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Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

2003; Elsevier BV; Volume: 279; Issue: 1 Linguagem: Inglês

10.1074/jbc.m308496200

ISSN

1083-351X

Autores

Qi Huang, Steven A. Fisher, Frank V. Brozovich,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3′-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p < 0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation. Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3′-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p < 0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation. Force regulation in smooth muscle is dependent on the activities of myosin light chain (MLC) 1The abbreviations used are: MLC, myosin light chain; P1-MLC20, monophosphorylated MLC20; P2-MLC20, diphosphorylated MLC20; PKG, cGMP-dependent protein kinase; PKGI, type I cGMP-dependent protein kinase; MBS, myosin-binding subunit; PP1cδ, protein phosphatase-1 catalytic subunit-δ; LZ, leucine zipper; SMC, smooth muscle cell; Br, bromo; CI, central insert; MYPT1, myosin-targeting subunit. 1The abbreviations used are: MLC, myosin light chain; P1-MLC20, monophosphorylated MLC20; P2-MLC20, diphosphorylated MLC20; PKG, cGMP-dependent protein kinase; PKGI, type I cGMP-dependent protein kinase; MBS, myosin-binding subunit; PP1cδ, protein phosphatase-1 catalytic subunit-δ; LZ, leucine zipper; SMC, smooth muscle cell; Br, bromo; CI, central insert; MYPT1, myosin-targeting subunit. kinase and MLC phosphatase (3.Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, Ltd., New York1987: 432-482Google Scholar, 4.Gong M.C. Cohen P. Kitazawa T. Ikebe M. Masuo M. Somlyo A.P. Somlyo A.V. J. Biol. Chem. 1992; 267: 14662-14668Abstract Full Text PDF PubMed Google Scholar). The activity of MLC kinase is regulated by Ca2+-calmodulin (3.Hartshorne D.J. Johnson L.R. Physiology of the Gastrointestinal Tract. Raven Press, Ltd., New York1987: 432-482Google Scholar), whereas MLC phosphatase was originally thought to be constitutively active and unregulated (5.Hartshorne D.J. Ito M. Erdîdi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (340) Google Scholar). However, there is abundant evidence that the activity of MLC phosphatase can be both inhibited to produce Ca2+ sensitization (reviewed in Refs. 5.Hartshorne D.J. Ito M. Erdîdi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (340) Google Scholar, 6.Somlyo A.P. Wu X. Walker L.A. Somlyo A.V. Rev. Physiol. Biochem. Pharmacol. 1999; 134: 201-234PubMed Google Scholar, 7.Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1715) Google Scholar) or an increase in force at a constant [Ca2+] and enhanced to produce Ca2+ desensitization (8.Lee M.R. Li L. Kitazawa T. J. Biol. Chem. 1997; 272: 5063-5068Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) or a decrease in force at a constant [Ca2+]. NO is the classical agent to produce Ca2+ desensitization (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar, 9.Lincoln T.M. Dey N. Sellak H. J. Appl. Physiol. 2001; 91: 1421-1430Crossref PubMed Scopus (409) Google Scholar, 10.Furchgott R.F. Zawadzki J.V. Nature. 1980; 288: 373-376Crossref PubMed Scopus (9872) Google Scholar). Recent evidence (11.Lincoln T.M. Pharmacol. Ther. 1989; 41: 479-502Crossref PubMed Scopus (236) Google Scholar) suggests that NO produces vasodilatation by activating the soluble pool of guanylate cyclase, which in turn produces cGMP and leads to the activation of type I cGMP-dependent protein kinase (PKGI). PKGI mediates smooth muscle cell relaxation by several mechanisms. It has been demonstrated that PKGI acts on the maxi K+ channel to produce hyperpolarization of the smooth muscle (12.Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), decreases Ca2+ flux (13.Schmidt H.H. