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Am J Physiol Gastrointest Liver Physiol 291: G630-G639, 2006. First published April 20, 2006; doi:10.1152/ajpgi.00350.2005
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NEUROREGULATION AND MOTILITY

Phosphorylated HSP27 modulates the association of phosphorylated caldesmon with tropomyosin in colonic smooth muscle

Sita Somara and Khalil N. Bitar

Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan

Submitted 26 July 2005 ; accepted in final form 17 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Thin-filament regulation of smooth muscle contraction involves phosphorylation, association, and dissociation of contractile proteins in response to agonist stimulation. Phosphorylation of caldesmon weakens its association with actin leading to actomyosin interaction and contraction. Present data from colonic smooth muscle cells indicate that acetylcholine induced a significant association of caldesmon with PKC{alpha} and sustained phosphorylation of caldesmon at ser789. Furthermore, acetylcholine induced significant and sustained increase in the association of phospho-caldesmon with heat-shock protein (HSP)27 with concomitant increase in the dissociation of phospho-caldesmon from tropomyosin. At the thin filament level, HSP27 plays a crucial role in acetylcholine-induced association of contractile proteins. Present data from colonic smooth muscle cells transfected with non-phospho-HSP27 mutant cDNA indicate that the absence of phospho-HSP27 inhibits acetylcholine-induced caldesmon phosphorylation. Our results further indicate that the presence of phospho-HSP27 significantly enhances acetylcholine-induced sustained association of phospho-caldesmon with HSP27 with a concomitant increase in acetylcholine-induced dissociation of phospho-caldesmon from tropomyosin. We thus propose a model whereby upon acetylcholine-induced phosphorylation of caldesmon at ser789, the association of phospho-caldesmon (ser789) with phospho-HSP27 results in an essential conformational change leading to dissociation of phospho-caldesmon from tropomyosin. This leads to the sliding of tropomyosin on actin thus exposing the myosin binding sites on actin for actomyosin interaction.

acetylcholine; heat-shock protein; protein kinase C

SMOOTH MUSCLE CONTRACTION is modulated by thick- and thin-filament regulatory pathways (53, 59). Contraction occurs when the myosin heads of the thick filament interact with the actin molecules of the thin filament. The actomyosin interaction is controlled by regulatory proteins that respond to changes in intracellular Ca2+levels. Increase in intracellular calcium levels leads to formation of calcium-calmodulin complex that modulates thick- and thin-filament regulatory pathways. Thick-filament regulation is mediated by Ca2+/calmodulin-dependent activation of myosin light chain (MLC) kinase (MLCK) leading to phosphorylation of MLC20. Inactivation of MLC phosphatase (MLCP) leads to maintenance of phosphorylation of MLC20 (12, 30, 44, 50, 51). Thin-filament regulation is achieved by the thin-filament binding proteins tropomyosin, caldesmon, and calponin (25, 40, 59). Caldesmon, a major thin-filament calcium-sensitizing regulatory protein, plays an important role in regulating smooth muscle contraction at the thin filament level (54, 56, 57).

Caldesmon, an ~87 kDa thin-filament associated protein, regulates smooth muscle contraction by preventing the binding of myosin heads to actin in a calcium/calmodulin-dependent manner (27, 36, 47). Caldesmon binds to actin and to tropomyosin in a conformation where the tropomyosin strand is positioned on the outer edge of the actin monomer. Such a conformation results in blocking the strong myosin-binding sites on actin (16, 34, 46). An increase in intracellular calcium levels results in two main events affecting caldesmon affinity towards actin: 1) binding of calcium/calmodulin to caldesmon results in the formation of a calcium-calmodulin-caldesmon complex (26), which results in weakening of caldesmon affinity for actin (36); 2) phosphorylation of caldesmon weakens the affinity of caldesmon toward actin (26).

Caldesmon can be phosphorylated on different putative phosphorylation sites by different pathways. Kinases such as PKC (Calcium/phospholipid-dependent enzyme) and MAP kinases (p38, ERK p42 and ERK p44) phosphorylate caldesmon at ser759 and ser789; calcium/calmodulin kinase II (CK II) phosphorylate caldesmon at ser73, Thr83 (1–3, 10, 22, 52). In most reported cases and in porcine carotid arteries, ser789 is the main phosphorylation site (21).

