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Am J Physiol Gastrointest Liver Physiol 286: G954-G963, 2004. First published January 15, 2004; doi:10.1152/ajpgi.00477.2003
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NEUROREGULATION AND MOTILITY

Direct association and translocation of PKC-{alpha} with calponin

Suresh B. Patil, Mercy D. Pawar, and Khalil N. Bitar

Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109

Submitted 1 November 2003 ; accepted in final form 9 January 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-{alpha} and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-{alpha}. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-{alpha} in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-{alpha}, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-{alpha} in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-{alpha}, indicating a direct association of GST-calponin with PKC-{alpha}. These data suggest that there is a direct association of translocated calponin and PKC-{alpha} in the membrane and a role for the complex calponin-PKC-{alpha}-HSP27, in contraction of colonic smooth muscle cells.

heat-shock protein 27; smooth muscle; contraction; cytoskeleton

AGONIST-INDUCED CONTRACTION of gastrointestinal smooth muscle results in activation of G protein-coupled receptors and in sequential signal-transduction events mediated through several enzymes. Two different contractile pathways have been identified in smooth muscle cells: 1) a transient contraction, which is calmodulin dependently mediated by inositol 1,4,5-trisphosphate-dependent calcium release (5), and 2) a sustained contraction (6, 19). On the basis of their functional behavior, visceral smooth muscles have been classified into sphincteric and nonsphincteric smooth muscles that represent tonic and phasic muscles (15, 17, 47). Phasic muscle shows a very wide range of contractile activity that varies from a fully relaxed basal state to a large-amplitude rapid contraction and rapid relaxation response. It is generally accepted that phosphorylation of 20-kDa MLC (MLC) by MLC kinase (MLCK) is the major mechanism responsible for contraction in smooth muscles. Phosphorylation of MLC by MLCK is counterbalanced by its dephosphorylation by MLC phosphatase (MLCP) (14).

Tonic muscles may have lower levels of MLCK and MLC phosphatase than phasic muscles (17). Differences in rates of phosphorylation/dephosphorylation of MLC cannot fully explain the divergent mechanical behavior of phasic and tonic smooth muscles (45, 47). Tonic contraction is activated by activation of PKC (33). Some excitatory agonists may act to inhibit MLCP via a G protein-coupled pathway that may involve certain PKCs and RhoA and Rho kinase (41, 52).

Our laboratory has previously shown (7) that PKC-{alpha} and heat-shock protein (HSP)27 are translocated to the membrane during agonist-induced contraction of smooth muscle cells from the rabbit colon. HSP27 is a small HSP that is relatively abundant in all types of tissues in various species. It colocalizes with actin filaments in cardiac and skeletal muscles (26). Results from our laboratory also have confirmed the association of HSP27 with contractile proteins such as actin, myosin, tropomyosin, and caldesmon (18) and with signaling proteins PKC-{alpha} and RhoA (19, 46). Thus, in smooth muscle cells, HSP27 appears to be the link between the signal-transduction cascade and the contractile machinery (7).

Studies (11, 58) suggest that calmodulin binding thin filament-associated proteins such as caldesmon and calponin plays an important role in smooth muscle contractility. Calponin, an actin-binding protein, inhibits actomyosin ATPase and slows the detachment of myosin from actin (16). In its unphosphorylated state, calponin binds to actin and inhibits Mg ATPase of myosin. On phosphorylation by PKC, its inhibiting activity is lost (55). Calponin can be phosphorylated by PKC, and its phosphorylation has been shown to modulate porcine arterial smooth muscle contraction (31, 37). PKC regulation of calponin phosphorylation is thought to be of physiological importance (24, 34, 39, 40).

Calponin was originally discovered in chicken gizzard smooth muscle as an F-actin-, calmodulin-, and tropomyosin-binding protein (57). Three isoforms of calponin, acidic, neutral, and basic calponin, have been classified on the basis of their isoelectric point (3, 46, 51, 57). Basic calponin is distributed relatively specifically in smooth muscle tissues (40) and has been well characterized in vitro (4, 18, 57, 58). Histochemical and digital imaging microscopy studies (38) have shown that calponin is distributed more toward the center rather than the periphery in a resting cell and is distributed with the cytoskeleton in chicken gizzard smooth muscle cells. In resting vascular smooth muscle cells of the ferret, calponin is distributed throughout the cytosol associated with filamentous structures, and on stimulation with a contractile agonist, the cellular distribution of calponin changed from primarily cytosolic to surface cortex (39). On stimulation with contractile agonists, calponin has also been shown to relocate to the membrane in coronary smooth muscle cells (40). Reports indicate that PKC/PKC-{epsilon} interacts with calponin (24, 37). Calponin is also shown to form a substrate for Rho-kinase in vitro (22). In addition, it was recently reported (25) that calponin may facilitate ERK-dependent signaling, thus playing a significant role in regulation of vascular smooth muscle contraction.