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (743) Google Scholar, 14.Fukao M. Mason H.S. Britton F.C. Kenyon J.L. Horowitz B. Keef K.D. J. Biol. Chem. 1999; 274: 10927-10935Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), and also activates MLC phosphatase (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar, 15.Etter E.F. Eto M. Wardle R.L. Brautigan D.L. Murphy R.A. J. Biol. Chem. 2001; 276: 34681-34685Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) to decrease the level of MLC20 phosphorylation and to produce smooth muscle relaxation. In addition, PKGI-dependent pathways for vasodilatation may also include phosphorylation of telokin (16.Walker L.A. Macdonald J.A. Liu X.P. Nakamoto R.K. Haystead T.A.J. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 2001; 276: 24519-24524Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 17.Wu X. Haystead T.A. Nakamoto R.K. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1998; 273: 11362-11369Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and HSP20 (18.Rembold C.M. Foster D.B. Strauss J.D. Wingard C.J. Van Eyk J.E. J. Physiol. (Lond.). 2000; 524: 865-878Crossref Scopus (122) Google Scholar). MLC phosphatase is a holoenzyme consisting of a catalytic subunit (PP1cδ); a myosin-binding subunit (MBS), which is also referred to as the myosin-targeting subunit (MYPT1); and a 20-kDa subunit of unknown function (5.Hartshorne D.J. Ito M. Erdîdi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (340) Google Scholar). The activation of MLC phosphatase by PKGI is hypothesized to be due to a leucine zipper-leucine zipper (LZ-LZ) interaction of the N-terminal LZ of PKGIα and the C-terminal LZ of the MBS of MLC phosphatase (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar, 2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The MBS has four major isoforms, which are produced by alternative RNA splicing of two different exons (5.Hartshorne D.J. Ito M. Erdîdi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (340) Google Scholar). Tissue-specific and developmentally regulated alternative splicing of a 123-bp central exon produces a 41-amino acid central insert (19.Dirksen W.P. Vladic F. Fisher S.A. Am. J. Physiol. 2000; 278: C589-C600Crossref PubMed Google Scholar). Alternative splicing of the 31-bp 3′-exon is responsible for the expression of LZ+ or LZ- MBS isoforms (5.Hartshorne D.J. Ito M. Erdîdi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (340) Google Scholar). Specifically, exclusion of the 3′-exon shifts the reading frame of the MBS transcript to encode a C-terminal LZ (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). We previously demonstrated that sensitivity to cGMP-mediated relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), which is consistent with the activation of MLC phosphatase activity resulting from a LZ-LZ interaction of PKGIα with the MBS (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar). In this study, we tested the hypothesis that cGMP-dependent activation of MLC phosphatase activity and smooth muscle vasodilatation are due to a LZ-LZ interaction of PKGIα and MBS by changing the expression of the MBS isoform, in isolation, and determining the effect on cGMP-mediated MLC20 dephosphorylation in primary cultured smooth muscle cells (SMCs). Cloning of the Chicken MBS of the MLC Phosphatase cDNA Fragment—Total RNA was isolated from fresh chicken gizzard and urinary bladder smooth muscle tissues using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Full-length cDNA encoding the MBS was synthesized by reverse transcription-PCR using the total RNA as template. We used primers 5′-GCGGCGATAGCGAGGGGGTCAG-3′ and 5′-CAGGTAAGAGGGCATTTGGCAGGATA-3′, flanking the MBS cDNA sequence between bp +86 and +3382 (20.Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar). The PCR products were cloned into the pCR2.1 plasmid vector (Invitrogen). cDNAs corresponding to four alternative splicing variants of the chicken smooth muscle MBS, differing in the inclusion or exclusion of a central exon and a 3′-exon, were screened (see Fig. 1) and confirmed by DNA sequencing. Note that the sequence at base 1822 is C instead of G, which encodes position 568; the GTT (valine) reported by Shimizu et al. (20.Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar) is CTT (leucine) in eight independent clones obtained by reverse transcription-PCR. Construction of Recombinant Adenoviruses Encoding MBS Isoforms—Recombinant adenoviruses were generated using the AdEasy system (21.He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3221) Google Scholar). The four MBS cDNAs were individually cloned into transfer vector pAdTrack-CMV (9.2 kb, kanamycin-resistant) downstream of a cytomegalovirus promoter. This vector has a second cytomegalovirus promoter expressing green florescent protein. Escherichia coli BJ5183 cells were first transformed with supercoiled adenoviral backbone DNA pAdEasy-1 (33.4 kb, ampicillin-resistant). The transformants were confirmed by restriction enzyme mapping and then prepared in competent cells by an acid salt method (22.Alexander D.C. Methods Enzymol. 1987; 154: 41-64Crossref PubMed Scopus (33) Google Scholar). For in vivo homologous recombination in the bacterial cell, ∼0.5-1 μg of linearized MBS-pAdTrack (12.5 kb) recombinant plasmid DNA was transformed into the above competent E. coli cells, followed by plating on LB agar medium containing 50 μg/ml kanamycin. The MBS-recombinant adenovirus plasmids were retransformed into a recA- strain of E. coli (JM109) for large-scale DNA preparation and verified by restriction enzyme mapping and PCR using two pairs of specific MBS primers: 5′-1620/3′-2069 (flanking the central alternative exon) and 5′-2888/3′-3203 (flanking the 3′-end alternative exon), respectively (see Fig. 1). To produce the MBS-recombinant adenovirus, 293 mammalian packaging cells (Quantum Biotechnology) were grown to ∼70% confluence in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 100 μg/ml penicillin, and 50 μg/ml streptomycin. 5 μg of each MBS-recombinant adenovirus plasmid DNA was linearized by the restriction enzyme PacI and used for transfection of 293 cells with LipofectAMINE™ 2000 reagent (Invitrogen) according to the manufacturer's protocol. The amplified MBS-recombinant viruses and their protein products in 293 cells were verified by PCR as described above and by Western blotting using anti-MYPT1 antibody (Upstate Biotechnology, Inc.), respectively (Fig. 1). After verification, a few rounds of amplification were performed in 293 cells to produce high titer viral stocks. All four MBS-recombinant adenoviruses and an adenovirus vector control were titrated by plaque forming units in 293 cell cultures. Preparation of Chicken Smooth Muscle Cells—Primary cultures of chicken gizzard SMCs were isolated from day 15 embryos using a modification of a previously described method (23.Huang Q.Q. Fisher S.A. Brozovich F.V. J. Biol. Chem. 1999; 274: 35095-35098Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 24.Hayashi K. Saga H. Chimori Y. Kimura K. Yamanaka Y. Sobue K. J. Biol. Chem. 1998; 273: 28860-28867Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Briefly, after being minced into fine pieces, the gizzard tissue was incubated in Hanks' balanced salt solution (Cellgro) containing 0.15% (w/v) collagenase type I (Worthington) at 37 °C for 20∼40 min. Dispersed single cells were collected by passing the cell suspension through a cell strainer (70 μm; BD Biosciences). The cells were then collected by centrifugation at 2000 × g for 5 min and washed twice with cell culture medium (50:50 mixture of Dulbecco's modified Eagle's medium and nutrient mixture F-12 medium plus 0.5% fetal bovine serum, 100 μg/ml penicillin, and 50 μg/ml streptomycin). The cells were suspended in cell culture medium and plated in culture dishes at 3-4 × 104 cells/cm2. Overnight cultures of the cells at ∼50-70% confluence were used for experiments. All cells used in this experiment were maintained in low concentration fetal bovine serum (0.5%) cell culture medium and did not undergo any further passage. MBS-Recombinant