A small heat-shock protein, HSP27, undergoes a significant and sustained phosphorylation in response to contractile agonists in colonic smooth muscle cells (6, 33). HSP27 phosphorylation has an impact on the signaling pathways as well as on the thin-filament regulation of colonic smooth muscle contraction. Stimulation of freshly isolated colonic smooth muscle cells with contractile agonists induces coimmunoprecipitation of HSP27 with contractile proteins such as actin, tropomyosin, and caldesmon (28). In colonic smooth muscle cells, in response to agonist stimulation, phosphorylated HSP27 has been shown to modulate ACh-induced association of HSP27 with tropomyosin (48). Colonic smooth muscle cells transfected with non-phospho-HSP27 mutant results in decrease in acetylcholine-induced association of tropomyosin with HSP27 (48). Thus phosphorylation of HSP27 plays an important role in the regulation of smooth muscle contraction at the level of thin-filament protein interaction.

Our data indicate that acetylcholine-induced phosphorylation of caldesmon at ser789. Furthermore, our data indicate the essential role of phosphorylated HSP27 in the association of phospho-caldesmon with HSP27 with concomitant dissociation of phospho-caldesmon from tropomyosin. We thus propose a model whereby acetylcholine induces phosphorylation of caldesmon at ser789. Association of ser789 phospho-caldesmon with phospho-HSP27 results in an essential conformational change leading to dissociation of phospho-caldesmon from tropomyosin. This results in the sliding of tropomyosin on actin thus exposing the myosin binding sites on actin.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Materials

The following reagents were purchased: monoclonal mouse anti-caldesmon antibody (immunogen: human uterus smooth muscle extract) from Sigma (St. Louis, MO); anti-phospho-caldesmon (ser789) antibody; anti-phospho-PKC{alpha} (ser657) antibody from Upstate Biotechnology (Lake Placid, NY); monoclonal anti-tropomyosin antibody (immunogen: chicken gizzard tropomyosin) and anti-caldesmon antibody from Sigma; and monoclonal mouse anti-huHSP27 antibody (2B4–123) previously described (7); polyvinylidene fluoride (PVDF) membranes from Bio-Rad Laboratories (Hercules, CA); protein G Sepharose and enhanced chemiluminescence (ECL) detection reagents from Amersham Biosciences (Piscataway, NJ); G-418, penicillin/streptomycin, FBS, collagen IV, and DMEM from GIBCO-BRL (Grand Island, NY); and collagenase type II from Worthington (Lakewood, NJ). All other reagents were purchased from Sigma.

Methods

Preparation of smooth muscle cells from the rabbit rectosigmoid. Smooth muscle cells of rabbit rectosigmoid were isolated as described previously (5). Briefly, the internal anal sphincter (IAS) from anesthetized New Zealand White rabbits, 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 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 collagenase containing 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 containing 0.1% (wt/vol), collagenase (150 U/mg), 0.01 (wt/vol) soybean trypsin inhibitor, and 0.184 (wt/vol) into DMEM. At the end of the second enzymatic incubation, the medium was filtered through 500-µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 10 ml 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 10–20 x 106 cells.

Preparation of particulate fractions. Particulate fractions from freshly isolated smooth muscle cells were prepared as described previously (43). Briefly, freshly isolated smooth muscle cells, incubated with or without inhibitors for 20 min, were treated with acetylcholine (0.1 µM) for 30 s and for 4 min and immediately frozen in slurry of acetone and dry ice. After stimulation, 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 Na4P2O7·10 H2O, 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 7.40)]. The sonicated cells were centrifuged at 100,000 g for 60 min. The supernatant was collected as a cytosolic fraction while the pellet was resuspended in the lysis buffer B with 1% Triton X-100 and sonicated twice for 30 s each time and collected as a particulate fraction. The protein content was determined using a protein assay reagent (Bio-Rad).

