed to bind to RhoGDI in vivo. A similar result was reported recently with non-GFP-tagged RhoA(T19N), and it was concluded that the failure of RhoA(T19N) to bind RhoGDI is due to its binding to GEFs that results in the inhibition of endogenous RhoA because of the elimination of GEFs (28). The present result is consistent with the earlier result and suggests that GFP-RhoA(T19N) functions as a dominant negative construct.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Binding of GFP-RhoAs to RhoGDI. The cell lysate of COS-7 cells transfected with GFP-RhoA, either wild type (WT), constitutively active form (Q63L), or dominant negative form (T19N) was immunoprecipitated with anti-GFP polyclonal antibody. Samples were subjected to Western blot analysis using anti-RhoGDI monoclonal antibody. S, supernatant (after immunoprecipitation); IP, immunoprecipitate.

Intracellular Localization of GFP-tagged RhoA and Its Mutants in Cultured Smooth Muscle Cells-- The GFP-tagged RhoA and its mutants were expressed in cultured porcine tracheal smooth muscle cells to detect any difference in localization between constitutively active and inactive/dominant negative forms of RhoA in a differentiated smooth muscle cell. Halayko et al. (29) previously showed that primary cultured smooth muscle cells show a differentiated phenotype, based upon expression of smooth muscle marker proteins as well as contractility. We employed this culture system and confirmed the expression of smooth muscle-specific markers such as calponin, the high molecular weight form of caldesmon, the smooth muscle isoform of myosin heavy chain, -actin, and desmin in the primary cultured cells by Western blot analysis (not shown). Furthermore, the cultured tracheal cells retained agonist-dependent signaling systems, as carbachol triggered an increase in myosin phosphorylation (see Figs. 6-8). The localization of GFP-RhoA expressed in tracheal cells was examined using high resolution three-dimensional imaging system (see "Experimental Procedures"). The images in the following figures were representative slices chosen from each restored through-focus three-dimensional image series.

GFP-tagged wild type RhoA distribution was mostly diffuse in the cytosol, but some localized to the plasma membrane (Fig. 4a). On the other hand, GFP-RhoA(Q63L), a constitutively active form, showed a clear localization on the plasma membrane, in addition to the localization to reticular like structure (Fig. 4b). In contrast, GFP-RhoA(T19N), a dominant negative form, was completely diffused (Fig. 4c) like the localization of the GFP alone control (Fig. 4d). These observations were consistent among the transfected cells with different GFP-RhoA expression levels.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   The localization of GFP-tagged proteins in primary cultured porcine tracheal smooth muscle cells (differentiated phenotype). Transfected porcine tracheal smooth muscle cells were cultured in serum-starved differentiation media. Three-dimensional digital images of living cells were taken and restored as described under "Experimental Procedures." Each panel shows a representative slice of a three-dimensional image series. a, GFP-tagged RhoA wild type transfectant; b, Q63L active mutant; c, T19N-inactive mutant; and d, pEGFP-C1 vector as a control. The arrowheads in b indicate the plasma membrane localization of the active form of RhoA.