Adenovirus Infection—18-24 h after plating, the SMC monolayers were infected with MBS-recombinant adenoviruses or adenovirus vector control at a multiplicity of infection of 40-100. This dosage was optimized to infect >50% of the SMCs without visible cytopathic effects. The viral stock was mixed with the same cell culture medium and added to the culture dish at 50 μl/cm2. The culture dishes were slowly rocked at 37 °C for 2 h. The infected cell cultures were supplemented with culture medium to 200 μl/cm2 and maintained at 37 °C for 48 h. The infection of SMCs was indicated by the expression of green florescent protein as visualized under a fluorescence microscope. The expression levels of exogenous and endogenous MBS isoforms in all experimental SMCs were verified by PCR and Western blotting as described below. Western Blot Analyses—Total protein was extracted from cultured SMCs or chicken tissue samples in SDS sample buffer and resolved by SDS-PAGE at an acrylamide/bisacrylamide ratio of 29:1. MBS was resolved using 6% gels, PKGI and calponin using 10% gels, and MLC20 and PP1cδ using 15% gels. Following SDS-PAGE separation, protein bands were electrophoretically transferred to 0.45-μm pore nitrocellulose membrane (Schleicher & Schüll) in buffer containing 25 mm Tris-HCl, 192 mm glycine, and 10% (v/v) methanol at 280 mA for 40 min. The protein bands were visualized on the blot either by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate reaction or by an ECL Plus Western blot detection system (Amersham Biosciences). The antibodies used were anti-MYPT1 (Upstate Biotechnology, Inc. and Covance), anti-PKGIα and anti-PKGIβ (Stressgen Biotech Corp.), anti-calponin CP3 (25.Jin J.-P. Walsh M.P. Resetar A. McMartin G.A. Biochem. Cell Biol. 1996; 74: 187-196Crossref PubMed Scopus (30) Google Scholar), anti-PP1cδ (Upstate Biotechnology, Inc.), and anti-MLC20 (Sigma). The secondary antibodies specific to the first antibody used were alkaline phosphatase-conjugated and horseradish peroxidase-conjugated for 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate reaction and ECL, respectively. Co-immunoprecipitation—Adherent SMCs (∼0.5-1 × 107 cells) were rinsed twice with phosphate-buffered saline and lysed on ice with 0.5 ml of ice-cold lysis buffer A (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar) containing 50 mm Tris-HCl (pH 7.5), 600 mm NaCl, 2 mm EDTA, 7 mm MgCl2, 2 mg/ml N-dodecyl-β-maltoside, 0.4 mg/ml cholesteryl hemisuccinate, 10 mm sodium molybdate, 10% (v/v) protease inhibitor, and 1 mm phenylmethylsulfonyl fluoride. Chicken aortic and gizzard tissues were homogenized as we described previously (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The cell or tissue lysate was gently rotated for 1 h at 4 °C, followed by centrifugation at 12,000 × g for 10 min at 4 °C. The cell lysate was then precleaned with ∼10% (v/v) protein G-Sepharose™ slurry (Amersham Biosciences) by rotating at 4 °C for 90 min and incubated with either 10 μl of anti-PP1cδ antibody or 5 μl of anti-PKGIα antibody with normal rabbit serum as a negative control. After rotating the lysate/antibody mixture overnight at 4 °C, the same quantity of protein G-Sepharose™ slurry was added and incubated by rotation at 4 °C for 2 h. After washing three times with ice-cold radioimmune precipitation assay buffer, the protein G-Sepharose beads were collected, suspended in 50 μl of SDS sample buffer, and analyzed by SDS-PAGE and Western blotting as described above. cGMP Stimulation of Cultured SMCs and Measurement of MLC20Phosphorylation—A less hydrolyzable, cell-permeable analog of cGMP, 8-bromo (Br)-cGMP (sodium salt; Calbiochem), was serially diluted from 10-3 to 10-6m in the medium of SMC monolayer cultures and incubated at 37 °C for 30 min to stimulate PKG. The reaction was stopped by removal of the culture medium and immediate addition of ice-cold 10% trichloroacetic acid (Sigma) in water containing 10 mm dithiothreitol, followed by freezing