Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109
Submitted 22 October 2003 ; accepted in final form 27 January 2004
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ABSTRACT |
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acetylcholine; fusion proteins; serine
Tropomyosin is an actin-binding protein widely distributed in virtually all eukaryotic cells. It is a crucial part of the contractile apparatus and of thin filament assemblies of both muscle and nonmuscle cells. Tropomyosin assembles into an -helical coiled-coil dimer with each molecule interacting with six or seven monomers of actin (21, 38). It also binds to itself and helps wrap around the actin molecule to stabilize thin filament assembly (52). The crucial role played by tropomyosin in regulation of contraction and the mechanism of its action have been studied extensively in cardiac and skeletal muscle, but its mode of action in smooth muscle is not clearly defined. Understanding the interaction of tropomyosin with other thin filament-binding contractile proteins may provide valuable information regarding the role of tropomyosin in smooth muscle contraction.
Contraction in smooth muscle is initiated by several signaling pathways that lead to the activation of myosin light chain kinase (MLCK) (23). MLCK phosphorylates the regulatory chain of myosin, which activates actomyosin adenophosphatase activity of smooth muscle, leading to contraction. Smooth muscle can maintain contraction even in the absence of myosin light chain phosphorylation, implicating the involvement of other regulatory pathways in smooth muscle contraction (7, 47). Protein kinase C (PKC) functions in an alternative signaling pathway leading to sustained smooth muscle contraction (42, 45, 49). PKC-mediated contraction results from PKC activation and translocation to the membrane, where it may activate kinases such as mitogen-activated protein (MAP) kinase, leading to the phosphorylation of several contractile proteins (9, 19, 27). Agonists that induce PKC-mediated contraction have been shown to be involved in the phosphorylation of heat shock protein (HSP)27, a thin filament-binding contractile protein involved in smooth muscle contraction (6).
HSP27 is an actin-associated protein that modulates actin filament dynamics (29, 33, 39). HSP27 undergoes rapid phosphorylation in response to a number of extracellular factors (3) such as heat shock, growth factors, phorbol esters, calcium ionophores, interleukin (IL)-1, and tumor necrosis factor (TNF)- (1, 53). In smooth muscle, during agonist-induced contraction p38 MAP kinase is activated, which in turn phosphorylates HSP27 (17, 31). HSP27 is phosphorylated rapidly at 30 s after stimulation, and the phosphorylation remains sustained at 4 min after stimulation. In smooth muscle, HSP27 has significant effects on the actin cytoskeleton and these effects are regulated by phosphorylation and dephosphorylation (17, 31). Phosphorylated HSP27 plays a crucial role in cytoskeletal reorganization by increasing cytoskeletal stability. HSP27 also appears to play an important role in smooth muscle contraction by forming a link between signaling cascade and contractile machinery as it colocalizes with the contractile proteins actin, myosin, caldesmon, and tropomyosin and also associates with the signaling proteins PKC-
and RhoA (25, 51). The phosphorylation sites in human HSP27 have been mapped to be Ser15, Ser78, and Ser82 (30), of which Ser82 appears to be the major site of in vivo phosphorylation, followed by Ser78 and Ser15, the minor sites (30, 48). Phosphorylation is accompanied by a decrease in the size of HSP27 oligomers (26). Small oligomers of HSP27 regulate and stabilize the cytoskeleton as phosphorylation of HSP27 leads to changes in the actin cytoskeleton and actin-dependent events (32, 33, 54).
The colocalization of tropomyosin with HSP27 and their individual roles in triggering and maintaining smooth muscle contraction led us to study the direct interaction of tropomyosin with HSP27 (25, 51). We previously proposed a model (5) whereby phosphorylated HSP27 could lead to actin-myosin interaction, possibly by interaction of HSP27 with tropomyosin. The objective of the present study was to investigate the possibility of direct interaction of HSP27 with tropomyosin and to study how phosphorylation of HSP27 modulates this interaction in/during agonist-induced sustained smooth muscle contraction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that, upon agonist-induced contraction, tropomyosin shows increased association to HSP27 phosphorylated at Ser78 and Ser82. Transfection of smooth muscle cells with the HSP27 phosphorylation mutants 3D-HSP27 mutant (Ser15, Ser78, and Ser82 mutated to aspartate to mimic the phosphorylated HSP27) and 3G-HSP27 mutant (Ser15, Ser78, and Ser82 phosphorylation sites mutated to nonphosphorylatable glycine) indicated that association of tropomyosin with HSP27 is affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to the phosphomimic HSP27 mutant compared with nonphosphomimic and wild-type HSP27. These data confirm our previously proposed model that phosphorylation of HSP27 may result in a conformational change, leading to greater direct interaction of phosphorylated HSP27 with tropomyosin. This interaction may help pull the tropomyosin away from thin filament, leading to a sustained actin-myosin interaction.