Immunoprecipitation and immunoblotting. One to two micrograms of antibody were added to 500 µg of sample protein in 500 µl of lysis buffer and rocked overnight at 4°C. Fifty microliters of 50% protein G-Sepharose bead slurry were then added, and the mixture was 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. The supernatant was discarded, and the bead pellet was washed three times at room temperature with TBS bead wash buffer (20 mM Tris·HCl, 150 mM NaCl, pH 7.6). The beads were then resuspended in 25 µl of 2 x sample buffer and boiled for 5 min. Proteins from the immunoprecipitates were separated on SDS-PAGE and transferred to PVDF membrane. The membrane was immunoblotted with the desired antibodies as described previously (48). Replicates of experiments were performed using completely separate sets of cells.

Western blot. The proteins were separated on SDS-PAGE and electrophoretically transferred to PVDF membrane as described previously (48). The membrane was blocked with 5% nonfat dry milk for 1 h and incubated in an appropriate dilution of primary antibody in 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. The membrane was washed thrice with TBST to remove unbound primary antibody for 15 min each wash at room temperature. 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 wash 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.

Transfection of smooth muscle cells with HSP27 mutants. Two HSP27 mutants were used in these studies: 1) 3GHSP27 mutant, where all three phosphorylation sites were mutated to glycine to mimic nonphosphorylatable HSP27 and 2) 3DHSP27 mutant, where all three phosphorylation sites were mutated to aspartate to mimic constitutively phosphorylated HSP27. The cDNAs encoding 3GHSP27 and 3DHSP27 cloned in vectors pcDNA3.1 were obtained from Dr. Benndorf (University of Michigan). Smooth muscle cells were transfected with HSP27 mutant cDNA using QiaGen Effectene transfection kit as described previously (48).

Data analysis. Data analysis was done as described previously (48). Briefly, Western blot bands were quantitated using 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 intensity was standardized to 100%. The band intensities of samples from treated cells were compared with the control and expressed as percent change from the control. "n" represents the number of different animals used to isolate different set of cells. The experimental protocol calls for cells isolated from circular muscle of the colon of each animal to be divided into three groups: 1) control unstimulated cells; 2) cells stimulated with acetylcholine for 30 s; and 3) cells stimulated with acetylcholine for 4 min. In each set of experiments, the response obtained on stimulation with acetylcholine at 30 s was compared with control unstimulated cells, and the response obtained on stimulation with acetylcholine at 4 min was compared with the same control unstimulated cells using Student’s t-test. Similarly, the cells transfected with phospho-HSP27 mutant cDNA were compared with the same control unstimulated cells. The response was considered significant at 0.05. The mean values are the mean of the response obtained from different set of cells isolated from different animal, which is represented by n.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Acetylcholine-Induced PKC-Mediated Phosphorylation of Caldesmon in Rabbit Colonic SMC

Phosphorylation of caldesmon has been known to activate myosin Mg2+-ATPase leading to contraction (41). Concurrent to previous studies suggesting a crucial role of phospho-caldesmon in smooth muscle contraction, we investigated the phosphorylation status of caldesmon in colonic smooth muscle cells in response to 0.1 µM acetylcholine. Rabbit colon smooth muscle cells were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Particulate fractions of stimulated and unstimulated colonic smooth muscle cells were separated on SDS-PAGE and transferred to a PVDF membrane. The membrane was then immunoblotted with anti-phospho-caldesmon (ser789) antibody. Stimulation of colonic smooth muscle cells with acetylcholine resulted in a significant and sustained increase in the phosphorylation of ser789 on caldesmon [278.89 ± 8.37% at 30 s and 299.99 ± 10.28% at 4 min compared with control (100); P ≤ 0.001; n = 3 (Fig. 1)].


Figure 1
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  		Fig. 1. ACh-induced PKC-mediated phosphorylation of caldesmon in rabbit colonic smooth muscle cells (SMC). Freshly isolated rabbit colon SMC were stimulated with 0.1 µM ACh for 30 s and 4 min with or without a 20-min preincubation with PKC inhibitor calphostin-C or GF109203X. Equal amounts (50 µg) of particulate fractions were separated on SDS-PAGE. Separated proteins were probed with anti-phospho-caldesmon (ser789) antibody. ACh induced a significant and sustained increase in the phosphorylation of ser789 on caldesmon compared with control. Preincubation with PKC inhibitors calphostin-C or GF109203X inhibited ACh-induced increase in phosphorylation of ser789 on caldesmon in freshly isolated rabbit colon SMCs.