at -70 °C to allow proteins to precipitate. The cells were then scraped into 10% trichloroacetic acid solution and transferred to microcentrifuge tubes. The protein precipitates were collected by centrifugation at 12,000 × g for 10 min. After washing twice with 100% acetone containing 10 mm dithiothreitol, the pellet was air-dried, resuspended in 50 μl of urea sample buffer (8 m urea, 10 mm dithiothreitol, 22 mm Tris-HCl (pH 8.6), 22 mm glycine, 5% (v/v) glycerol, and 0.1% (w/v) bromphenol blue), and incubated at room temperature for 45 min to dissolve the protein. The samples were then cleaned by passing through an Ultrafree-MC centrifugal filter (Amicon, Inc.) at 12,000 × g for 5 min. Non-phosphorylated MLC20, monophosphorylated MLC20 (P1-MLC20), and diphosphorylated MLC20 (P2-MLC20) were resolved by electrophoresis using a urea/glycerol-PAGE system with 10% polyacrylamide (acrylamide/bisacrylamide ratio of 19:1), 40% (v/v) glycerol, 22 mm Tris-HCl (pH 8.6), and 22 mm glycine (26.Richards C.T. Ogut O. Brozovich F.V. J. Biol. Chem. 2002; 277: 4422-4427Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27.Hathaway D.R. Haeberle J.R. Am. J. Physiol. 1985; 249: C345-C351Crossref PubMed Google Scholar), running at 300 V for 5-6 h in buffer containing 22 mm Tris-HCl, 22 mm glycine, 10 mm dithiothreitol, and 0.1% thioglycolic acid (pH 8.6). The gel was then transferred to nitrocellulose membrane for Western blotting as described above. MLC20 bands were identified using anti-MLC20 antibody and quantified by Scion Image Beta Version 4.0.2 software. The percentage of phosphorylated MLC20 was calculated by the ratio of phosphorylated MLC20 to total MLC20 (P1-MLC20 or P2-MLC20/(P1-MLC20 + P2-MLC20 + MLC20)). The data are expressed as a percentage of phosphorylated MLC20 (P1-MLC20 or P2-MLC20) against unstimulated controls. All values are means ± S.D., and p < 0.05 was taken as the level for significance. The full-length chicken smooth muscle MBS cDNA is 4.7 kb, with the coding region from base 121 to 3251 (20.Shimizu H. Ito M. Miyahara M. Ichikawa K. Okubo S. Konishi T. Naka M. Tanaka T. Hirano K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1994; 269: 30407-30411Abstract Full Text PDF PubMed Google Scholar). Alternative RNA splicing of a central exon and the 3′-exon produces several different protein isoforms, and the expression of these isoforms is both developmentally regulated and tissue-specific (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19.Dirksen W.P. Vladic F. Fisher S.A. Am. J. Physiol. 2000; 278: C589-C600Crossref PubMed Google Scholar). The MBS isoforms differ by the presence or absence of the central insert (CI+ or CI-) and the C-terminal LZ (LZ+ or LZ-). In this study, four distinct cDNAs encoding the CI+/LZ+, CI+/LZ-, CI-/LZ+ and CI-/LZ- isoforms, respectively, were cloned and successfully expressed in both a mammalian cell line and primary cultured avian SMCs. It has been well documented that primary cultured SMCs under conventional serum-stimulated conditions quickly convert from a differentiated to dedifferentiated state. The degree of dedifferentiation is related to the time and passage in culture (28.Kashiwada K. Nishida W. Hayashi K. Ozawa K. Yamanaka Y. Saga H. Yamashita T. Tohyama M. Shimada S. Sato K. Sobue K. J. Biol. Chem. 1997; 272: 15396-15404Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and it has been documented that the expression of PKG is rapidly down-regulated with the passage of SMCs (29.Chiche J.D. Schlutsmeyer S.M. Bloch D.B. de la Monte S.M. Roberts Jr., J.D. Filippov G. Janssens S.P. Rosenzweig A. Bloch K.D. J. Biol. Chem. 1998; 273: 34263-34271Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In our experiments, the number of freshly isolated single cells obtained during isolation from the tissue was sufficient, so the cells did not need to be passaged; and the chicken gizzard SMCs were always kept in low serum (0.5%) medium to maintain a differentiated