We postulate that in rabbit colonic smooth muscle, calponin associates with HSP27 in the particulate fraction and that the association of the translocated proteins is mediated through PKC-{alpha}. We also hypothesize that calponin interacts directly with PKC-{alpha}. To test this hypothesis, we have examined the possible interaction of calponin with HSP27 and with PKC-{alpha} in smooth muscle cells from the rabbit colon. Immunoprecipitation of cell particulate fractions with calponin antibody followed by immunoblotting with either HSP27 antibody or with PKC-{alpha} antibody indicated that acetylcholine (10–7 M) induced an increased immunocomplexing of calponin-HSP27 and of calponin-PKC-{alpha} in the particulate fraction. Furthermore, Western blot analyses of glutathione eluates from the GST-calponin fusion protein-PKC-{alpha} slurry show a coelution of GST-calponin and of PKC-{alpha}, indicating a direct association of GST-calponin with PKC-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Acetylcholine was purchased from Sigma (St. Louis, MO); collagenase type II was purchased from Worthington Biochemical (Freehold, NJ). Protein G-sepharose was from Pharmacia Biotechnology (Uppsala, Sweden). Polyvinylidene fluoride (PVDF) membranes were from Bio-Rad (Hercules, CA); enhanced chemiluminescence detection reagents were from Amersham (Arlington Heights, IL). Monoclonal mouse anti-HSP27 antibody (2B4–123) was previously described (5). Monoclonal mouse anti-calponin (cp-93) and anti-tropomyosin antibody were from Sigma, and polyclonal rabbit anti-PKC-{alpha} was from Panvera (Madison, WI); Soybean trypsin inhibitor (STI) was from Santa Cruz Biotechnology (Santa Cruz, CA); creatinine phosphatase, creatinine phosphokinase, and ATP were obtained from Sigma. Protease inhibitor tablets were purchased from Roche (Germany). All other reagents were purchased from Sigma. Isopropyl thio-{beta}-D-galactoside (IPTG) was purchased from Invitrogen (Carlsbad, CA).

Isolation of smooth muscle cells from rabbit colon. New Zealand rabbits were euthanized according to our institution's animal care guidelines. The internal anal sphincter, consisting of the distal-most 3 mm of the circular muscle layer, ending at the junction of skin and mucosa was removed by sharp dissection. A 10-cm length of the colon to the junction of jejunum was dissected and used for further isolation of smooth muscle cells. Cells were isolated as previously described (7). Circular muscle tissue was incubated for two successive 1-h periods at 31°C in 15 ml of HEPES buffer (pH 7.4) (in mM): 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, and 5.6 glucose containing 0.1% (wt/vol) collagenase type II, 0.01% (wt/vol) STI, and 2 mg/ml DMEM. At the end of the second enzymatic incubation period, the medium was filtered through a 500-µm Nitex filter. 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 cell count, the harvested cells were diluted in collagenase-free HEPES buffer (pH 7.4). Each colon yielded 10–20 x 106 cells.

Preparation of particulate fractions. Freshly isolated smooth muscle cells were counted on a hemocytometer and diluted with HEPES buffer as needed. Cells were then treated with agonists and/or antagonist for the indicated periods. After the treatment, the cells were washed twice with buffer A (in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate, pH 7.4) and sonicated in buffer B (in mM: 1 Na3VO4, 1 NaF, 2 phenylmethylsulfonyl fluoride, 5 EDTA, 1 Na4MoO4, 1 dithiothreitol, 20 NaH2PO4, 20 Na2HPO4, and 20 Na4P2O7·10H2O with 50 µl/ml DNase-RNase, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain-HCL, pH 7.4; 0.08 mg/ml STI; 60 µg/ml phosphor-amidon; 5 mg/ml pefbloc). The cell sonicates were centrifuged at 100,000 g for 60 min. The supernatant material from the high-speed centrifugation was collected as soluble cytosolic fraction. The pellet material was resuspended by sonication twice for 30 s in buffer B plus 1% Triton X-100 and collected as particulate fraction. The protein content was determined by using Bio-Rad protein assay reagent.

Immunoprecipitation and immunoblotting. Each sample (400–500 µg protein) obtained as described above was subjected to immunoprecipitation with monoclonal anti-calponin antibody overnight at a ratio of 1:250. The G protein sepharose beads were then added and rocked for 2 h. The beads were washed in Tris-buffered saline twice and boiled in 2x Laemmli sample buffer with 2-mercaptoethanol.

SDS-PAGE and electrophoretic transfer. For one-dimensional SDS-PAGE, the samples were mixed in an equal volume of 2x sample buffer [50 mM Tris, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.1% (wt/vol) bromophenol blue, pH 6.8]. The proteins were separated by 12.5 or 15% SDS-PAGE and transferred onto nitrocellulose or PVDF membranes. Proteins were identified by chemiluminescence.

Western immunoblotting of immunoprecipitates. Immunoprecipitates of calponin were size separated by SDS-PAGE and electrophoretically transferred to PVDF membranes. Immunoblotting was performed using antibodies specific to each of the proteins tested, namely monoclonal anti-calponin antibody, monoclonal anti-HSP27 antibody (1:5,000), monoclonal anti-tropomyosin antibody (1:2,000), or a polyclonal anti-PKC-{alpha} antibody (1:200), as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG as the case may be (1:2,500 dilution) for 1 h at 24°C. The enzymes on the membrane were detected with luminescent substrates. As a negative control, blots were incubated in the secondary antibody only.