 
We also investigated the effect of the PKC inhibitors, calphostin C (interacts with the protein’s regulatory domain by competing the binding site of diacylglycerol and phorbol esters) and GF109203X (acts as a competitive inhibitor for the ATP-binding site of PKC), on acetylcholine-induced phosphorylation of caldesmon. Freshly isolated rabbit colon smooth muscle cells were preincubated either with calphostin C (10 µM) or with GF109203X (1 µM) for 20 min before stimulation with 0.1 µM acetylcholine. Preincubation with calphostin C or with GF109203X inhibited the acetylcholine-induced increase in phosphorylation of ser789 on caldesmon (calphostin C: 53.50 ± 4.04% at 0 s; 70.77 ± 1.70% inhibition at 30 s and 75.41 ± 2.21% at 4 min; P ≤ 0.05; n = 3; GF109203X: 76.08 ± 4.74% at 0 s, 88.11 ± 2.68% at 30 s and 97.52 ± 3.76% at 4 min; P ≤ 0.05; n = 3) in freshly isolated rabbit colon smooth muscle cells (Fig. 1). The data indicate that acetylcholine-induced caldesmon phosphorylation at ser789 is PKC-mediated in rabbit colonic smooth muscle cells.

Acetylcholine-Induced Translocation of Phosphorylated PKC{alpha} in Rabbit Colonic SMC

We have previously shown that acetylcholine induces translocation of PKC{alpha} to the particulate fraction (43). Immunoblotting cytosolic and particulate fractions of freshly isolated rabbit colonic smooth muscle cells against anti-phospho-PKC{alpha} (ser657) indicate that acetylcholine induced a significant increase in the amount of phospho-PKC{alpha} (ser657) in the particulate fraction (149.61 ± 8.10% at 30 s and 176.15 ± 12.58% at 4 min; P ≤ 0.05, n = 3) while there was a decrease in the amount of phospho-PKC{alpha} in the cytosolic fraction (91.93 ± 0.08% at 30 s and 84.06 ± 2.47% at 4 min; P ≤ 0.1, n = 3) (Fig. 2). Representative blot is presented to show a sustained increase in the amount of phospho-PKC{alpha} in the particulate fraction.


Figure 2
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  		Fig. 2. ACh-induced translocation of phosphorylated PKC{alpha} in rabbit colonic SMC. SMCs isolated from rabbit colon were stimulated with 0.1 µM ACh for 30 s and 4 min. Particulate fractions and the cytosolic fractions were separated on SDS-PAGE and analyzed by Western blot analysis with anti-phospho-PKC{alpha} (ser657) antibody. Stimulation with the contractile agonist ACh (0.1 µM) resulted in a significant and sustained increase of phospho-PKC{alpha} in the particulate fraction, whereas there was no significant change in the amount of phospho-PKC{alpha} in the cytosolic fraction. *P ≤ 0.05.

 
Acetylcholine-Induced Association of Either Caldesmon or Phosphorylated Caldesmon with PKC{alpha} in Rabbit Colonic SMC

Since both PKC{alpha} and caldesmon seem to be phosphorylated and caldesmon phosphorylation is PKC dependent, we studied the direct association of both proteins in phospho and nonphospho states. A significant increase in PKC{alpha} was detected in caldesmon immunoprecipitate (152.10 ± 2.03% at 30 s and 166.32 ± 5.50% at 4 min; P ≤ 0.01; n = 3) in acetylcholine-induced freshly isolated rabbit colon smooth muscle cells. We further noticed that there was a significant decrease of PKC{alpha} in phospho-caldesmon (ser789) immunoprecipitate (91.66 ± 7.08% at 30 s and 73.85 ± 3.76% at 4 min; P ≤ 0.01; n = 3) (Fig. 3A). Similar results occurred when caldesmon and phospho-caldesmon immunoprecipitate were probed for phospho-PKC{alpha} (ser657) (IP caldesmon: 168.90 ± 7.35% at 30 s and 172.79 ± 4.09% at 4 min; P ≤ 0.005; n = 3; IP phospho-caldesmon: 79.92 ± 4.50% at 30 s and 62.98 ± 0.94% at 4 min; P ≤ 0.02; n = 3) (Fig. 3B). The data indicate that caldesmon binds to phospho-PKC{alpha} resulting in phosphorylation of caldesmon at ser789. On phosphorylation of caldesmon, phospho-PKC{alpha} dissociates from caldesmon. The association seems to be dynamic between the signaling molecule PKC{alpha} and the contractile protein caldesmon. This association seems to depend on the translocation and phosphorylation of PKC{alpha}. The dynamic association of PKC{alpha} and caldesmon may modulate the association of caldesmon with tropomyosin and HSP27.