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MATERIALS AND METHODS |
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The following reagents were purchased: monoclonal -smooth muscle actin antibody and monoclonal mouse anti-tropomyosin antibody (developed against chicken gizzard tropomyosin) and tropomyosin from chicken gizzard from Sigma (St. Louis, MO); monoclonal F-actin antibody from Abcam (Cambridge, MA); monoclonal mouse anti-myosin antibody (MAB1670) from Chemicon International (Temecula, CA); monoclonal mouse anti-human HSP27 antibody (2B4123) as previously described (4); polyclonal rabbit p-HSP27 (Ser82), polyclonal rabbit p-HSP27 (Ser78), and polyclonal goat p-HSP27 (Ser15) from Santa Cruz Biotechnology (Santa Cruz, CA); protein G Sepharose from Amersham Biosciences Sweden; Silver-Plus staining kit and polyvinylidene fluoride (PVDF) membranes from Bio-Rad (Hercules, CA); QiaGen Effectene transfection kit from QiaGen (Valencia, CA); enhanced chemiluminescence (ECL) detection reagents from Amersham Biosciences UK; G-418, penicillin/streptomycin, fetal bovine serum (FBS), collagen IV, and Dulbecco's (DMEM) from GIBCO-BRL (Grand Island, NY); actin-binding protein spin-down kit from Cytoskeleton (Denver, CO); all other reagents from Sigma.
Methods
Preparation of smooth muscle cells from rabbit rectosigmoid. All procedures were performed according to the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Smooth muscle cells of rabbit rectosigmoid were isolated as described previously (4). Briefly, the internal anal sphincter (IAS) from anesthetized New Zealand White rabbits, consisting of the distalmost 3 mm of the circular muscle layer, ending at the junction of skin and mucosa, was removed by sharp dissection. A 5-cm length of the rectosigmoid orad to the junction was dissected and digested to yield isolated smooth muscle cells. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml of HEPES buffer (pH 7.4). The composition of the buffer was (in mM) 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, 5.6 glucose with 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II), 0.01% (wt/vol) soybean trypsin inhibitor, and 0.184% (wt/vol) in DMEM. At the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 10 ml of collagenase-free buffer solution. Tissue was then transferred into 15 ml of fresh collagenase-free buffer solution, and cells were gently dispersed. After a hemocytometric cell count, the harvested cells were resuspended in collagenase-free HEPES buffer (pH 7.4). Each rectosigmoid yielded 1020 x 106 cells.
Transfection of smooth muscle cells with HSP27 mutants.
Smooth muscle cells were cultured in DMEM with 10% FBS and 3% penicillin/streptomycin on collagen IV-coated dishes. Cells were passed on the day before transfection and allowed to reach 70% confluence on the day of transfection. Cells were washed twice with PBS. 3D or 3G human HSP27 mutant cDNA was transfected into the cells with a QiaGen Effectene transfection kit. Briefly, the cDNA was diluted with buffer EC (QiaGen) and mixed with enhancer followed by incubation at room temperature for 5 min. The DNA-enhancer mixture was well mixed with Effectene transfection reagent followed by incubation at room temperature for 10 min to allow complex formation. The transfection complex was then mixed with cell culture medium and overlaid on the cells. After 2 days of transfection, the cells were selected with G-418 (3 mg/ml) for 12 days. The estimated transfection efficiency was 40% as measured by the number of cells remaining attached to the plate upon G-418 treatment. These cells were maintained as a stable transfection and were grown to confluence for further studies. Expressions of mutant HSP27 proteins were confirmed by immunoblotting of the whole cell lysates with human HSP27-specific antibody (Stress Gen) (5). The protein has been shown not to cross-react with rabbit and mouse antibody. Overexpression of HSP27 in transfected cells was confirmed by immunoblot with HSP27 antibody (2B4123). Furthermore, phenotypic characterization of the stable HSP27-expressing cell lines was done by studying the expression of
-actin and smooth muscle-myosin heavy chain by Western blot. All the cells used for the experiments were from passage 1.