Translocation of GFP-RhoA in Living Porcine Tracheal Smooth Muscle Cells-- The results shown in Fig. 4 suggest that the localization of RhoA in tracheal cells changes based upon its activation state, because the inactive forms are completely diffuse whereas the constitutively active forms are on the surface membrane or an intracellular membrane structure. Therefore, it is reasonable to assume that RhoA translocates during its activation process. To verify this notion, we monitored the translocation of GFP-RhoA in tracheal cells after agonist stimulation. The best approach to follow such a translocation is to monitor translocation in a single living cell (see "Discussion"). We achieved this approach by monitoring GFP-tagged RhoA under the three-dimensional digital imaging microscope. As shown in Fig. 5, A and B, the localization of GFP-RhoA on the plasma membrane increased within a minute after the carbachol stimulation and reached to maximum around 5 min after stimulation. On the other hand, the signal intensity in the cytosol was not increased significantly. These results were consistent in repeated independent experiments. To ascertain that the translocation was not due to a GFP-tagging effect, the translocation of endogenous RhoA to the membrane was confirmed by biochemical experiments. The non-transfected tracheal differentiated cells were stimulated with carbachol and then the reaction was terminated at given times as described in Fig. 5C. The cell extract was fractionated and the content of RhoA in each fraction was determined by Western blot analysis using anti-RhoA antibodies (Fig. 5C). The amount of RhoA in the particulate fraction increased greatly after carbachol stimulation, although a predominant amount of RhoA was still cytosolic. The time required for the increase of endogenous RhoA in the particulate fraction was similar to that for the translocation of GFP-RhoA observed by imaging analysis (Fig. 5, A and B). The result showing the translocation of the endogenous RhoA was consistent with the carbachol-induced translocation of GFP-tagged RhoA (Fig. 5A), indicating that the translocation and localization of GFP-tagged RhoA in tracheal cells represent those of endogenous RhoA distribution. The addition of 200 µM GTPS to the -toxin-permeabilized cells induced more prominent localization in particulate fraction (Fig. 5C), although a significant amount of RhoA was in cytosolic fraction.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Translocation of GFP-tagged RhoA in a living porcine tracheal smooth muscle cell. A, GFP images of the living tracheal cells expressing GFP-tagged RhoA. Three-dimensional digital images of living cells taken at the indicated times after agonist stimulation (50 µM carbachol). Upper panels show the time course images of a GFP-RhoA (wild type) transfected cell, and lower panels show those of a GFP-RhoA(T19N)-transfected cell. Each panel shows the same z slice of the three-dimensional image series during the time course. Bar, 5 µm. B, after restoration of the three-dimensional time series, a quantitative analysis of the fluorescence distribution was performed. Average fluorescence as a function of distance from the plasma membrane was calculated. The plasma membrane was outlined manually (this is possible because most of the cytoplasm contains at least some GFP). Average fluorescence was calculated as the total fluorescence within a distance range (0-0.08 µm from plasma membrane, closed triangle; 0.24-0.32 µm from plasma membrane, closed square; >0.72 µm from plasma membrane, closed circle) divided by the cell volume (i.e. number of voxels) at that distance. C, Western blot analysis of the cell fractions of untransfected tracheal cells probed by anti-RhoA antibodies. C, cytosolic fraction; P, particulate fraction. Cultured differentiated smooth muscle cells were stimulated with carbachol, and the cell lysate was fractionated by centrifugation (see "Experimental Procedures"). On the right panel (as a positive control), cells were permeabilized by incubating with intracellular solution (42), containing 5000 units/ml of -toxin for 30 min at room temperature and stimulated with 200 µM GTPS for 30 min. Carbachol stimulation induced translocation of endogenous RhoA to the plasma membrane.

Effects of GFP-RhoA on the Phosphorylation Level of Myosin in Porcine Tracheal Smooth Muscle Cells-- The tracheal cells were challenged by carbachol, and changes in myosin phosphorylation were examined by Western blot using anti-phospho-MLC₂₀ antibody. The cells were treated with carbachol, and then the reaction was terminated at a given times as described. The samples were subjected to SDS-PAGE, and the phosphorylated regulatory light chains were detected by Western blot analysis. The phosphorylation level of myosin regulatory light chain increased after carbachol. The peak of myosin phosphorylation was within 1 min after stimulation, and the maximum was about 1.5-2.0 times higher than the resting level (Fig. 6). Diphosphorylation of the regulatory light chain of myosin in our sample was not detected in alkali-urea gel probed by anti-MLC₂₀ antibodies (not shown). The result indicates that the cultured smooth muscle cells used in this study retain the signaling system linking agonist stimulation to myosin phosphorylation.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Western blot of the phosphorylated myosin light chain at Ser-19 of tracheal cells after carbachol stimulation. Cultured porcine tracheal smooth muscle cells were stimulated with 20 µM carbachol (lane 1, resting; lane 2, for 10 s; lane 3, for 1 min; lane 4, for 5 min; lane 5, for 15 min) and stopped the reaction with ice-cold 5% trichloroacetic acid. Each sample was subjected to Western blot using either anti-phospho-MLC20 (upper) or anti-MLC20 (lower) antibody.