Figure 3
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  		Fig. 3. ACh-induced association of either caldesmon or phosphorylated caldesmon with PKC{alpha} in rabbit colonic SMC. Freshly isolated rabbit colon SMCs were stimulated with 0.1 µM ACh for 30 s and 4 min. Particulate fractions (500 µg) were immunoprecipitated (IP) with anti-caldesmon antibody and anti-phospho-caldesmon (ser789) antibody separately. A: immunoblotting (IB) of immunoprecipitates with anti-PKC{alpha} antibody (1:500) showed a significant and sustained increase in ACh-induced association of caldesmon with PKC{alpha} with a concomitant decrease in association of phosphorylated caldesmon with PKC{alpha} in the particulate fractions compared with control. *P ≤ 0.05. B: immunoblotting of immunoprecipitates with anti-phospho-PKC{alpha} antibody showed similar significant and sustained increase in ACh-induced association of caldesmon with phospho-PKC{alpha} in the particulate fractions with concomitant decrease in association of phosphorylated caldesmon with phospho-PKC{alpha} in the particulate fractions compared with control. *P ≤ 0.05.

 
Acetylcholine-Induced Association of Phosphorylated Caldesmon with HSP27 in Rabbit Colonic SMC

We have previously reported that tropomyosin shows an increased association with phosphorylated HSP27 (48) and also that caldesmon associates with HSP27 in colonic smooth muscle cells (28). We investigated the effect of acetylcholine stimulation on the association of phosphorylated caldesmon with HSP27. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Particulate and cytosolic fractions were separated as described in METHODS. Particulate fractions were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200). The proteins in the immunoprecipitates were separated on SDS-PAGE and were transferred onto PVDF membrane. The membrane was then immunoblotted with anti-HSP27 antibody (1:3,000). Stimulation of colonic smooth muscle cells with 0.1 µM acetylcholine resulted in a significant and sustained increase in the association of phosphorylated caldesmon with HSP27 in the particulate fraction [277.52 ± 11.59% at 30 s and 306.83 ± 10.39% at 4 min compared with control (100); P ≤ 0.01; n = 3] (Fig. 4). The data indicate that in rabbit colonic smooth muscle cells, the association of phosphorylated caldesmon with HSP27 increases in response to acetylcholine stimulation.


Figure 4
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  		Fig. 4. ACh-induced association of phosphorylated caldesmon with HSP27 in rabbit colonic SMC. Freshly isolated rabbit colon SMCs were stimulated with 0.1 µM ACh for 30 s and 4 min. Particulate fractions (500 µg) were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200) followed by immunoblotting with anti-HSP27 antibody (1:3,000). Graph represents the significant and sustained increase in ACh-induced association of phosphorylated caldesmon with HSP27 in the particulate fractions compared with control.

 
Acetylcholine-Induced Association of Phosphorylated Caldesmon with Tropomyosin in Rabbit Colonic SMC

Caldesmon is associated with actin-tropomyosin in smooth muscle (36). Phosphorylation of caldesmon has been reported to weaken the association of tropomyosin with caldesmon (8). We investigated the effect of acetylcholine-induced caldesmon phosphorylation on the association of caldesmon with tropomyosin in colonic smooth muscle cells. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Particulate and cytosolic fractions were separated as described in METHODS. Particulate fractions were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200). The proteins in the immunoprecipitates were separated on SDS-PAGE and were transferred onto PVDF membrane. The membrane was then immunoblotted with anti-tropomyosin antibody (1:500). Stimulation of colonic smooth muscle cells with 0.1 µM acetylcholine resulted in a significant and sustained decrease in the association of tropomyosin with phospho-caldesmon in the particulate fraction [66.79 ± 0.47% at 30 s and 58.73 ± 0.78% at 4 min compared with control (100), P ≤ 0.01; n = 3] (Fig. 5). The data indicate that tropomyosin dissociates from phospho-caldesmon during acetylcholine-induced contraction of rabbit colon smooth muscle cells.