Immunoprecipitation and immunoblotting. Smooth muscle cells isolated from rabbit colon were diluted in HEPES buffer as needed. Rabbit colon smooth muscle cells and confluent smooth muscle cells in culture were separately divided into three sets for stimulation: the first set was treated with 0.1 µM acetylcholine for 30 s; the second set was treated with 0.1 µM acetylcholine for 4 min; and the third set was untreated and served as control. After treatment, the cells were washed with PBS (in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4; pH 7.4). The cells were then suspended in lysis buffer [in mM: 20 Tris·HCl, pH 7.4, 150 NaCl, 2 phenylmethylsulfonyl (PMSF), 1 Na3VO4, 1 NaF, 1 Na4MoO4, 1 dithiothreitol (DTT), 20 Na2HPO4, 20 NaH2PO4, 20 Na4P2O7·10H2O, and 50 EDTA with 5 µg/ml DNase/RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain]. The suspended cells were lysed by sonication for 30 s followed by vortexing for 30 s and incubated on ice for 30 min. The cells were centrifuged for 15 min at 14,000 g, and the supernatant was collected. Protein content was estimated by Bio-Rad protein assay solution. Antibody (12 µg) was added to 500 µg of cell lysate protein in a total of 500 µl of lysis buffer and rocked overnight at 4°C. Fifty microliters of 50% protein G Sepharose bead slurry were added to the overnight mixture and rocked at 4°C for 2 h. The beads bound with proteins were then collected by centrifuging at 14,000 g for 3 min at 4°C. Supernatant was discarded, and the bead pellet was washed three times at room temperature with Tris-buffered saline (TBS) bead wash buffer (20 mM Tris·HCl, 150 mM NaCl, pH 7.6). The beads were then resuspended in 25 µl of 2x sample buffer and boiled for 5 min. Immunoprecipitates were immunoblotted with polyclonal anti-HSP27s78 (1:500), anti-HSP27s82 (1:500), anti-HSP27s15 (1:500), and anti-tropomyosin antibody (1:500). Replicates of experiments were performed with completely separate sets of cells.
Silver staining. The proteins were separated on SDS-PAGE, and the gel was silver stained following the manufacturer's protocol.
Western blot. The proteins were separated on SDS-PAGE and electrophoretically transferred to PVDF membrane. The PVDF membrane was then blocked with 5% nonfat dry milk for 1 h. After blocking, the membrane was incubated in an appropriate dilution of primary antibody in 5% nonfat dry milk in TBS-Tween 20 (TBST) for 1 h. The membrane was washed three times for 15 min each with TBS at room temperature to remove unbound primary antibody. The membrane was then incubated in an appropriate dilution of secondary antibody in 5% nonfat dry milk in TBST for 1 h at room temperature. The membrane was washed three times with TBST for 15 min each at room temperature to remove unbound secondary antibody. The membrane was then incubated with ECL reagent for 1 min. The proteins were detected on the membrane by immediately exposing the membrane to the film for 30 s and 1 min.
Cloning of HSP27 mutants in GST-tagged expression vector.
Three isoforms of human HSP27 were used in these studies: 1) wild-type (wt)HSP27, which has three phosphorylation sites, Ser15, Ser78, and Ser82; 2) 3G-HSP27 mutant, in which all three phosphorylation sites were mutated to glycine to mimic nonphosphorylatable HSP27; and 3) 3D-HSP27 mutant, in which all three phosphorylation sites were mutated to aspartate to mimic constitutively phosphorylated HSP27. The cDNAs encoding the human wtHSP27, 3G-HSP27, and 3D-HSP27 cloned in vectors pcDNA3.1 (Dr. R. Benndorf, University of Michigan) were used as template for PCR amplification of mutant HSP27 cDNAs. The sense and antisense primers used were 5'-TTT GGA TCC ATG ACC GAG CGC CGC GT-3' (restriction site BamHI) and reverse primer 5'-TTT GAA TTC GCT AAG GCT TTA CTT G-3' (restriction site EcoRI), respectively. The amplified PCR products of 700 bp were digested with the restriction enzymes BamHI and EcoRI. The digested PCR products were cloned in frame with the NH2-terminal GST tag of the vector pGEX-KT (Dr. J. Dixon, University of Michigan) (18). Recombinant pGEX KT was transformed into Escherichia coli DH5
. After confirmation of the clones by double digest release of the inserts, recombinant pGEX KT-HSP27 isoforms were transformed into expression host BL21 (DE3) pLysS cells. All plasmid constructs were further verified by sequencing.
Purification of recombinant GST-HSP27 fusion protein isoforms.