Expression and purification of recombinant calponin. Recombinant calponin expressing amino acids (aa) 92–298 was generated with the glutathione S-transferase (GST) fusion protein system. We have taken the advantage of specific affinity of GST with glutathione agarose beads to immobilize the fusion protein GST-calponin. The use of GST enabled easier and quicker purification for the bacterial extracts and also could be used to immobilize the fusion protein on agarose for studying protein-protein interactions. A 1.3-Kb donor cDNA (pBS KS+; gift from Dr. J. M. Miano, University of Rochester Medical Center Rochester, NY) was used as the source for amplifying coding regions of the human basic calponin gene. The donor cDNA comprised ~0.7 Kb of the coding region and ~0.6-Kb of 3'-noncoding region of the human basic calponin gene (30). The following sense and antisense oligonucleotide primers were designed to amplify the coding region of the donor cDNA; 5'-TTT GGA TTC ATC AAG GCC ATC ACC (nt 352–367) and 3'-TTT GAA TTC GTG GCC CTA GGC GGA ATT GTA (nt 951–972) with stringent annealing temperature of 57°C. The primers were engineered such that the PCR product would have adapter sequences that match the insertion sites BamHI and EcoRI of the vector pGEX-KT (a gift from Dr. J. E. Dixon, University Michigan, Ann Arbor, MI). The vector consisted of GST DNA sequences immediately following an IPTG inducible promoter region and a transcription start codon ATG. The newly inserted DNA would fuse with the GST at its 3'-end such that when a protein is expressed, it would form as an NH2-terminal GST fusion protein. The amplification products were digested with BamHI and EcoRI and inserted in frame into the BamHI and EcoRI sites of pGEX-KT. The GST fusion proteins were expressed, then they were gel-purified with glutathione-agarose column (Amersham) as described by Smith and Johnson (44). Briefly, Eschericia coli (BL21.DE3) cells containing pGEX-KT were grown to an optical density (OD 595{lambda}) of 0.6 at 37°C and were induced with IPTG for 3 h. At the end of 3 h, cells were harvested and lysed. The lysates were centrifuged at 4,000 rpm for 20 min, and the pellet containing cell debris was discarded. The supernatant was applied to a 10-ml glutathione agarose column. The beads were washed three times with PBS (pH 7.4) containing 2% Triton X-100 and were eluted with 10 mM reduced glutathione buffer. The eluates were concentrated by ultra filtration using Millipore filter system, and the protein contents were determined by using Bio-Rad reagent at OD 595{lambda} as suggested by the manufacturer. The products were tested by running on a 12% SDS PAGE followed by Western blot analysis for both anti-GST and anti-calponin antibody. The recombinant proteins were then subjected to in vitro binding experiments.

Direct protein-protein interaction. Twenty-four micrograms of GST-calponin or GST alone were incubated with 200 µl of 50% suspension of glutathione-agarose in PBS containing 2% Triton X-100 (PBST)/0.1% {beta}-mercaptoethanol at 4°C for 30 min. The mixture was centrifuged at 1,000 g for 5 min, and the supernatant was collected as unbound fraction. The mixture was further washed twice with PBS (pH 7.4), and the fractions were retained. Twenty-four micrograms of recombinant hPKC-{alpha} were added to the washed beads and incubated at room temperature for 1–3 h. The beads were then washed three times with PBST. The mixture was then incubated with 10 mM reduced glutathione buffer for 15 min with shaking. The mixture was again centrifuged, and the eluates were collected. The procedure was repeated at least three times. All of the fractions were then spotted in three sets of duplicate on nitrocellulose membrane. The membranes were blocked with nonfat milk and probed with either anti-GST, anti-calponin, or anti-PKC-{alpha} antibody and were detected by chemiluminescence.

Measurement of contraction. Smooth muscle cells isolated from the rabbit colon were permeabilized by incubation for 4 min in saponin (75 µg/ml). The cells were washed free of saponin and resuspended in the cytosol-like buffer (in mM: 20 NaCl, 100 KCl, 5 MgSO4, 0.96 NaH2PO4, 25 NaHCO3, 10 antimycin, 1.5 ATP, and 5 creatine phosphate plus 10 U/ml creatine phosphokinase). The cells were allowed to rest for 30 min at 37°C. Aliquots (~2.5 x 104 cells/0.5 ml) were preincubated with either anti-calponin antibody (1:1,000) or anti-HSP27 antibody (1:5,000) or anti-PKC-{alpha} antibody (1:100) for 20 min and were stimulated with acetylcholine (10–7 M) for 30 s or 4 min. In separate experiments, a combination of both anti-PKC-{alpha} and anti-calponin antibody was used. The reaction was stopped by the addition of 0.1 ml of acrolein at a final concentration of 1% (vol/vol). Individual cell length was measured by computerized image micrometry. The average length of cells in the control state or after addition of test agents was obtained from 50 cells encountered randomly in successive microscopic fields. The contractile response is defined as the decrease in the average length of the 50 cells and is expressed as the absolute change or the percent change from control length (8).

Data analysis. Bands from the Western blot spots were quantitated using a densitometer (Bio-Rad model GS-700, Bio-Rad Laboratories), and the band densities (absorbance units x mm2) were calculated and expressed as percent of total density. The control band intensities were standardized to 100%, and the band intensities of samples from treated cells were compared with the control and expressed as percent change from the control. Each experiment had its own control. All the means were compared and analyzed using Student's t-test. Band data are within the linear range of detection for each antibody used.

Spots were quantitated using a densitometer (model GS-700, Bio-Rad Laboratories), and spot densities (absorbance units x mm2) were calculated and expressed as a percentage of the total density. Spot data are within the linear range of detection for each antibody used. In addition, spots for standard proteins (of GST and PKC-{alpha}) in serial dilutions of 500–1,000 ng were analyzed. The combined intensity of the eluted fractions of the fusion protein (GST-calponin) and the combined intensity of the eluted fractions of the binding protein (PKC-{alpha}) were converted to molar quantities by the plot drawn against the standard protein intensities. Hence, the molar ratios of the proteins interacting were calculated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of acetylcholine on the association of calponin with HSP27. We have previously shown that acetylcholine induces translocation of HSP27 to the membrane as revealed under confocal microscopy (18). We investigated the association of calponin with translocated HSP27. Smooth muscle cells isolated from the rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate and cytosolic preparations were immunoprecipitated with anti-calponin antibody, further separated by SDS-PAGE, and Western blot analyzed with anti-HSP27 antibody. Stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase in the association in the particulate fraction of calponin with HSP27 (236.51 ± 32.28 and 275.39 ± 41.66%, respectively, at 30 s and 4 min compared with controls; P <= 0.05, n = 3; Fig. 1A). No significant changes in the association of calponin with HSP27 were observed at 30 s and 4 min in the cytosolic fractions (Fig. 1B).