Figure 5
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  		Fig. 5. ACh-induced association of phosphorylated caldesmon with tropomyosin in rabbit colonic SMC. Freshly isolated rabbit colon SMCs were stimulated with 0.1 µM ACh for 30 s and 4 min. Particulate fractions (500 µg) were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200) followed by immunoblotting with anti-tropomyosin antibody (1:500). ACh induced a significant and sustained decrease in the association of phosphorylated caldesmon with tropomyosin in the particulate fractions compared with control.

 
Acetylcholine-Induced Phosphorylation of Caldesmon in Rabbit Colonic SMC transfected with phospho-HSP27 Mutants

Since phosphorylation of HSP27 modulates the affinity of tropomyosin to HSP27 (48), we investigated whether the phosphorylated state of HSP27 affects acetylcholine-induced phosphorylation of caldesmon. Confluent colonic smooth muscle cells transfected with phospho-HSP27 mutants were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Proteins from particulate fractions of stimulated and unstimulated transfected colonic smooth muscle cells were separated on SDS-PAGE. Separated proteins were transferred to PVDF membrane and the membrane was immunoblotted with anti-phospho-caldesmon (ser789) antibody (1:200). A significant decrease in phosphorylation of caldesmon was observed in the cells transfected with non-phospho-HSP27 (3G) (99.97 ± 0.51% at 0 s, 130.36 ± 2.70% at 30 s and 139.63 ± 3.45% at 4 min; P ≤ 0.01; n = 3), whereas enhanced caldesmon phosphorylation was observed in colonic smooth muscle cells transfected with phospho-HSP27 (3D) (176.08 ± 4.19% at 0 s, 425.89 ± 3.01% at 30 s and 444.51 ± 7.27% at 4 min; P ≤ 0.001; n = 3) compared with normal cells (0.47 ± 3.32% and 258.58 ± 3.32% at 30 s and 4 min; P ≤ 0.01; n = 3) (Fig. 6). Because both HSP27 and caldesmon phosphorylation are PKC dependent and caldesmon phosphorylation seems to be affected by HSP27 phosphorylation, caldesmon seems to play a crucial role in PKC-dependent rabbit colonic smooth muscle contraction.


Figure 6
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  		Fig. 6. ACh-induced phosphorylation of caldesmon in rabbit colonic SMC transfected with phospho-HSP27 mutants. Confluent colonic SMCs transfected with phospho-HSP27 mutants were stimulated with 0.1 µM ACh for 30 s or 4 min. Equal amount (50 µg) of particulate fractions of stimulated and unstimulated transfected colonic SMCs were separated on SDS-PAGE. Separated proteins were immunoblotted with anti-phospho-caldesmon (ser789) antibody (1:200). Colonic SMCs transfected with non-phospho-HSP27 (3G) showed a significant inhibition of ACh-induced phosphorylation of caldesmon, whereas enhanced caldesmon phosphorylation was observed in colonic SMCs transfected with phospho-HSP27 (3D) compared with normal cells.

 
Acetylcholine-Induced Association of Phosphorylated Caldesmon with HSP27 in Rabbit Colonic SMC Transfected with phospho-HSP27 Mutants

