FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023410176> ?p ?o ?g. }

• W2023410176 endingPage "734" @default.
• W2023410176 startingPage "725" @default.
• W2023410176 abstract "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. 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. myosin light chain kinase green fluorescent protein guanosine 5′-(3-O-thio) triphosphate GTP-binding protein Rho-associated serine/threonine kinase myosin regulatory light chain of 20 kDa guanine nucleotide dissociation inhibitor guanine nucleotide exchange factor phosphate-buffered saline indocarbocyanine indodicarbocyanine 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 (1Somlyo A.P. Somlyo A.V. Acta Physiol. Scand. 1998; 164: 437-448Crossref PubMed Scopus (132) Google Scholar, 2Somlyo A.P. Wu X. Walker L.A. Somlyo A.V. Rev. Physiol. Biochem. Pharmacol. 1999; 134: 201-234PubMed Google Scholar). Takai and co-workers (3Hirata K. Kikuchi A. Sasaki T. Kurada S. Kaibuchi K. Matsuura Y. Seki H. Saida K. Takai Y. J. Biol. Chem. 1992; 267: 8719-8722Abstract Full Text PDF PubMed Google Scholar) originally reported that GTPγS 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 (4Noda M. Yasuda-Fukazawa C. Moriishi K. Kato T. Okuda T. Kurokawa K. Takuwa Y. FEBS Lett. 1995; 367: 246-250Crossref PubMed Scopus (171) Google Scholar). 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 (5Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2433) Google Scholar) 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 (5Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2433) Google Scholar). It was also suggested that Rho kinase can directly phosphorylate MLC20 at serine 19 in vitro, thus activating actomyosin ATPase activity (6Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar, 7Feng J. Ito M. Kureishi Y. Ichikawa K. Amano M. Isaka N. Okawa K. Iwamatsu A. Kaibuchi K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 3744-3752Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). 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 (8Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar) and by electron microscopy (9Robertson D. Paterson H.F. Adamson P. Hall A. Monaghan P. J. Histochem. Cytochem. 1995; 43: 471-480Crossref PubMed Scopus (67) Google Scholar) 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 (10Gong M.C. Fujihara H. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1997; 272: 10704-10709Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and fibroblasts (11Fleming I.N. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 33067-33073Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). 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. 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). [γ-32P]GTP was obtained from Amersham Biosciences. 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 (12Komatsu S. Yano T. Shibata M. Tuft R.A. Ikebe M. J. Biol. Chem. 2000; 275: 34512-34520Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), 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). 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.). Porcine tracheae were obtained from adult pigs, and smooth muscle cell primary culture was performed with a modification of the method as described previously (13Halayko A.J. Salari H. Ma X. Stephens N.L. Am. J. Physiol. 1996; 270: L1040-L1051Crossref PubMed Google Scholar). 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. 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 (14Der C.J. Finkel T. Cooper G.M. Cell. 1986; 44: 167-176Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 15Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 2472-2478Crossref PubMed Scopus (155) Google Scholar). Threonine 19 was mutated to asparagine, thus generating a dominant negative mutant (T19N) (16Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). Site-directed mutagenesis was done as described previously (17Yano K. Araki Y. Hales S.J. Tanaka M. Ikebe M. Biochemistry. 1993; 32: 12054-12061Crossref PubMed Scopus (34) Google Scholar). NRK-52E and COS-7 cells (about 106cells/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). 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). 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 Nα-p-tosyl-l-lysine chloromethyl ketone, 120 μg/mlN-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 (18Kikuchi A. Yamashita T. Kawata M. Yamamoto K. Ikeda K. Tanimoto T. Takai Y. J. Biol. Chem. 1988; 263: 2897-2904Abstract Full Text PDF PubMed Google Scholar). Subcellular fractionation was performed as described previously (11Fleming I.N. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 33067-33073Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). 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 mmphenylmethylsulfonyl fluoride, 500 μm sodium orthovanadate, 10 mm sodium fluoride, and 1 mmdithiothreitol). 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. 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. 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. 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-μmz 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 (19Carrington W.A. Fogarty K.E. Fay F.S. Foster K. Grinstein S. Non-invasive Techniques in Cell Biology. Wiley-Liss, Inc., New York1990: 53-72Google Scholar). 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). 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 mmCaCl2, followed by addition of 4% formaldehyde for fixation. Cells were permeabilized and double-stained with anti-phospho-MLC20 monoclonal IgG antibody (12Komatsu S. Yano T. Shibata M. Tuft R.A. Ikebe M. J. Biol. Chem. 2000; 275: 34512-34520Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), 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. 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. 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 (20Drummond R.M. Tuft R.A. J. Physiol. (Lond.). 1999; 516: 139-147Crossref Scopus (102) Google Scholar). The time required for [Ca2+] to resume the resting level after carbachol stimulation was measured on each tracheal smooth muscle cell. 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 (21Narumiya S. Morii N. Cell. Signal. 1993; 5: 9-19Crossref PubMed Scopus (50) Google Scholar); 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 (22Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2684) Google Scholar); and T19N, expected to bind to guanine nucleotide exchange factors (GEFs) and inactivate endogenous Rho family homologues (16Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar), 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.Figure 2Western 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 Pi/min/mg, which is comparable with the GTPase activity of naturally isolated RhoA (23Anderson P.S. Lacal J.C. Mol. Cell. Biol. 1987; 7: 3620-3628Crossref PubMed Scopus (21) Google Scholar,24Hoshijima M. Kondo J. Kikuchi A. Yamamoto K. Takai Y. Mol. Brain Res. 1990; 7: 9-16Crossref PubMed Scopus (23) Google Scholar), 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 Pi/min/mg (for Q63L) and 10.7 nmol of Pi/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 (22Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2684) Google Scholar) 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 (16Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar) 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 (16Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). 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 (25Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar, 26Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). 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 (27Sasaki T. Kato M. Takai Y. J. Biol. Chem. 1993; 268: 23959-23963Abstract Full Text PDF PubMed Google Scholar). 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 (28Strass" @default.