The GST-HSP27 fusion proteins were expressed and purified with glutathione-agarose beads as described by Hakes and Dixon (18) with slight modification. Five milliliters of overnight culture were inoculated in five hundred milliliters of fresh LB medium with 100 µg/ml ampicillin. The culture was grown with vigorous shaking at 37°C until the culture reached 0.40.5 optical density at 600 nm (3 h). Isopropyl-
-d-1-thiogalactopyranoside (IPTG) was then added to the culture at 1 mM final concentration, and the cultures were grown for another 23 h with vigorous shaking at 37°C. The cells were then collected by centrifugation at 6,000 rpm for 5 min. The cell pellet was resuspended in 1/10th volume of ice-cold mercaptoethanol-Triton X-100-PBS (MTPBS; 150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.3, and 1% Triton X-100 plus 0.1%
-mercaptoethanol), and lysozyme at a final concentration of 100 µg/ml (freshly prepared from 10 mg/ml stock) was added, followed by an incubation on ice for 30 min. The cells were lysed by mild sonication and centrifuged at 10,000 rpm for 15 min. The supernatant was mixed with glutathione-agarose beads (50 µl of each 500-µl supernatant) and rocked at 4°C for 2 h. The beads were washed three times with 5 bead volumes of MTPBS. The fusion protein was eluted with 1 bed volume of elution buffer (50 mM Tris·HCl, pH 8.0 containing 10 mM reduced glutathione). The fusion proteins were analyzed by immunoblotting with anti-GST and anti-HSP27 antibody.
In vitro binding of different GST-HSP27 fusion proteins isoforms with tropomyosin. Different isoforms of GST-HSP27 (50 µg each) were mixed with 200 µl of glutathione-agarose beads separately and rocked for 2 h at 4°C. Unbound GST-HSP27 fusion proteins were removed by washing five times with MTPBS. All the washes were retained for further analysis. Twenty-five micrograms of tropomyosin were added to the different GST-HSP27 isoform-bound beads and rocked for 1 h at 4°C. Unbound tropomyosin was removed by washing five times with MTPBS. All the washes were retained for further analysis. The bound proteins were eluted twice with 1 bed volume of elution buffer (50 mM Tris·HCl, pH 8.0, containing 10 mM reduced glutathione). Ten microliters each of all the washes and the eluates were spotted on a PVDF membrane. The membrane was blocked with 5% nonfat skim milk in TBS and immunoblotted with anti-GST antibody, anti-tropomyosin antibody, and anti-HSP27 antibody. The spots were detected by chemiluminescence.
Dot blot. Dry blotting paper and the PVDF membrane were cut according to the dot blot apparatus size. The cut PVDF membrane was soaked in 100% methanol for 23 min followed by equilibration in 1x PBS for 5 min at room temperature. The cut dry blotting paper was incubated in 1x PBS for 5 min. The wet blotting paper was placed on the dot blot apparatus, followed by the equilibrated PVDF membrane onto the blotting paper. The dot blot apparatus was clasped with a clamp on the sides and was attached to the vacuum pipe. The vacuum was passed at the rate of 1 ml/min. Ten microliters of the sample were added into the each slot. The slots were then washed with 100 µl of 1x PBS. The clamps were opened to remove the membrane. The membrane was incubated in 5% nonfat dry milk in TBST with rocking for 1 h at room temperature. The membrane was washed twice for 10 min each with TBST. The membrane was then incubated in the appropriate dilution of primary antibody for 1 h with rocking at room temperature. The membrane was washed three times for 15 min each with TBST. The membrane was then incubated in the appropriate dilution of secondary antibody for 1 h with rocking at room temperature followed by washing three times for 15 min each with TBST. The membrane was then incubated with Amersham's ECL reagent for 1 min. The proteins were detected on the membrane by immediately exposing the membrane to the film.
F-actin cosedimentation assay. F-actin cosedimentation assays were performed as described by the actin manufacturer (Cytoskeleton) with slight modification. Briefly, whole cell lysates were incubated in F-actin buffer (in mM: 20 Tris·HCl, pH 7.5, 75 KCl, 10 NaCl, 2 DTT, and 2.5 MgCl2) for 30 min at room temperature and subjected to ultracentrifugation at 110,000 g for 1 h at 24°C to pellet out the F-actin from the cell lysate. The supernatant was collected, and an equal amount of F-actin prepared per the manufacturer's protocol was added to each sample, followed by incubation at room temperature for 1 h. After 1 h, the samples were subjected to ultracentrifugation at 110,000 g to pellet F-actin and proteins bound to F-actin. After solubilization of the pellet fraction in a volume equal to the initial incubation volume, 20 µl of the pellet fractions was loaded onto SDS-PAGE.