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  		Fig. 1. A: effect of acetylcholine on association of HSP27 with calponin in the particulate fraction. Immunoprecipitates of calponin (anti-calponin antibody) from 500 µg of particulate fractions were subjected to SDS-PAGE and Western blot analysis with anti-heat shock protein (HSP)27 antibody (1:1,000; anti-HSP27 antibody). Inset: representative blot showing an increase in the association of calponin with HSP27 when cells were stimulated with acetylcholine. Stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase (236.51 ± 32.28 and 275.39 ± 41.66%, respectively, at 30 s and 4 min compared with controls *P <= 0.05, n = 3) in the association of calponin with HSP27 at 30 s and 4 min compared with control. B: cytosolic fractions (500 µg) were immunoprecipitated with anti-calponin antibody and were subjected to SDS-PAGE and Western blot analysis with anti-HSP27 antibody (1:1,000). Representative blot (n = 3) shows lack of significant change in association of HSP27 with calponin in the cytosolic fraction of smooth muscle cells stimulated with acetylcholine.

 
Effect of acetylcholine on the association of calponin with tropomyosin. Calponin was originally discovered in chicken gizzard smooth muscle as an F-actin-, calmodulin-, and tropomyosin-binding protein (57). Here, we tested whether calponin associates with tropomyosin on induction of contraction with acetylcholine. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate and cytosolic preparations were immunoprecipitated with anti-calponin antibody, further separated by SDS-PAGE, and Western blot analyzed with anti-tropomyosin antibody. Stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase in the association of calponin with tropomyosin, in the particulate fraction, at 30 s and 4 min (131.38 ± 9.88 and 133.31 ± 9.88%; P <= 0.05, n = 6, respectively), compared with control (Fig. 2A). A decrease in the association of calponin and tropomyosin was observed in the cytosolic fractions (76.89 ± 12.71 and 88.49 ± 4.25%; P <= 0.04, n = 6 at 30 s and 4 min, respectively; Fig. 2B). The observed increases in association in the particulate fraction and the concomitant decrease in the association in the cytosolic fraction indicated translocation calponin and tropomyosin to the particulate fraction.



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  		Fig. 2. A: coimmunoprecipitation of tropomyosin with calponin in the particulate fraction in response to stimulation with acetylcholine in rabbit colon smooth muscle cells. Cells were either untreated or stimulated with acetylcholine (0.1 µM) for 30 s or 4 min. Immunoprecipitates of calponin (anti-calponin antibody) from 500 µg of particulate fraction were subjected to SDS-PAGE and Western blot analysis with anti-tropomyosin antibody (1:500; tropomyosin antibody). Inset: representative blot showing an increase in the association of tropomyosin with calponin when stimulated with acetylcholine. Stimulation with the contractile agonist resulted in a significant and sustained increase (131.38 ± 9.88 and 133.31 ± 9.88%; *P <= 0.05, n = 6 respectively, compared with control) in the association of tropomyosin with calponin at 30 s and 4 min compared with control. B: reduced coimmunoprecipitation of tropomyosin with calponin in the cytosolic fraction in response to agonist stimulation in rabbit colon smooth muscle cells. Cytosolic fractions (500 µg) were immunoprecipitated with calponin antibody and were subjected to SDS-PAGE and Western blot with anti-tropomyosin antibody (1:500). Representative blot showing reduced association (76.89 ± 12.71 and 88.49 ± 4.25%; P <= 0.04, n = 6 at 30 s and 4 min, respectively, compared with control) of tropomyosin with calponin in the cytosolic fraction of smooth muscle cells stimulated with acetylcholine.

 
Effect of acetylcholine on the association of calponin with PKC-{alpha}. It has previously been shown (7) that acetylcholine induces translocation of PKC-{alpha} to the particulate fraction. We investigated the effect of acetylcholine on the association of PKC-{alpha} with calponin. Stimulation of isolated smooth muscle cells with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase in the association of calponin with PKC-{alpha} in the particulate fraction (Fig. 3A; 128.85 ± 10.95 and 137.21 ± 5.1%, respectively, at 30 s and 4 min P <= 0.05, n = 3) compared with control. There was a concomitant decrease (84.62 ± 7.76 and 86.64 ± 13.56%; P <= 0.15, n = 10) in the association of calponin with PKC-{alpha} in the cytosolic fractions at 30 s and 4 min compared with control cells (Fig. 3B).



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  		Fig. 3. A: effect of acetylcholine on association of PKC-{alpha} with calponin in the particulate fraction. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate fractions, as described in the MATERIALS AND METHODS, were immunoprecipitated with anti-calponin antibody, further separated by SDS-PAGE, and Western blot analyzed with anti-PKC-{alpha} antibody. Stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase (128.85 ± 10.95 and 137.21 ± 5.1%, respectively, at 30 s and 4 min compared with controls *P <= 0.05, n = 3) in the association of calponin with PKC-{alpha} at 30 s and 4 min compared with control. B: cytosolic fractions (500 µg) were immunoprecipitated with anti-calponin antibody and were subjected to SDS-PAGE and Western blot analysis with anti-PKC-{alpha} antibody (1:200). Representative blot shows no significant change (84.62 ± 7.76 and 86.64 ± 13.56%; P <= 0.15, n = 10) in the association of PKC-{alpha} with calponin in the cytosolic fraction of smooth muscle cells stimulated to acetylcholine.