A question is whether or not myosin phosphorylation levels change upon expression of RhoA and its active or inactive variants. If such changes occur, then the next question is whether the kinetics of the changes in phosphorylation agrees with the RhoA translocation kinetics. To address these issues, we studied changes in myosin phosphorylation in tracheal cells expressing GFP-RhoA and its variants. The cells were fixed at various times after stimulation with carbachol and subjected to immunofluorescence staining. The relative phosphorylation level of myosin at Ser-19 of MLC₂₀ was determined by the ratio of the signal intensities detected by anti-phospho-MLC₂₀ antibody and anti-smooth muscle myosin heavy chain antibody. Fig. 7 demonstrates the time course of myosin phosphorylation in transfected smooth muscle cells (see "Experimental Procedures"). MLC₂₀ phosphorylation levels promptly increased after adding carbachol and almost reached a peak at 10 s and were sustained for more than 15 min in both control GFP-, GFP-RhoA(wild type)-, and GFP-RhoA(Q63L)-expressing cells. The time course of the initial increase in MLC₂₀ phosphorylation correlates with the increase in cytosolic [Ca²⁺] (Table I). In contrast, in the GFP-RhoA(T19N)-expressing cells, MLC₂₀ phosphorylation level promptly decreased after 10 s, and the sustained phosphorylation was significantly attenuated.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Myosin phosphorylation level of porcine tracheal smooth muscle cells stimulated by carbachol. A, Western blot of the smooth muscle lysate. The whole lysate of porcine tracheal smooth muscle cells (10 µg) was applied to the Western blot analysis detected by anti-smooth muscle myosin heavy chain antibody. B, time course of the change in myosin phosphorylation. Differentiated smooth muscle cells were transfected with GFP-RhoA wild type (closed circles), GFP-RhoA(Q63L) (open and closed squares), GFP-RhoA(T19N) (triangles), and GFP (open circles). The transfectants with GFP-RhoA(Q63L) were treated with (open squares) or without (closed squares) 10 µM Y-27632 for 30 min. The transfected cells were fixed immediately at each indicated time point after the stimulation of carbachol (20 µM) before co-staining with anti-phosphorylated MLC20 monoclonal antibody and anti-myosin heavy chain polyclonal antibody. The MLC20 phosphorylation level was presented as the ratio of the total fluorescent intensity of phosphorylated MLC20 and that of myosin heavy chain (see "Experimental Procedures"). Error bars represent S.E. *, p < 0.05 versus control. C, differential change in myosin phosphorylation between the cortical region (closed symbols) and the central region (open symbols) of the cells. The change in myosin phosphorylation level after the stimulation was analyzed in each region of the GFP-expressing (squares) and GFP-RhoA-expressing (circles) cells. The mean intensity per pixel of a square measuring 10 × 10 pixels in either cortical or central region of each cell was determined. The measurement was performed on 10 different places chosen randomly in each region of the cells, and their mean values were obtained as the intensity for each region of the cell. Eight transfected cells per each indicated time were selected, and their intensity values were determined. The intensity ratio of phospho-MLC20 (pLC) to myosin heavy chain (HC) was calculated in each region as a measure of the relative phosphorylation level. Similar results were obtained for three independent experiments. Error bars represent S.E.