Data analysis. Western blot bands were quantitated with a densitometer (model GS-700, Bio-Rad Laboratories), and band volumes (absorbance units x mm2) were calculated and expressed as a percentage of the total volume. Band data are within the linear range of detection for each antibody used. The control band intensities were standardized to 100%. The band intensities of samples from treated cells were compared with the control and expressed as percent change from the control. Blotting data are within the linear range of detection for each antibody used. All the means were compared and analyzed with Student's t-test.
Dot blot spots were quantitated with a densitometer (model GS-700, Bio-Rad Laboratories), and spot volumes (absorbance units x mm2) were calculated and expressed as a percentage of the total volume. Spot data are within the linear range of detection for each antibody used. The control spot intensity was standardized to 100%. The spot intensities of eluted fractions of the tropomyosin were compared with the control and expressed as percent change from the control. All the means were compared and analyzed with Student's t-test.
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RESULTS |
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Tropomyosin was previously reported by our laboratory (25) to colocalize with HSP27 in colonic smooth muscle cells. In the present study, we characterized the form of HSP27 associated with tropomyosin upon acetylcholine stimulation. HSP27 has been reported to be phosphorylated at three sites, Ser15, Ser78, and Ser82. We have thus studied the phosphorylation state of HSP27 that is associated with tropomyosin, in response to acetylcholine stimulation. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Whole cell lysates were immunoprecipitated with anti-tropomyosin antibody. The proteins in the immunoprecipitates were separated on SDS-PAGE and were transferred onto PVDF membrane. The membrane was then immunoblotted with different phospho-HSP27 antibodies, i.e., anti-HSP27s15, anti-HSP27s78, and anti-HSP27s82 antibodies separately. No bands were observed when the membrane was probed with anti-HSP27s15 antibody, whereas bands were observed with anti-HSP27s78 and anti-HSP27s82 antibodies (Fig. 1A). Acetylcholine induced a significant and sustained increase in the association of tropomyosin with HSP27s78 (182 ± 4 and 146 ± 19% of control at 30 s and 4 min, respectively; P 0.001, n = 4; Fig. 1B) and HSP27s82 (185 ± 7 and 158 ± 28% of control at 30 s and 4 min, respectively; P
0.001, n = 4; Fig. 1C) compared with the unstimulated control. The data thus suggest that in rabbit colon smooth muscle cells acetylcholine-induced contraction is associated with an increase in the association of tropomyosin with HSP27 phosphorylated at Ser78 and Ser82.
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Phenotypic characterization of the stably maintained HSP27 mutant-transfected smooth muscle cells was carried out by immunoblotting the whole cell lysate with anti--smooth muscle actin and smooth muscle-myosin heavy chain. No difference in expression of smooth muscle-specific actin and myosin between normal and transfected cells was observed (Fig. 2A).
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To check whether there were proteins other than actin and HSP27 coimmunoprecipitating with tropomyosin, proteins separated on SDS-PAGE were silver stained. The silver-stained gel showed that, in addition to actin and HSP27, there were other proteins coimmunoprecipitating with tropomyosin (Fig. 3).
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To study the gain of function and loss of function of phosphorylated HSP27, cultured smooth muscle cells were transfected with HSP27 mutants. Cells were transfected with 3G-HSP27, in which all the potential serine phosphorylation sites, i.e., Ser15, Ser78, and Ser82, were replaced with glycine to mimic the nonphosphorylatable HSP27, and with 3D-HSP27, in which all the potential serine phosphorylation sites, i.e., Ser 15, Ser78, and Ser82, were replaced with aspartate to mimic the constitutively phosphorylated HSP27. These mutant constructs were expressed in transfected smooth muscle cells, and the expressions of mutant HSP27 proteins were confirmed by immunoblotting the whole cell lysates. Confluent transfected and nontransfected rabbit colon smooth muscle cells were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Whole cell lysates from these cells were immunoprecipitated with anti-tropomyosin antibody. The immunoprecipitates were then subjected to SDS-PAGE, and the separated proteins were transferred onto PVDF membrane. The membrane was immunoblotted with anti-HSP27s78 and anti-HSP27s82 antibodies.
HSP27s78.
An increase in band intensity was observed in phosphomimic-transfected cells upon stimulation with acetylcholine when probed with anti-HSP27s78 compared with nonphosphomimic-transfected cells and nontransfected cells (Fig. 4A). The densitometric analysis of these bands showed a significant increase in association of tropomyosin with phospho-Ser78-HSP27 in response to stimulation with acetylcholine at 30 s in the cells transfected with 3D-HSP27 mutant (287 ± 11% of control; P 0.01, n = 3) compared with cells transfected with 3G-HSP27 mutant (174 ± 25% of control; P
0.01, n = 3) and nontransfected cells (172 ± 4% of control; P
0.01, n = 3) above unstimulated cells (Fig. 4B). The increased association was observed to be sustained at 4 min after acetylcholine stimulation in cells transfected with 3D-HSP27 mutants (172 ± 7% of control; P
0.01, n = 3) compared with cells transfected with 3G-HSP27 mutant (145 ± 21% of control; P
0.01, n = 3) and nontransfected cells (140 ± 7% of control; P
0.01, n = 3) above unstimulated cells (Fig. 4B).