 
Translocation of calponin, PKC-{alpha} and HSP27 to the particulate fraction in response to acetylcholine. To confirm that the increase in association of calponin with PKC-{alpha} and of calponin with HSP27 is due to agonist-induced translocation of each of the proteins (calponin, PKC-{alpha}, and HSP27) per se to the particulate fraction, cells were stimulated with acetylcholine (0.1 µM) for 30 s or 4 min, and equal amounts (50 µg) of particulate fractions and cytosolic fractions were separated by SDS-page and subjected to Western blot analysis against anti-calponin antibody, anti-PKC-{alpha} antibody, or anti-HSP27 antibody.

Western blot analysis against anti-calponin antibody indicate that acetylcholine induced an increase in the amount of calponin in the particulate fraction (118.87 ± 10.14 and 144.31 ± 14.54% increase at 30 s and 4 min, respectively; P <= 0.05, n = 3; Fig. 4A). There was either no change or a decrease in the calponin in cytosolic fractions (110.28 ± 8.74 and 81.69 ± 12.06% at 30 s and 4 min, respectively; P <= 0.15, n = 3; Fig. 4B). Furthermore, immunoprecipitates of anti-calponin antibody subjected to Western blot analysis against anti-calponin antibody indicates an increase in the amount of calponin immunoprecipitated from the particulate fractions of cells treated with acetylcholine for 30 s or 4 min (Fig. 4C).



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  		Fig. 4. Effect of acetylcholine on translocation of calponin to the particulate fraction of rabbit colon smooth muscle cells. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate fractions and the cytosolic fractions were separated by SDS-page and Western blot analyzed with anti-calponin antibody. A: stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant sustained increase of calponin in the particulate fraction (118.87 ± 10.14 and 144.31 ± 14.54% increase at 30 s and 4 min, respectively; *P < 0.05, n = 3). B: there was no significant change and a decrease in the amount of calponin in the cytosolic fractions (110.28 ± 8.74 and 81.69 ± 12.06%; P <= 0.15, n = 3% at 30 s and 4 min, respectively). C: immunoprecipitates of anti-calponin antibody subjected to Western blot analysis against anti-calponin antibody confirmed the previous results of sustained increase in calponin (lanes 1–3, arrow) in the particulate fraction. Note that the IGG (heavy chain) bands indicate that equal amount of antibody was used in immunoprecipitation experiments.

 
Similarly, Western blot analyses against anti-PKC-{alpha} antibody indicate that acetylcholine induced an increase in the amount of PKC-{alpha} in the particulate fraction (155.55 ± 40.04 and 120.66 ± 20.0% at 30 s and 4 min, respectively; P <= 0.05, n = 3; Fig. 5A). There was no change of PKC-{alpha} in the cytosolic fractions (97.45 ± 18.67 and 87.32 ± 17.96% at 30 s and 4 min, respectively; P <= 0.5, n = 3; Fig. 5B). Furthermore, immunoprecipitates of anti-PKC-{alpha} antibody subjected to Western blot analysis against anti-PKC-{alpha} antibody indicated an increase in the amount PKC-{alpha} immunoprecipitated from the particulate fractions of cells treated with acetylcholine for 30 s or 4 min (Fig. 5C).



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  		Fig. 5. Effect of acetylcholine on translocation of PKC-{alpha} to the particulate fraction of rabbit colon smooth muscle cells. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate fractions and the cytosolic fractions were separated by SDS-page and Western blot analyzed with anti-PKC-{alpha} antibody. A: stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase of PKC-{alpha} in the particulate fraction (155.55 ± 40.04 and 120.66 ± 20.0% at 30 s and 4 min, respectively; P <= 0.05, n = 3). B: there was no significant change in the amount of PKC-{alpha} in the cytosolic fractions (97.45 ± 18.67 and 87.32 ± 17.96% at 30 s and 4 min, respectively; P <= 0.1, n = 3). C: immunoprecipitates of anti-PKC-{alpha} antibody were subjected to Western blot analysis against anti-PKC-{alpha} antibody; there was an observed increase in the amount of PKC-{alpha} immunoprecipitated from the particulate fractions of cells treated with acetylcholine for 30 s or 4 min. Note that the IGG bands indicate that an equal amount of antibody was used in immunoprecipitation experiments.

 
Similarly, Western blots against anti-HSP27 antibody indicate that acetylcholine induced an increase in the amount of HSP27 in particulate fraction (161.27 ± 41.55 and 129.98 ± 17.90 at 30 s and 4 min, respectively; P <= 0.05, n = 3; Fig. 6A). There was a decrease in the amount of HSP27 in the cytosolic fractions (80.20 ± 15.09 and 72.27 ± 18.14 at 30 s and 4 min, respectively; P <= 0.1, n = 3; Fig. 6B). It has been previously reported (7) that the increase in association of HSP27 with PKC-{alpha} was due to the increase in the amount of HSP27 translocated to the particulate fraction.



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  		Fig. 6. Effect of acetylcholine on translocation of HSP27 to the particulate fraction of rabbit colon smooth muscle cells. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate fractions and the cytosolic fractions were separated by SDS-page and Western blot analyzed with anti-HSP27 antibody. A: stimulation with the contractile agonist acetylcholine (0.1 µM) resulted in a significant and sustained increase of HSP27 in the particulate fraction (161.27 ± 41.55 and 129.98 ± 17.90% at 30 s and 4 min, respectively; P <= 0.05, n = 3). B: there was no significant change in the amount of HSP27 in the cytosolic fractions (80.20 ± 15.09 and 72.27 ± 18.14% at 30 s and 4 min, respectively; P <= 0.1, n = 3).