state. The SMCs for these conditions expressed ∼60% of the PKG compared with tissue at this stage and markers of the differentiated state such as calponin and smooth muscle myosin light chain and heavy chain (data not shown). Recombinant adenoviruses are currently used for a variety of purposes, including gene transfer. We chose this technique as the delivery vehicle to bring the MBS expression construct into SMCs because it has a high transfection efficiency, and SMCs do not need to be passaged for transfection. Normally, 60-100% of the cells are infected. SMCs infected by recombinant adenovirus express predominantly the expected exogenous MBS isoform. The exogenous MBS was overexpressed at levels 5-10 times higher than the endogenous MBS (Fig. 2), without changing the level of expression of other major smooth muscle proteins, including PKGI (both α and β isoforms), calponin, PP1cδ, and MLC20, compared with the uninfected control SMCs or SMCs infected with a adenovirus vector alone (Fig. 2). Co-immunoprecipitation assays using anti-PP1cδ antibody were performed to capture PP1cδ from the lysate of SMCs overexpressing a single MBS isoform to determine whether the exogenous MBS replaced the endogenous isoform of MBS in the MLC phosphatase holoenzyme. The results show that the immunoprecipitates of PP1cδ in all groups of the cell lysates contained MBS isoform patterns similar to those in the corresponding whole cell lysate (Fig. 3A), indicating that the overexpressed exogenous MBS isoform was proportionally assembled into the MLC phosphatase complex to replace the endogenous MBS isoform in the holoenzyme. Co-immunoprecipitation by anti-PKGI antibody was used to determine whether PKGI was associated with the MBS. Our results demonstrate that, in cultured SMCs, neither the LZ+ nor LZ- MBS isoforms bound to PKGI (Fig. 3B). This contrasts with the results obtained with both embryonic and day 7 chicken aortic and gizzard tissues, where we found that both LZ+ and LZ- MBS isoforms associated with PKGI, and although the association did not depend on the presence of cGMP, cGMP appeared to increase the binding of the MBS to PKGI in the aorta (Fig. 3C). Comparing adult gizzard, aorta, and SMCs, the PKG/MBS ratio was highest in adult gizzard and lowest in cultured SMCs. It has been reported that the interaction of the N-terminal LZ of PKGIα (but not PKGIβ) mediates cGMP-stimulated smooth muscle relaxation through interaction with the C-terminal LZ of the MBS of MLC phosphatase (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar). We used both reverse transcription-PCR with PKGIα or PKGIβ isoform-specific primers and Western blotting with both a PKGI-nonspecific antibody and a PKGIβ-specific antibody to show the expression of PKGIα versus PKGIβ in embryonic and adult chicken aortic and gizzard smooth muscle tissues as well as in cultured SMCs (Fig. 4). Expression of PKGI in embryonic day 15 chicken aortic and gizzard smooth muscle tissues was similar; and after 72 h in culture, PKGI expression in the SMCs was 50-60% of the level at embryonic day 15. In addition, the expression of PKGI was up-regulated in aortic and down-regulated in gizzard smooth muscle tissues during development and after hatching (Fig. 4). The Western blots confirm that, in the smooth muscle tissue, only PKGIα was expressed (Fig. 4). In cultured SMCs, PKGIα expression predominated, as PKGIβ was barely detected (Fig. 4). MLC20 phosphorylation levels were determined after stimulation of infected monolayer cultures of SMCs with 8-Br-cGMP. The control cultured SMCs expressed an endogenous MBS isoform that was CI+ and LZ+. 