Furthermore, Y-27632 (10 µM), a Rho kinase-specific inhibitor (30), completely abolished this prolonged myosin phosphorylation in the cells expressing GFP-RhoA-(Q63L)-active form, suggesting that the sustained increase in myosin phosphorylation involves Rho/Rho kinase pathway. It should be noted that the MLC₂₀ phosphorylation level in the presence of Y-27632 returned to the basal level at 60 s, when the cytosolic [Ca²⁺] returned to the basal level (Table I). On the other hand, the dominant negative form of RhoA(T19N) suppressed the sustained myosin phosphorylation but not the peak phosphorylation level at 10 s after stimulation. As shown in Table I, RhoA variants did not influence the transient [Ca²⁺] increase after carbachol stimulation; therefore, this effect of RhoA on MLC₂₀ phosphorylation is due to a Ca²⁺/calmodulin-independent pathway. The important issue is that the increase in phosphorylation by the RhoA cascade is not in the fast phase (within 10 s) but during the prolonged phase, when the translocation of RhoA takes place (Fig. 5).

The cells transfected with GFP-RhoA or GFP-RhoA(Q63L) retained a high level of myosin phosphorylation after the peak phosphorylation, whereas in the control cells (GFP-transfected cells) the phosphorylation level decreased over the prolonged time, although a significant amount is maintained (Fig. 7B). We found that for the GFP-RhoA-overexpressing cells, the phosphorylation level at the peripheral region over the prolonged time was higher than the 10 s after stimulation, whereas the phosphorylation at the central region was decreased over the prolonged phase (Fig. 7C). Similar results were also obtained for the GFP-RhoA(Q63L)-expressing cells (not shown). When the overall phosphorylation was monitored for these cells, the phosphorylation level was apparently less changed (Fig. 7B).

Spatio-temporal Localization of the Myosin Phosphorylation after Agonist Stimulation-- A critical question is whether or not the Rho pathway and the MLCK pathway change myosin phosphorylation differently in cells in terms of spatial and temporal manner. To answer this question, we observed the cellular localization of phosphorylated myosin in smooth muscle cells at various times after carbachol stimulation. As shown in Fig. 8, we clearly demonstrated that myosin phosphorylation is not even throughout the cell bodies during agonist stimulation. Challenged by carbachol, the myosin phosphorylation was immediately increased within 10 s both at the cortical and the central region of the cells. After 5 min and sometimes even after 15 min, myosin phosphorylation was still sustained at the cortical region of the smooth muscle cells, whereas it was markedly decreased at the central fibers (Fig. 8A). It should be noted that the length of the cells shortened after stimulation, including the contraction of the cells.

View larger version (54K): [in this window] [in a new window]   Fig. 8.   Distribution of the myosin phosphorylation in smooth muscle cells after carbachol stimulation. Porcine tracheal smooth muscle cells were grown on coverslips in the differentiation media and challenged by carbachol stimulation (20 µM). Each sample was fixed at the indicated time after the stimulation and was immunostained using anti-phosphorylated MLC20 monoclonal antibody, which is visualized by fluorescein isothiocyanate-labeled anti-mouse IgG secondary antibody (green), and anti-smooth muscle myosin heavy chain antibody, visualized by Cy3-conjugated anti-rabbit IgG secondary antibody (red). A shows smooth muscle cells without any inhibitors; B shows the cells pretreated with ML-9 (15 µM) for 15 min; and C shows the cells with Y-27632 (10 µM) for 15 min. Bar, 20 µm.