 
Expression and purification of GST-calponin fusion protein. PCR amplification of the donor cDNA, pBS KS+ containing 1.3 kb of calponin cDNA yielded a product of ~0.7-kb corresponding to nt 352–972 (coding for aa 92–298 of human basic calponin) as confirmed by DNA sequencing. This DNA was cloned into the vector pGEX-KT at BamH1 and EcoR1 restriction sites "in-reading-frame" with GST. The clones were selected against ampicillin. The bacterial clones containing pGEX-KT calponin were induced with IPTG. The expressed protein was a product of ~56-kDa molecular mass that could be detected both by anti-GST antibody or anti-calponin antibody (Fig. 7; only GST antibody shown). A yield of 700 µg of the fusion protein per 500 ml of the culture was obtained by IPTG induction (0.2 mM) at 37°C for 3 h.



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  		Fig. 7. Expression and purification of glutathione S-transferase (GST)-calponin fusion protein. Eschericia coli cells transformed with the plasmid pGEX-KT containing GST-calponin at EcoRI and BamHI were grown to 0.6 OD and were induced with 0.2 mM isopropyl thio-{beta}-D-galactoside at 37°C for 3 h. Cells were harvested and lysed. The lysate was applied to a 10-ml glutathione agarose column. The flow through was collected as unbound. The column was washed with PBS (pH 7.4) containing 1% Triton X-100. The bound proteins were then eluted with 10 mM reduced glutathione buffer. The fractions collected were subjected to SDS-PAGE followed by Western blot analysis against anti-GST antibody. Lane 1: standard GST protein; lane 2: unbound fraction of cell lysates; lane 3–4: fractions after PBS wash; lanes 5–7: elution with reduced glutathione buffer. note that the standard gst protein runs at 27 kDa (lane 1, arrow). The fusion protein runs at 56 kDa (lanes 5–7, arrow).

 
Direct association of recombinant PKC-{alpha} with GST-calponin fusion protein. To examine whether the observed interaction of the proteins in smooth muscle cells is direct, the recombinant calponin fusion protein conjugated to glutathione agarose was incubated with recombinant PKC-{alpha}. Examination of the dot blots of the unbound, the wash, and the eluted fractions indicated that GST-calponin fusion protein and PKC-{alpha} coeluted, indicating a direct interaction of calponin and PKC-{alpha} at a molar ratio of 0.269 ± 0.028 (Fig. 8; n = 4). Glutathione agarose beads conjugated to GST alone were used as controls. In these experiments, PKC-{alpha} and GST did not coelute, thus not revealing any binding of PKC-{alpha} with GST alone (fractions 7–10), indicating that the association of calponin-PKC-{alpha} was specific and not due to an association of PKC-{alpha} with GST (Fig. 9).



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  		Fig. 8. Dot blots showing direct association of calponin and PKC-{alpha}. GST-calponin (24 µg) was incubated with 200 µl of 50% suspension of glutathione-agarose in PBST/0.1% {beta}-mercaptoethanol at 4°C for 30 min. All the washes and the eluates were spotted on nitrocellulose membrane as detailed in MATERIALS AND METHODS, and they were Western blot analyzed with antibodies specific for GST, calponin, or PKC-{alpha}. A-C are dot blots in duplicates with antibodies specific for GST, calponin, and PKC-{alpha}, respectively. Fractions 1–3 are washings of unbound GST-calponin or calponin alone. Recombinant human PKC-{alpha} (24 µg) was added to the washed beads and incubated at room temperature. Fractions 4–6 are washings of unbound PKC-{alpha} and indicate washing of unbound PKC-{alpha}. Fractions 7–10 are eluates of the agarose beads with 10 mM glutathione. In fractions 7–10, there was coelution of PKC-{alpha} with GST-calponin, an indication of direct association of recombinant PKC-{alpha} with GST-calponin.

 


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  		Fig. 9. For control experiments, 24 µg of GST alone were incubated with 200 µl of 50% suspension of glutathione-agarose in PBST/0.1% {beta}-mercaptoethanol at 4°C for 30 min. All the washes and the eluates were spotted on nitrocellulose membrane as detailed in MATERIALS AND METHODS and were Western blot analyzed with antibodies specific for GST, PKC-{alpha}, or calponin. A-C are dot blots in duplicates with antibodies specific for GST, PKC-{alpha}, and calponin, respectively. Fractions 1–3 are washings of unbound GST alone. Recombinant human PKC-{alpha} (24 µg) was added to the washed beads and incubated at room temperature. Fractions 4–6 are washings of unbound PKC-{alpha}. Fractions 7–10 are eluates of the agarose beads with 10 mM glutathione. There was no coelution in fractions 7–10. Note that in C, GST-calponin was spotted as a positive control for the anti-calponin immunoblot.

 
Effect of preincubation with anti-calponin antibody, anti-PKC-{alpha} antibody, or anti-HSP27 antibody on acetylcholine-induced contraction of colonic smooth muscle cells. To examine the physiological involvement of calponin, HSP27, or PKC-{alpha} in acetylcholine-induced contraction of smooth muscle cells, isolated smooth muscle cells from the rabbit colon permeabilized with saponin (75 µg/ml) were preincubated with anti-calponin antibody (1:1,000), anti-PKC-{alpha} antibody (1:100), or anti-HSP27 antibody (1:5,000) and were stimulated with acetylcholine (10–7 M) for 30 s or 4 min. Control saponified cells exhibited sustained contraction in response to acetylcholine (50.81 ± 0.55% decrease in cell length at 30 s and 56.05 ± 3.09% at 4 min; Fig. 10). Preincubation of cells with anti-calponin antibody resulted in a reduced contractile response to acetylcholine (9.02 ± 2.01 and 8.03 ± 1.75% decrease in cell length at 30 s and at 4 min, respectively; Fig. 10). Preincubation of cells with anti-PKC-{alpha} antibody alone or in combination with anti-calponin antibody inhibited acetylcholine-induced decreases in cell length (6.52 ± 0.06 and 5.37 ± 1.07% at 30 s and 4 min, respectively, with anti-PKC-{alpha} antibody alone; 4.63 ± 1.08 and 2.60 ± 0.73% decrease at 30 s and 4 min, respectively, in combination of anti-PKC-{alpha} antibody and anti-calponin antibody). Preincubation of cells with anti-HSP27 antibody also inhibited the acetylcholine-induced decrease in the cell length (4.01 ± 2.02 and 3.61 ± 0.34% decrease at 30 s and 4 min, respectively). Nonsaponified cells showed normal response to acetylcholine (42.86 ± 1.67 and 43.01 ± 2.65% decrease in cell length at 30 s and 4 min, respectively).