8-Br-cGMP stimulation resulted in a dose-dependent decrease in the level of phosphorylation of MLC20 (P2-MLC20) in SMCs expressing a LZ+ MBS (Fig. 5). In uninfected control SMCs as well as in SMCs infected with adenovirus vector or MBS-recombinant adenoviruses expressing an exogenous LZ+ MBS, the level of P2-MLC20 decreased to ∼65% of the unstimulated control at 0.01-1 mm 8-Br-cGMP. However, in cells overexpressing LZ- MBS isoforms, 8-Br-cGMP produced a significant decrease in MLC20 phosphorylation only at the highest concentration of 8-Br-cGMP (10-3m) (Fig. 5). These results suggest that the expression of the C-terminal LZ of the MBS may be required for PKG to activate MLC phosphatase during cGMP-mediated smooth muscle relaxation. In this study, we have shown that we could change the expression of the MBS isoform of the MLC phosphatase, in isolation, without changing the expression of other contractile proteins (Fig. 2), and the exogenous MBS isoform replaced the endogenous isoform in the MLC phosphatase holoenzyme (Fig. 3A). Our data show that cGMP stimulation of MLC phosphatase containing a LZ+ MBS isoform produced a dose-dependent decrease in MLC20 phosphorylation, whereas cGMP stimulation of MLC phosphatase containing LZ- MBS isoforms did not produce dose-dependent dephosphorylation of MLC20 (Fig. 5). These data demonstrate that, in cultured SMCs, a LZ+ MBS is required for PKG activation of MLC phosphatase activity. Previous studies have suggested that the activation of MLC phosphatase by PKG is due to a LZ-LZ interaction of PKGIα and the MBS (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar). A series of PKG constructs were used to show that only PKG with the LZ would co-immunoprecipitate with the MBS. Our co-immunoprecipitation studies with the cultured chicken SMCs did not detect an association of either LZ+ or LZ- MBS isoforms with PKG. However, in both embryonic and adult smooth muscle tissues, we observed the association of both LZ+ (aortic) and LZ- (adult gizzard) MBS isoforms with PKG (Fig. 3C). These results suggest that the MBS LZ is not required for an association of PKG and the MBS. We also found that the interaction of PKG and the MBS was unrelated to the presence of cGMP, although cGMP appeared to enhance the association in aortic tissue (Fig. 3C). This result is similar to our previous report (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), where the LZ+ MBS was associated with PKG. In this study (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), we could not co-immunoprecipitate the LZ- MBS and PKG from adult chicken gizzard, possibly because the expression of PKG was rapidly down-regulated after hatching (Fig. 4). These results suggest that a LZ-LZ interaction does not mediate the interaction of PKG with the MBS. It is unclear why we did not observe association of the MBS and PKG in cultured SMCs, whereas the MBS and PKG were associated in both the presence and absence of cGMP in chicken tissue expressing either LZ+ or LZ- MBS isoforms. However, in the cultured SMCs, we can rule out that the absence of an association of the MBS with PKG is due to a lack of PKGIα expression (Fig. 4). Our data show that an association between PKG and the MBS is not required for cGMP to activate MLC phosphatase activity, although a LZ+ MBS isoform is necessary for cGMP to stimulate MLC20 dephosphorylation. PKG has multiple targets in smooth muscle (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar, 13.Schmidt H.H. Lohmann S.M. Walter U. Biochim. Biophys. Acta. 1993; 1178: 153-175Crossref PubMed Scopus (743) Google Scholar, 14.Fukao M. Mason H.S. Britton F.C. Kenyon J.L. Horowitz B. Keef K.D. J. Biol. Chem. 1999; 274: 10927-10935Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 15.Etter E.F. Eto M. Wardle R.L. Brautigan D.L. Murphy R.A. J. Biol. Chem. 2001; 276: 34681-34685Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and a cofactor or an anchoring protein maybe required for PKG to interact with or target to the MBS. Our data suggest that an association of PKG and the MBS is needed for PKG to efficiently activate MLC phosphatase activity, and the lack of this interaction in cultured SMCs could explain the difference in sensitivity to cGMP in chicken tissue with a LZ+ MBS, where force relaxation is seen at 0.1 μm 