To test whether the phosphorylated state of HSP27 modulates its association with phosphorylated caldesmon, confluent muscle cells in culture were stimulated with 0.1µM acetylcholine for 30 s or 4 min. Particulate fractions of stimulated and unstimulated colonic smooth muscle cells were immunoprecipitated with anti-caldesmon antibody (1:200) followed by protein separation on SDS-PAGE. Separated proteins were transferred to PVDF membrane, and the membrane was immunoblotted with anti-HSP27 antibody (1:3,000). Stimulation with acetylcholine significantly decreased the association of phospho-caldesmon with HSP27 in the cells transfected with non-phospho-HSP27 (3G) (84.81 ± 0.54% at 0 s, 105.62 ± 2.34% at 30 s and 109.70 ± 2.86% at 4 min; P ≤ 0.01; n = 3), whereas enhanced association of phosphorylated caldesmon with HSP27 was observed in colonic smooth muscle cells transfected with phospho-HSP27 (3D) (161.61 ± 2.11% at 0 s, 400.15 ± 5.07% at 30 s and 444.90 ± 7.11% at 4 min; P ≤ 0.01; n = 3) compared with normal cells (245.35 ± 3.57% and 274.94 ± 1.53% at 30 s and at 4 min, respectively, P ≤ 0.01; n = 3) (Fig. 7). The data indicate that phosphorylated HSP27 is essential for acetylcholine-induced association of phosphorylated caldesmon with HSP27 in colonic smooth muscle cells.


Figure 7
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  		Fig. 7. ACh-induced association of phosphorylated caldesmon with HSP27 in rabbit colonic SMC transfected with phospho-HSP27 mutants. Confluent colonic SMCs transfected with phospho-HSP27 mutants in culture were stimulated with 0.1 µM ACh for 30 s or 4 min. Particulate fractions (500 µg) of stimulated and unstimulated colonic SMCs were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200) followed by immunoblotting with anti-HSP27 antibody (1:3,000). Colonic SMCs transfected with non-phospho-HSP27 (3G) showed a significant inhibition of ACh-induced association of phosphorylated caldesmon with HSP27, whereas enhanced association of phosphorylated caldesmon with HSP27 was observed in colonic SMCs transfected with phospho-HSP27 (3D) compared with normal cells.

 
Acetylcholine-Induced Association of Phosphorylated Caldesmon with Tropomyosin in Rabbit Colonic SMC Transfected with phospho-HSP27 Mutants

To test whether phosphorylated HSP27 influences the association of phosphorylated caldesmon with tropomyosin, confluent phospho-HSP27 mutant-transfected colonic smooth muscle cells in culture were stimulated with 0.1 µM acetylcholine for 30 s or 4 min. Particulate fractions of stimulated and unstimulated phospho-HSP27 mutant-transfected colonic smooth muscle cells were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200), and proteins were separated on SDS-PAGE. Separated proteins were transferred to PVDF membrane, and the membrane was immunoblotted with anti-tropomyosin antibody (1:500). Significantly decreased association of phosphorylated caldesmon (ser789) with tropomyosin (Fig. 8) is seen in the cells transfected with phospho-HSP27 (3D) (80.78 ± 2.48% at 0 s, 54.76 ± 2.19% at 30 s, and 46.24 ± 1.47% at 4 min; P ≤ 0.05; n = 3) similar to normal cells (69.14 ± 2.53% at 30 s and 59.44 ± 3.86% at 4 min; P ≤ 0.05; n = 3), but there was no significant difference in the association of phosphorylated caldesmon (ser789) with tropomyosin in non-phospho-HSP27 (3G)-transfected smooth muscle cells [93.27 ± 1.26% at 0 s, 94.58 ± 1.40% at 30 s and 96.64 ± 1.25% at 4 min; P ≤ 0.08 (NS); n = 3]. The data indicate that, upon acetylcholine stimulation, phosphorylated HSP27 increases the dissociation of phosphorylated caldesmon from tropomyosin leading to sustained contraction in colonic smooth muscle cells.


Figure 8
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  		Fig. 8. ACh-induced association of phosphorylated caldesmon with tropomyosin in rabbit colonic SMC transfected with phospho-HSP27 mutants. Confluent phospho-HSP27 mutant transfected colonic SMCs in culture were stimulated with 0.1 µM ACh for 30 s or 4 min. Particulate fractions (500 µg) of stimulated and unstimulated colonic SMCs were immunoprecipitated with anti-phospho-caldesmon (ser789) antibody (1:200) followed by immunoblotting with anti-tropomyosin antibody (1:500). Colonic SMCs transfected with non-phospho-HSP27 (3G) showed no significant change in ACh-induced association of phosphorylated caldesmon (ser789) with tropomyosin, whereas phospho-HSP27 (3D) cells transfected SMCs showed a significant decrease in ACh-induced association of phosphorylated caldesmon (ser789) with tropomyosin similar to normal cells.