To clarify whether or not the signaling pathways responsible for the myosin phosphorylation are different between at the cortical and central area, we examined the effects of a MLCK-specific inhibitor, ML-9 and a Rho kinase-specific inhibitor, Y-27632 on the cellular distribution of myosin phosphorylation. As shown in Fig. 8B, ML-9 significantly diminished myosin phosphorylation at the central area both at 10 s and 1 min after the stimulation, whereas it did not diminish the phosphorylation at the cortical area. On the other hand, Y-27632 diminished the myosin phosphorylation at the cortical but not central region (Fig. 8C). The results clearly indicate that Rho/Rho kinase pathway increases myosin phosphorylation at the cortical region, whereas MLCK pathway is responsible for the phosphorylation at the central region. The phosphorylation of myosin at the cortical region was sustained whereas that at the inner space was rather transient, indicating that Rho pathway and MLCK pathway are responsible for a prolonged and transient increase in myosin phosphorylation, respectively, after agonist stimulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dynamics of Translocation of RhoA in a Smooth Muscle Cell-- One of the most important questions addressed in this paper is the temporal and spatial correlation of RhoA activation and myosin phosphorylation. To obtain conclusive evidence for the correlation between RhoA activation/translocation and myosin phosphorylation in differentiated smooth muscle cells, the analysis using a single living cell system with high temporal and spatial resolution is critical. We achieved this goal by employing GFP technology. We expressed fluorescently tagged RhoA variants having distinct intrinsic activity in cultured smooth muscle cells that retained a differentiated contractile phenotype, and we used these RhoA variants as probes for localization analysis as well as for functional analysis (effects on myosin phosphorylation). The technique of GFP-tagging for localization studies has a number of advantages. First, artifacts due to nonspecific antibody labeling are common problems in conventional immunostaining methods but can be avoided with the GFP-tagging approach. More importantly, GFP tagging makes it possible to follow any changes in localization thus allowing monitoring of the translocation kinetics in the same cells. This is a clear advantage over conventional immunostaining methods because they require cell fixation, making it difficult to evaluate the actual movement of molecules in cells. A potential problem of the GFP-tagging approach is that the tagged GFP might interfere with the proper function of the molecule. In the present study, the functional authenticity of GFP-RhoA was confirmed by the following criteria. 1) GFP-RhoA showed GTPase activity similar to non-tagged RhoA. 2) The mutations GFP-RhoA(Q63L) and GFP-RhoA(T19N) showed a marked decrease and increase in GTPase activity, respectively, as is observed with non-tagged molecules. 3) GFP-tagged RhoA(wild type) can bind to RhoGDI thus suggesting the presence of C-terminal geranylgeranylation critical for the membrane targeting of Rho. 4) The expression of GFP-RhoA induced stress fiber is known for non-tagged RhoA. 5) GFP-RhoA co-localized with endogenous RhoA was probed by an anti-RhoA antibody (not shown).

The subcellular translocation of RhoA has been demonstrated with cultured cell lines using a cell fractionation approach (11), which can only show the change in the amount of Rho in the cytosolic fraction versus a particulate fraction that is composed of various membrane components including plasma membrane, endoplasmic reticulum, and Golgi apparatus. Therefore, this methodology cannot reveal the specific nature of the translocation. In the present study, a three-dimensional digital imaging system with a high resolution CCD camera was utilized to clarify the detailed intracellular localization of GFP-RhoA in differentiated smooth muscle cells. The constitutively active form of GFP-tagged RhoA was found on the surface membrane, but the dominant negative form was not found there. The GFP signal was detected in a very thin layer in restored images, suggesting that it localizes to the membrane rather than a submembranous domain. It was reported previously that geranylgeranylation of the C terminus of RhoA is essential for its translocation, and this lipid tail could bind to the lipid bilayer (21, 31). The present result is consistent with this notion.

A significant amount of RhoA is present in cytosol even after stimulation. The cytosolic fraction of RhoA could be due to the presence of the GDP form even after stimulation, but the possible presence of the GTP form in cytosol cannot be eliminated. However, based upon the result showing that myosin near the plasma membrane, not the central portion of the cell, is phosphorylated by Rho/Rho kinase cascade, it is anticipated that even if the GTP form of RhoA is present in cytosol, such molecules are not directly related to the activation of Rho kinase and thus the phosphorylation of myosin.