8-Br-cGMP (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and in cultured SMCs expressing LZ+ MBS isoforms, where MLC20 dephosphorylation begins between 1 and 10 μm 8-Br-cGMP (Fig. 5). However, both in our previous report performed with smooth muscle strips (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and in the present study of cultured SMCs, a LZ+ MBS is required for cGMP-mediated smooth muscle relaxation. Taken together, these results suggest that the interaction of PKG with the MBS is not due to a LZ-LZ interaction, but could be due to an interaction of a coiled-coil domain of PKG with the coiled-coil domain of the MBS. The MBS has a predicted coiled-coil domain between amino acids 647-705 and amino acids 888-928 (30.Langsetmo K. Stafford III, W.F. Mabuchi K. Tao T. J. Biol. Chem. 2001; 276: 34318-34322Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar); and similarly, the N-terminal 10-46 residues of PKGI have a 100% probability of a coiled-coil domain structure. 2T. Tao, personal communication. It should be noted that all of the constructs of PKG used by Surks et al. (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar) containing the LZ also contained the coiled-coil domain, and mutations that disrupted the coiled-coil domain of PKGI inhibited binding of PKG and the MBS. Thus, the results of Surks et al. could also be consistent with an interaction of the coiled-coil domain of PKG with the coiled-coil domain of the MBS. The mechanism for activation of MLC phosphatase by PKG is unknown. It is possible that PKG and the MBS interact via coiled-coil domains, and activation of MLC phosphatase activity could be due to phosphorylation of the MBS by PKG. It has been reported that PKG phosphorylates the MBS (1.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (434) Google Scholar). PKG binding to substrates is mediated by an Arg-Arg or Arg-Lys sequence (31.Dostmann W.R. Taylor M.S. Nickl C.K. Brayden J.E. Frank R. Tegge W.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14772-14777Crossref PubMed Scopus (144) Google Scholar), and PKG phosphorylates at a Ser-Ser sequence (32.MacDonald J.A. Walker L.A. Nakamoto R.K. Gorenne I. Somlyo A.V. Somlyo A.P. Haystead T.A. FEBS Lett. 2000; 479: 83-88Crossref PubMed Scopus (34) Google Scholar). There are consensus sites for PKG binding at residues 841 and 842 (Arg-Arg) and residues 847 and 848 (Arg-Arg), and a Ser-Ser phosphorylation site lies at residues 790-794 of the MBS, all of which are between the two predicted coiled-coil domains of the MBS. Another PKG-binding site can be found at residues 916 and 917 (Arg-Lys); and similarly, another Ser-Ser sequence lies at residues 895 and 896 of the MBS, which are within one of the predicted coiled-coil domains (amino acids 888-928) and ∼100 residues from the beginning of the C-terminal LZ (amino acid 1002). It could be that the presence of the C-terminal LZ in the MBS exposes the more C-terminal binding and phosphorylation sites to PKG and allows for PKG to bind to and phosphorylate the MBS and thus to increase MLC phosphatase activity. In LZ- MBS isoforms, PKG could bind to the MBS, but the phosphorylation sites may not be accessible to PKG, and MLC phosphatase with a LZ- MBS would not be activated by PKG. In summary, the results of this study suggest that PKG binds to the MBS, possibly via coiled-coil domain interactions, and the activation of MLC phosphatase activity by PKG requires an LZ+ MBS. The expression of LZ+ MBS isoforms is developmentally regulated and tissue-specific (2.Khatri J.J. Joyce K.M. Brozovich F.V. Fisher S.A. J. Biol. Chem. 2001; 276: 37250-37257Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); and thus, the tissue diversity in the sensitivity to NO-mediated vasodilatation in smooth muscle could be determined, in part, by the relative expression of LZ+/LZ- MBS isoforms. We thank Dr. Ozgur Ogut for comments on the manuscript.

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