• W2023410176 created "2016-06-24" @default.
• W2023410176 creator A5008707497 @default.
• W2023410176 creator A5027440746 @default.
• W2023410176 creator A5034677413 @default.
• W2023410176 creator A5046858181 @default.
• W2023410176 creator A5056761543 @default.
• W2023410176 creator A5056974250 @default.
• W2023410176 creator A5062493641 @default.
• W2023410176 creator A5063052973 @default.
• W2023410176 creator A5075683543 @default.
• W2023410176 date "2002-01-01" @default.
• W2023410176 modified "2023-09-27" @default.
• W2023410176 title "Rho-dependent Agonist-induced Spatio-temporal Change in Myosin Phosphorylation in Smooth Muscle Cells" @default.
• W2023410176 cites W1485670014 @default.
• W2023410176 cites W1514066364 @default.
• W2023410176 cites W1561770236 @default.
• W2023410176 cites W1581768673 @default.
• W2023410176 cites W1597787921 @default.
• W2023410176 cites W1801147305 @default.
• W2023410176 cites W1966210349 @default.
• W2023410176 cites W1977598234 @default.
• W2023410176 cites W1978039310 @default.
• W2023410176 cites W1991489939 @default.
• W2023410176 cites W1997087774 @default.
• W2023410176 cites W2003268498 @default.
• W2023410176 cites W2021453901 @default.
• W2023410176 cites W2032045788 @default.
• W2023410176 cites W2032295238 @default.
• W2023410176 cites W2038028591 @default.
• W2023410176 cites W2045033550 @default.
• W2023410176 cites W2054978371 @default.
• W2023410176 cites W2062129029 @default.
• W2023410176 cites W2083822658 @default.
• W2023410176 cites W2085947939 @default.
• W2023410176 cites W2089741493 @default.
• W2023410176 cites W2096791952 @default.
• W2023410176 cites W2101496796 @default.
• W2023410176 cites W2105338223 @default.
• W2023410176 cites W2112832183 @default.
• W2023410176 cites W2127279692 @default.
• W2023410176 cites W2137528739 @default.
• W2023410176 cites W2140619884 @default.
• W2023410176 cites W2142083272 @default.
• W2023410176 cites W2155369806 @default.
• W2023410176 cites W2158253639 @default.
• W2023410176 cites W2169881041 @default.
• W2023410176 cites W4231194613 @default.
• W2023410176 cites W4247855969 @default.
• W2023410176 cites W4249820641 @default.
• W2023410176 cites W4250102918 @default.
• W2023410176 doi "https://doi.org/10.1074/jbc.m108568200" @default.
• W2023410176 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11673466" @default.
• W2023410176 hasPublicationYear "2002" @default.
• W2023410176 type Work @default.
• W2023410176 sameAs 2023410176 @default.
• W2023410176 citedByCount "49" @default.
• W2023410176 countsByYear W20234101762012 @default.
• W2023410176 countsByYear W20234101762015 @default.
• W2023410176 countsByYear W20234101762017 @default.
• W2023410176 countsByYear W20234101762018 @default.
• W2023410176 countsByYear W20234101762021 @default.
• W2023410176 crossrefType "journal-article" @default.
• W2023410176 hasAuthorship W2023410176A5008707497 @default.
• W2023410176 hasAuthorship W2023410176A5027440746 @default.
• W2023410176 hasAuthorship W2023410176A5034677413 @default.
• W2023410176 hasAuthorship W2023410176A5046858181 @default.
• W2023410176 hasAuthorship W2023410176A5056761543 @default.
• W2023410176 hasAuthorship W2023410176A5056974250 @default.
• W2023410176 hasAuthorship W2023410176A5062493641 @default.
• W2023410176 hasAuthorship W2023410176A5063052973 @default.
• W2023410176 hasAuthorship W2023410176A5075683543 @default.
• W2023410176 hasBestOaLocation W20234101761 @default.
• W2023410176 hasConcept C11960822 @default.
• W2023410176 hasConcept C12554922 @default.
• W2023410176 hasConcept C134018914 @default.
• W2023410176 hasConcept C170493617 @default.
• W2023410176 hasConcept C185592680 @default.
• W2023410176 hasConcept C2778938600 @default.
• W2023410176 hasConcept C2992686903 @default.
• W2023410176 hasConcept C55493867 @default.
• W2023410176 hasConcept C6997183 @default.
• W2023410176 hasConcept C8035138 @default.
• W2023410176 hasConcept C86803240 @default.
• W2023410176 hasConcept C95444343 @default.
• W2023410176 hasConceptScore W2023410176C11960822 @default.
• W2023410176 hasConceptScore W2023410176C12554922 @default.
• W2023410176 hasConceptScore W2023410176C134018914 @default.
• W2023410176 hasConceptScore W2023410176C170493617 @default.
• W2023410176 hasConceptScore W2023410176C185592680 @default.
• W2023410176 hasConceptScore W2023410176C2778938600 @default.
• W2023410176 hasConceptScore W2023410176C2992686903 @default.
• W2023410176 hasConceptScore W2023410176C55493867 @default.
• W2023410176 hasConceptScore W2023410176C6997183 @default.
• W2023410176 hasConceptScore W2023410176C8035138 @default.
• W2023410176 hasConceptScore W2023410176C86803240 @default.
• W2023410176 hasConceptScore W2023410176C95444343 @default.
• W2023410176 hasIssue "1" @default.

• next