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  		Fig. 10. Effect of preincubation with anti-calponin antibody, anti-PKC-{alpha} antibody, or anti-HSP27 antibody on acetylcholine-induced contraction of colonic smooth muscle cells. Isolated smooth muscle cells were permeabilized by saponification and were incubated with anti-calponin antibody (1:1,000) for 20 min. The cells were further stimulated with acetylcholine (10–7 M) for 30 s or 4 min. The reaction was arrested by 1% acrolein (vol/vol). Cell lengths were measured by computerized image micrometry. Sustained contraction was greatly inhibited ({blacksquare}; 9.02 ± 2.01 and 8.03 ± 1.75% decrease in cell length at 30 s and at 4 min, respectively; n = 3) in saponified cells preincubated with anti-calponin antibody (1:1,000) or PKC-{alpha} antibody alone (1:100 {blacktriangleup}) or in combination with anti-calponin antibody ({circ}). Preincubation of cells with anti-HSP27 antibody similarly inhibited acetylcholine-induced contraction (X; 1:5,000) compared with control cells ({blacklozenge}; 50.81 ± 0.55% decrease in cell length at 30 s and 56.05 ± 3.09% at 4 min; n = 3).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Because of its ability to bind to actin, calponin is hypothesized to be a cytoskeleton regulatory protein functioning as a bridge between actin and the intermediate filament network in smooth muscles (28). The smooth muscle specific variant of calponin was originally identified as actin-associated protein from chicken gizzard (49). Potential binding partners for calponin include calmodulin, s100 proteins, tropomyosin, myosin, and caldesmon (43). A direct interaction of calponin with phospholipids and with HSP90 has also been suggested (9, 13).

Immunoprecipitation with anti-calponin antibody followed by Western blot analysis against either anti-HSP27 or anti-PKC-{alpha} antibody indicated an increase in the amount of associated proteins in the particulate fraction. The increase was evident at 30 s after stimulation with acetylcholine and was sustained at 4 min (Figs. 1A and 3A). There is a concomitant decrease in the acetylcholine-induced association of calponin with PKC-{alpha} in the cytosolic fractions. This is indicative of increased association and translocation of calponin with PKC-{alpha} in response to acetylcholine in the smooth muscle cells.

In the present studies, we have shown that calponin associates with tropomyosin also in the particulate fraction. In skeletal and cardiac muscle, tropomyosin plays a central role in the regulation of contraction by binding to the myosin-binding site on actin in the resting state (1). The physiological role of tropomyosin in smooth muscle cells is poorly understood. Calponin is a smooth muscle tropomyosin-binding protein that is antigenically related to troponin T (48, 50). Present results show acetylcholine-induced sustained increase in the association of calponin with tropomyosin. This is in corroboration with earlier reports indicating an interaction between calponin and tropomyosin (12). Calponin has been shown to be associated with tropomyosin in toad stomach smooth muscles, and their distribution is uniform throughout the cell as shown by fluorescent microscopy (10). Phosphorylation of calponin decreases its affinity toward actin and tropomyosin in the cytoplasm (37). In the present observations, association of calponin with tropomyosin in the particulate fraction increased at 30 s and remained sustained 4 min after stimulation with acetylcholine (Fig. 2A). It has previously been shown (54, 60) that during agonist-induced contraction, HSP27 phosphorylates and redistributes to the particulate fraction. It has also been shown (18) that HSP27 associates with tropomyosin. Thus, during agonist-induced contraction, translocation of HSP27 would result in the formation of a functional complex among tropomyosin, calponin, and PKC-{alpha} in smooth muscle. Whether tropomyosin or HSP27 interacts directly with calponin in rabbit colon smooth muscle is presently not clear.

Association and redistribution of calponin along with PKC on agonist stimulation has been previously reported in vascular smooth muscle (39, 40). In the relaxed state of the cell, calponin is distributed throughout the cell cytoplasm of vascular smooth muscle cells (40). In the present studies, reduction of the associated proteins in the cytosolic fraction indicated a translocation of the protein. What mediated the translocation of calponin was not clear. In previous studies (60), results have indicated that contractile agonists induce a sustained contraction of smooth muscle cells through a pathway that involves the activation of MAP kinase, and that HSP27 is phosphorylated during agonist-induced contraction. It has also been shown (7) that there are translocation and sustained increase in the association of HSP27 with PKC-{alpha} in the particulate fractions of rabbit colon smooth muscle cells, which were inhibited on preincubation of the cells with the PKC inhibitor calphostin C. Current results suggest a sustained agonist-induced complexing of calponin with HSP27 and of calponin with PKC-{alpha} in smooth muscle from the rabbit colon.