The Relation between Translocation of RhoA and Myosin Phosphorylation-- One of the most important physiological roles of the Rho pathway in smooth muscle is the enhancement of agonist-induced contraction. It has been shown by using permeabilized smooth muscle tissue that GTPS, an activator of G-proteins, enhances contraction at a given [Ca²⁺], and this was shown to be due to an increase in myosin phosphorylation (32). Although the identity of the G-protein responsible for GTPS-induced activation of contraction was undetermined, evidence has accumulated indicating that the Rho pathway is involved in this process. In the present study, we succeeded in monitoring the translocation of RhoA in living cells and changes in myosin phosphorylation levels in individual single cells. The relative level of myosin light chain phosphorylation was estimated by the ratio of the total fluorescence of anti-phosphorylated MLC₂₀ signal to anti-myosin heavy chain signal.

Diphosphorylation of the regulatory light chain of myosin was not detected in our sample as measured by an alkali-urea gel probed with anti-MLC₂₀ antibodies, which can recognize diphosphorylation of MLC₂₀ (data not shown); therefore, the extent of phosphorylation as determined by the phospho-Ser-19-specific antibody reflects the amount of phosphorylated myosin molecules. The extent of myosin phosphorylation estimated by this technique in single cells promptly increased after carbachol stimulation and was sustained for more than 15 min with a slight decrease. The kinetics of changes in myosin phosphorylation detected with this method at a single cell level agrees well with that obtained by Western blot analysis of a large number of the cells (Fig. 6). The observed time course of the response to the agonist showed that tracheal cells retain agonist-coupled signaling systems to regulate myosin phosphorylation and that the method used is appropriate to follow changes in myosin phosphorylation at the single cell level.

By using this system, we examined the effects of RhoA on myosin phosphorylation after carbachol stimulation. Whereas myosin phosphorylation level was sustained for more than 15 min in control cells expressing only GFP, the dominant negative form of RhoA abolished the sustained myosin phosphorylation during prolonged phase. Preincubation with Y-27632 completely abolished this prolonged myosin phosphorylation even in the cells expressing the constitutively active form of RhoA. This sustained phosphorylation is consistent with the kinetics of GFP-RhoA translocation toward the plasma membrane, which occurred 30 s after stimulation (Fig. 5B). The expression of RhoA variants did not alter the transient [Ca²⁺] increase, suggesting that the MLC₂₀ phosphorylation during the prolonged phase is not due to Ca²⁺/calmodulin-dependent pathway. Consistently, the inhibition of Rho kinase, a target of RhoA by Y-27632, abolished the sustained phase of MLC₂₀ phosphorylation even in the cells expressing constitutively active form of RhoA.

One of the most important findings of the present study is that there are at least two distinct mechanisms of myosin phosphorylation in smooth muscle cells during agonist stimulation, which are distinguished both temporally and spatially. Our conclusion is that MLCK phosphorylates myosin at the inner space of the cell in the early phase and that Rho kinase induces myosin phosphorylation at the cortical area in the sustained manner. This view is supported by the following findings. 1) Although the myosin phosphorylation increases immediately after agonist stimulation at both cortical region and inner space, only the cortical region shows sustained phosphorylation. 2) MLCK-specific inhibitor abolished myosin phosphorylation at the inner space of the cells but not the cortical region. 3) On the other hand, Rho kinase-specific inhibitor diminished myosin phosphorylation at the cortical region but not the inner space of the cells. The result is consistent with the finding that agonist stimulation initiates the translocation of RhoA to the plasma membrane within a minute in the sustained manner (Fig. 5). Translocation of Rho kinase to particulate fraction has also been suggested (33). Therefore, the proposed scenario is that agonist stimulates RhoA translocation to membrane and the recruited Rho kinase at the plasma membrane plays a role in the maintenance of high phosphorylation level of myosin at the cortical region. This may be attributed to the down-regulation of myosin light chain phosphatase by Rho kinase (5) or direct phosphorylation of myosin by Rho kinase (6, 7), although we cannot exclude either possibility at this point. Because the translocation of RhoA to the membrane is sustained, the phosphorylation of myosin at the cortical region lasts for a prolonged time. On the other hand, Ca²⁺ diffuses throughout the cells and activates MLCK localized on actin (34, 35) throughout the cells, thus increasing myosin phosphorylation. The phosphorylated myosin at the inner space would be promptly dephosphorylated by myosin light chain phosphatase.