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The data suggest that acetylcholine induced an increased association of tropomyosin with HSP27 in phosphomimic (3D-HSP27)-transfected smooth muscle cells and this increased association remained sustained at 4 min after stimulation, whereas in nonphosphomimic (3G-HSP27)-transfected smooth muscle cells and nontransfected cells the association was relatively lower.
Acetylcholine-Induced Coimmunoprecipitation of Tropomyosin with F-actin in Whole Cell Lysates of Smooth Muscle Cells Transfected with HSP27 Mutants
Interaction of tropomyosin with F-actin in the presence or absence of phosphorylated HSP27 was studied in smooth muscle cells transfected with different HSP27 mutants. Whole cell lysates from acetylcholine-stimulated transfected and nontransfected cells were immunoprecipitated with anti-tropomyosin antibody, and the immunoprecipitates were subjected to SDS-PAGE. The separated proteins were then immunoblotted with F-actin-specific antibody. The data indicate that transfection of smooth muscle cells with the nonphosphomimic mutant of HSP27 resulted in an increase in the association of tropomyosin with actin in the relaxed cells (137 ± 1% of control; P 0.01, n = 3) compared with phosphomimic mutant-transfected cells (84 ± 1% of control; P
0.01, n = 3). The levels of interaction of tropomyosin with actin remained significantly higher in the nonphosphomimic-transfected cells in response to stimulation with acetylcholine at 30 s (121 ± 1% of control; P
0.001, n = 3) or 4 min (120 ± 1% of control; P
0.001, n = 3). The cells transfected with the phosphomimic mutant of HSP27 maintained the same lower level of interaction of actin with tropomyosin after stimulation with acetylcholine at 30 s (82 ± 1% of control; P
0.01, n = 3) and at 4 min (80 ± 1% of control; P
0.01, n = 3) (Fig. 5). The data suggest that in the presence of phosphomimic HSP27 less actin is associated with tropomyosin compared with the amount of actin associated with tropomyosin in the presence of nonphosphomimic HSP27. The decreased association of tropomyosin with actin in phosphomimic-transfected cells correlated with the concomitant increased association of tropomyosin with HSP27 in these cells. This suggested a possibility of direct interaction of tropomyosin with HSP27.
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To study whether the association of tropomyosin with HSP27 was direct or indirect, in vitro binding studies were carried out with GST-tagged HSP27 mutant fusion proteins. Fragments (652 bp) containing mutant HSP27 cDNAs were amplified from the respective HSP27 mutant pcDNA3.1 clones. The PCR-amplified fragments from each mutant were inserted into pGEX-KT at BamH1 and EcoRI sites to express an NH2-terminal GST-fused protein. The clones were confirmed to be in the correct open reading frames by sequencing. The GST-fused mutant HSP27 proteins were expressed in response to induction with 1 mM IPTG.
The expressed fusion protein was purified with the glutathione-agarose beads as described in Methods and analyzed by Western blot with anti-HSP27 antibody. A band of 53-kDa (27-kDa HSP27 + 26-kDa GST) molecular mass was detected on the blot by both anti-HSP27 antibody and anti-GST antibody (Fig. 6).
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The purified GST-fused HSP27 mutant proteins were conjugated to glutathione-agarose beads and incubated with commercially available tropomyosin protein. The GST-HSP27 fusion proteins bound with tropomyosin were eluted as described in Methods. Eluted fractions were analyzed on the Western blot by probing with anti-tropomyosin and anti-HSP27 antibodies. Increased band intensity of tropomyosin was observed in elution from the glutathione agarose-bound GST-3D-HSP27 fraction (Fig. 7B). All the unbound fractions, washes, and eluted fractions were analyzed on dot blot. Glutathione-agarose beads conjugated to GST alone were used as controls and showed no binding of tropomyosin to GST (Fig. 7A). The binding affinity of tropomyosin with GST-3D HSP27 fusion protein, which represents a phosphomimic HSP27 protein, was 333 ± 76% of control (P 0.001, n = 6) compared with nonphosphomimic HSP27 mutant fusion protein, which was 168 ± 17% of control (P
0.001, n = 6), and with wild-type HSP27 fusion protein, which was 200 ± 44% of control (P
0.001, n = 6) above the control GST (Fig. 7, A and C). The data suggest that tropomyosin is associated with HSP27 directly and this direct binding of tropomyosin with HSP27 varies depending on the phosphorylation state of HSP27.