To examine whether the association between calponin and PKC-{alpha} seen in rabbit colon smooth muscle is direct, we produced GST-calponin fusion protein in E. coli using plasmid expression vectors. Although the fusion protein contained aa 92–298, it included the putative phosphorylation site (Ser175) (21). The fusion protein could also be identified by anti-calponin monoclonal antibody, indicating that the expressed fusion protein was functional. The fusion protein was immobilized on suitable affinity gel substrate (Glutathione agarose) and was tested for its interaction with recombinant PKC-{alpha}. Examination of the dot blots of the fractions collected from the in vitro binding studies indicated a direct association of recombinant PKC-{alpha} with GST-calponin fusion protein (Fig. 8). Furthermore, to test whether the association was specific, we used GST-alone as control, and the eluates did not reveal any binding of PKC-{alpha} with GST alone (Fig. 9). Direct association of calponin with PKC-{alpha} explains the basal association of calponin with PKC-{alpha} in unstimulated smooth muscle cells.

It has previously been reported (7) that HSP27 modulates association of translocated PKC-{alpha} and RhoA in rabbit colon smooth muscle cells. Therein, it was demonstrated that the increased association of HSP27 with PKC-{alpha} is due to increased translocation of the individual proteins per se. Present results suggest that calponin does not significantly interact with HSP27 or with PKC-{alpha} in the cytosolic fraction (Figs. 1B and 3B). However, the present results also suggest that calponin interacts directly with PKC-{alpha} in vitro. This would support the fact that there is always a basal association of these proteins in the cytosolic fraction. However, results indicate an increased association of calponin with PKC-{alpha} and with HSP27 in the particulate fraction on stimulation with acetylcholine. It is possible that the phosphorylation of HSP27 results in a greater affinity for binding of PKC-{alpha} with calponin.

Leinweber et al. (24) showed that calponin interacts with PKC-{epsilon} at the regulatory domain in vitro and that aa 160–182 of calponin seem to be necessary for its interaction with PKC-{epsilon}. Calponin has been shown to inhibit actin-activated myosin ATPase activity in reconstituted contractile protein systems, and this inhibition is reversed by phosphorylation catalyzed in vitro by PKC or Ca+2/calmodulin-dependent protein kinase II (CaM kinase II) (35–37, 58). Calponin is also a well-established in vitro substrate for PKC as well as a possible in vivo substrate for PKC (56). Rokolya et al. (42) demonstrated that the physiological kinase for calponin phosphorylation is PKC. Studies (32) from other laboratories have indicated that PKC activity is related to its subcellular localization. Many investigators (2, 7, 23) have described association of PKC to the plasma membrane on stimulation of smooth muscles. Membrane association is reflected in a shift in subcellular localization and translocation from cytosolic PKC to membrane compartments. This process is controlled by protein-protein interactions that play an important role in localization and function of PKC isozymes. The interaction between PKCs and cytoskeletal proteins is isozyme selective. Current results indicating a translocation and direct association of calponin with PKC-{alpha} suggest that calponin may form a substrate for PKC-{alpha} in colon smooth muscle cells.

We propose that of the low molecular weight HSPs, HSP27, which is known for its chaperon activity, could participate in the interaction with calponin and its transportation for proper alignment in the cytoskeleton. Immunoprecipitation with calponin antibody followed by Western blot analysis with HSP27 antibody (Fig. 1A) indicated that calponin interacts with HSP27. HSP90 have been shown to interact with calponin (27). The literature does not provide evidence for a direct interrelationship between HSP27 and calponin. Data presented here show that both PKC-{alpha} and HSP27 coimmunoprecipitate with calponin in the particulate fraction. A possible explanation could be due to a direct association of calponin with PKC-{alpha} and that HSP27 possibly mediates the association of translocated PKC-{alpha} with calponin.

Agonist-induced smooth muscle contractions are not entirely dependent on MLC phosphorylation (19). MLC phosphorylation did not change in ferret aorta smooth muscles loaded with antisense calponin RNA during phenylephrine-induced contraction. Ferret aorta smooth muscle strips loaded with antisense calponin RNA also showed decreases in the amplitude of phenylephrine-induced contraction (20). Matthew et al. (29) reported an increase in shortening velocity of smooth muscle from the bladder and vas deference of calponin knockout mouse. Agonist-induced contraction was not addressed in mice lacking calponin (29). In the present studies, we have used isolated permeabilized smooth muscle cells to examine the involvement of calponin during agonist-induced contraction. Present results suggest that preincubation of cells with anti-calponin antibody inhibits agonist-induced smooth muscle contraction. Walsh (53) suggested that calponin might play an important role in the regulation of agonist-induced contraction of tonic smooth muscles. Thus our results are in agreement with other reports indicating an important role for calponin in agonist-induced smooth muscle contraction. Furthermore, both calponin and HSP27 being actin-binding proteins, the direct interaction between calponin and PKC-{alpha} would suggest the possibility that HSP27 could mediate the association between signaling and contractile molecules. Present results confirm our previously published data indicating a role for both PKC-{alpha} and HSP27 in agonist-induced smooth muscle contraction (5, 8, 59). In summary, interaction of PKC-{alpha} with calponin and with HSP27 is indicative of interplay between signaling and contractile proteins. Whether HSP27 interacts directly with calponin is not certain at present. The mechanism by which HSP27 may play a role in association with calponin to regulate smooth muscle contraction needs to be examined.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-57020.


   ACKNOWLEDGMENTS
 
We thank Dana Thomas for assistance with technical editing and figure development.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Division of Pediatric Gastroenterology, Univ. of Michigan Medical School, 1150 West Medical Center Dr., MSRB 1, Rm. A520, Ann Arbor, MI 48109-0656 (E-mail: bitar{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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