The previous reports (36-38) describe that the phosphorylation of myosin at the peripheral region is dependent on MLCK, and the phosphorylation of myosin in the stress fibers at the central region is mediated by Rho kinase in fibroblasts. We think that there are several possibilities to account for this apparent discrepancy between the previous reports and the present study. First, the amount and distribution of myosin is quite different between these cell types. In differentiated smooth muscle cells, myosin is abundantly expressed and present volume fillingly throughout the cells. On the other hand, for fibroblasts, myosin is concentrated at the cortical region and the stress fibers. Smooth muscle MLCK is associated with actin filaments that are adjacent to myosin filaments and phosphorylates myosin upon activation by Ca²⁺, thus increasing myosin phosphorylation throughout the cells (39, 40). It is known that non-muscle MLCK shows more discrete localization that is different from smooth muscle MLCK (36, 41), and it is shown that non-muscle MLCK localized at the cortical region of the actin structures in fibroblasts.

Second, differentiated smooth muscle cells are cylindrically shaped, and Fig. 8 shows the distribution of the phosphorylated myosin at the center of the cells in terms of z axis, where the Rho/Rho kinase pathway-induced phosphorylation was found near the peripheral region of the cells. On the other hand, fibroblasts have flat morphology, and the stress fibers are present near the bottom of the cells, thus close to the basolateral cell membrane where active Rho/Rho kinase is present. Therefore, it is likely that the myosin in the stress fibers can be phosphorylated by Rho/Rho kinase system that is localized near the plasma membrane.

In conclusion, we visualized the translocation of RhoA to the plasma membrane during agonist stimulation in single living differentiated smooth muscle cells. The Rho-dependent pathway is responsible for the myosin phosphorylation at the cortical region of the cells, whereas MLCK pathway increases myosin phosphorylation at the inner space of the cells. The Rho/Rho kinase pathway plays an important role in the increase in myosin phosphorylation during the tonic phase of smooth muscle contraction, which is due to the increase in myosin phosphorylation at the cortical region of the cells.

	ACKNOWLEDGEMENTS

We thank Dr. Alan Hall (University College of London, London, UK) for pEXV/Myc-tagged RhoAVal14 vector and Dr. M. Uehata and A. Yoshimura (Yoshitomi Pharmaceutical Industries, Japan) for Y-27632. We also thank Dr. Andrew J. Halayko (The University of Chicago, Chicago, IL) for his valuable suggestions.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL60831 and HL61426.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.

Recipient of an American Heart Association postdoctoral fellowship (New England Affiliate).

Present address: Department of Thoracic Surgery, Mie University, School of Medicine, Tsu, Mie 514, Japan

^** To whom correspondence should be addressed: Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655. Tel.: 508-856-1954; Fax: 508-856-4600; E-mail: Mitsuo.Ikebe@umassmed.edu.

Published, JBC Papers in Press, October 22, 2001, DOI 10.1074/jbc.M108568200

	ABBREVIATIONS

The abbreviations used are: MLCK, myosin light chain kinase; GFP, green fluorescent protein; GTPS, guanosine 5'-(3-O-thio) triphosphate; G-protein, GTP-binding protein; Rho kinase, Rho-associated serine/threonine kinase; MLC₂₀, myosin regulatory light chain of 20 kDa; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; PBS, phosphate-buffered saline; Cy3, indocarbocyanine; Cy5, indodicarbocyanine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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