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Interaction of tropomyosin with F-actin in the presence or absence of phosphorylated HSP27 was also studied by cosedimentation of tropomyosin with actin in smooth muscle cells transfected with different HSP27 mutants. Whole cell lysates from acetylcholine-stimulated transfected and nontransfected cells were incubated in F-actin polymerization buffer and subjected to high-speed centrifugation to remove all the F-actin. The supernatant collected was incubated with equal amounts of F-actin stock and centrifuged to sediment F-actin and F-actin binding proteins. The pellet was then subjected to SDS-PAGE and immunoblotted with tropomyosin- and F-actin-specific antibody. The data indicate that transfection of smooth muscle cells with the phosphomimic 3D-HSP27 mutant resulted in a decrease in the cosedimentation of tropomyosin with actin (86 ± 1% of control; P 0.01, n = 2) compared with nonphosphomimic 3G-HSP27 mutant-transfected cells (119 ± 4% of control; P
0.01, n = 2) (Fig. 8). The data suggest that in the presence of phosphomimic HSP27 less actin is associated with tropomyosin.
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DISCUSSION |
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Muscle contraction depends on the sliding of thick filaments past thin filaments driven by myosin cross bridges. The actomyosin ATPase, which energizes muscle contraction, is switched "on" and "off" by changes in intracellular free Ca2+ concentration (11). However, the mechanism that leads to the activation of actomyosin ATPase and the sliding of the filaments by intracellular free Ca2+ concentration varies in different muscle types (15). In skeletal and cardiac muscle, "on-off" switching is mediated by the binding of Ca2+ to the troponin C subunit of troponin-tropomyosin complex on thin filaments. In smooth muscle, troponin is absent and Ca2+ regulates contraction by binding to calmodulin, which links the MLCK phosphorylation cascade to activation of actomyosin ATPase (46, 50).
Actin-myosin interaction during smooth muscle contraction is mediated by tropomyosin (10, 35). Tropomyosin is localized on thin filament, sterically blocking myosin-binding sites on actin in all types of muscle cells (12, 28). The role of tropomyosin in contraction is well studied and appears to be similar in all types of muscle cells. Upon elevation of intracellular free Ca2+ concentration in skeletal and cardiac muscle, troponin aids in the displacement of tropomyosin off the actin filament and exposes myosin-binding sites on actin (14, 43, 52), whereas in smooth muscle cells, which lack troponin, the mechanism that initiates displacement of tropomyosin off the actin is not clearly understood. Tropomyosin is one of the most conserved of all the actin-binding proteins identified in organisms ranging from yeast to humans, and it is expressed in nearly all eukaryotic cells (24). Multiple genes as well as alternatively spliced RNA transcripts have been implicated in expressing several different isoforms in a tissue-specific manner (34). Differential expression has been found during cell transformation and differentiation, suggesting that different isoforms are required for specific roles in regulating both the actin filament structure and the interaction of other proteins with the actin cytoskeleton (13). Tropomyosin is a two-chained parallel coiled-coil 284-amino acid-containing protein and is aligned head to tail along the helical actin filament (20). Head-to-tail alignment of tropomyosin is achieved by nine amino acids at the NH2 terminal interacting with nine amino acids at the COOH terminal to form an overlap complex (40). The NH2- and COOH-terminal ends of tropomyosins play a major role in modulating actin affinity and in the cooperative regulation of contraction (16, 37).
Sustained smooth muscle contraction is associated with activation of MAP kinase (8, 45). p38 MAP kinase has been implicated in the maintenance of force in gastrointestinal and vascular smooth muscle cells (27). p38 MAP kinase is known to activate MAPKAP2 kinase, which in turn phosphorylates HSP27 (17, 30, 31). We previously showed (5) that ceramide-induced contraction is associated with sustained increase in the di- and triphosphorylated forms of HSP27. In freshly isolated smooth muscle cells from the rabbit colon, the data indicate that acetylcholine induces a significant and sustained increase in association of tropomyosin with phosphorylated HSP27, more specifically, to the HSP27 that is phosphorylated at Ser78 and Ser82 but not at Ser15. The sustained interaction of tropomyosin with HSP27 is thus affected by the sustained phosphorylation state of HSP27. Sustained association of tropomyosin with phospho-HSP27 is in concurrence with previous results indicating that contraction is associated with sustained phosphorylation of HSP27 (25).
Previous data from our lab (25) showed that transfection of smooth muscle cells with the nonphosphomimic form of HSP27 resulted in a reduced association of actin-myosin. Our data suggest that transfection of smooth muscle cells with the nonphosphomimic mutant of HSP27 resulted in an increase in the association of tropomyosin with actin in the relaxed unstimulated cells compared with stimulated nontransfected cells or cells transfected with the phosphomimic mutant of HSP27. Upon sustained stimulation with acetylcholine, the levels of interaction of tropomyosin with actin remained significantly higher in the nonphosphomimic-transfected cells, whereas they remained unchanged at the same lower levels in the cells transfected with the phosphomimic mutant of HSP27. The present data suggest, in light of the previously proposed model, that transfection of smooth muscle cells with the phosphomimic form of HSP27 resulted in an increased association of HSP27 with tropomyosin. This significant increase in binding with tropomyosin was sustained for 4 min, whereas the association of tropomyosin with actin decreased in cells transfected with phosphomimic HSP27 and remained at the same lower levels after stimulation with acetylcholine. The data indicate that upon stimulation, tropomyosin associates strongly to phospho-HSP27, whereby the association of tropomyosin to actin is reduced. The data were also confirmed by cosedimentation of tropomyosin with actin. In the presence of the phosphomimic mutant, there was reduced cosedimentation of tropomyosin with F-actin. The binding of tropomyosin to phosphorylated HSP27 is increased, with a concomitant reduction in the association of tropomyosin to F-actin in the presence of phosphorylated HSP27. The observed decreased association of tropomyosin with F-actin in the phosphomimic HSP27-transfected cells can be related to a decrease in the total amount of F-actin or to the length of F-actin filaments. Other proteins that cosediment with tropomyosin could also be involved.
To test the possible direct interaction of tropomyosin with HSP27, recombinant GST-HSP27 mutant fusion proteins were constructed. In vitro binding studies using recombinant GST-HSP27 mutant fusion proteins show that tropomyosin binds directly with HSP27. Tropomyosin shows significantly higher binding affinity to phosphorylated HSP27, whereas a reduced binding affinity with nonphosphorylatable HSP27 is observed compared with control wild-type HSP27. However, the data suggest that, in vitro, tropomyosin has greater direct affinity to phosphorylated HSP27 in the absence of F-actin.
The data suggest that, in smooth muscle cells, HSP27 is rapidly phosphorylated upon agonist stimulation that may result in structural change of HSP27. This possible change may be responsible for the stronger association of tropomyosin to phosphorylated HSP27, thereby reducing the association of tropomyosin with actin. Marston and Huber (36) have suggested polymerization-depolymerization of actin as a part of the contraction-relaxation cycle of smooth muscle. Actin polymerization-depolymerization is regulated by a number of mechanisms, in which HSP27 is likely to contribute to the equilibrium between polymerization and depolymerization (2, 17, 32). The reduced association of tropomyosin with actin may be due to an increased association of tropomyosin with phosphorylated HSP27 or may be due to the equilibrium between polymerized and depolymerized actin, i.e., the ratio of F-actin to G-actin. The increased association of tropomyosin with phosphorylated HSP27 may result in displacement of the tropomyosin, thus exposing the myosin-binding sites on actin. How this phosphorylation is achieved is being investigated. The serine residues in HSP27 might be phosphorylated in a certain order, i.e., sequentially: Ser82, being the major phosphorylated site, could be phosphorylated first, leading to phosphorylation of the other two minor sites, Ser78 and Ser15 (25, 48). It is also possible that phosphorylation sites on HSP27 are phosphorylated simultaneously or that each site is phosphorylated specifically depending on the signal. The data also suggest that upon phosphorylation at Ser82 and Ser78, HSP27 undergoes a conformational change leading to a stronger association of HSP27 with tropomyosin. Conformational change may be just the structural change, i.e., dissociation of oligomers into monomers and dimers that may expose certain amino acids that are responsible for greater binding of tropomyosin to phospho-HSP27. Colocalization of tropomyosin with HSP27 upon agonist-induced contraction is primarily due to the direct association of tropomyosin with phospho-HSP27 as suggested by in vitro binding experiments. We thus propose that upon phosphorylation HSP27 undergoes structural changes resulting in greater binding to tropomyosin. This strong binding may help in the displacement of tropomyosin off the F-actin filament, exposing the myosin-binding site for actin-myosin interaction and leading to contraction. Thus we propose that agonist-induced phosphorylation of HSP27 is of physiological relevance and that it is involved in modulation of actin-myosin interaction in smooth muscle contraction.
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