 

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 ABSTRACT
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Smooth muscle contraction occurs when cross bridges are formed by the interaction of myosin S1 heads with actin molecules (15). The formation of cross bridges is regulated by phosphorylation and dephosphorylation of MLC20 (4, 29). However, research suggests a role for the thin-filament proteins calponin, caldesmon, and tropomyosin (40, 59) in maintaining contraction. Of these thin-filament regulatory proteins, caldesmon appears to be an important calcium-dependent modulator of the actin-myosin interaction in smooth muscle contraction (36).

Caldesmon is found in muscle and nonmuscle cells (47). There are two isoforms of caldesmon: h-caldesmon (120 kDa; high molecular weight) and l-caldesmon (70 kDa; low molecular weight). The h-caldesmon is predominantly expressed in smooth muscles, whereas the l-caldesmon is widely distributed in nonmuscle tissues and cells (18). Caldesmon is an integral part of the contractile machinery and exclusively localizes with the contractile apparatus in smooth muscle cells (9, 36). Significant reduction in the levels of caldesmon in muscle fibers has been shown to reduce the ability of the fibers to develop force and hydrolyze ATP (53). In the present study, we have explored the role of caldesmon in relation to tropomyosin and HSP27 in modulating acetylcholine-induced contraction of colonic smooth muscle cells.

Caldesmon binds to thin-filament actin as well as to thick-filament myosin (23, 45). Caldesmon has three functionally distinct domains: an NH2-terminal domain that harbors the major myosin-binding sites (35), a rigid {alpha}-helical middle domain, and a COOH-terminal domain that houses the binding sites for actin and tropomyosin (14, 55). Two actin-binding clusters are present on the COOH-terminal domain of caldesmon (26). Caldesmon has been known to inhibit actin-activated myosin ATPase activity by inhibiting actomyosin interaction, suggesting that caldesmon may act in modulation of relaxation of smooth muscle (36).

An increase in intracellular calcium results in the binding of calcium-calmodulin complex to caldesmon and/or phosphorylation of caldesmon. The binding of calcium-calmodulin to caldesmon reduces the binding affinity of caldesmon to myosin as well as actin leading to actomyosin interaction and contraction (23). Phosphorylation of caldesmon causes conformational change whereby one of the two actin-binding clusters on caldesmon becomes detached from actin thus weakening the affinity of caldesmon to actin (13).

It has been suggested that both caldesmon phosphorylation and the binding of calcium-calmodulin complex to caldesmon cooperate in modulating the binding of caldesmon to actin-tropomyosin leading to actomyosin interaction and activation of actomyosin ATPase (23). Although caldesmon phosphorylation has been shown to regulate smooth muscle contraction by weakening its affinity for actin, the mechanism of regulation at the thin-filament level by phosphorylated caldesmon is not clear. Most of the studies done on phosphorylation of caldesmon have been performed in vitro, and several kinases have been implicated in its phosphorylation. Different kinases phosphorylate caldesmon at different sites. ser789 Is the predominant phosphorylation site on caldesmon involved in smooth muscle contraction (11). One class of enzymes known to act on caldesmon are the MAP kinases (2), which add a phosphate group at ser759 and ser789 near the COOH terminus of mammalian caldesmon (3). Present data show that acetylcholine induces a significant and sustained phosphorylation of caldesmon at ser789 in colonic smooth muscle cells. Acetylcholine-induced caldesmon phosphorylation was significantly inhibited upon preincubation with the PKC inhibitors calphostin-C and GF109203X, indicating PKC-dependent phosphorylation of caldesmon. PKC is a known activator of the MAPK/ERK pathway (39). ERK pathway has been suggested to be involved in phosphorylation of caldesmon (21). Thus it is possible that inhibition of PKC may inhibit activation of ERK, which in turn inhibits caldesmon phosphorylation. Coimmunoprecipitation studies were performed to investigate the direct involvement of PKC{alpha} in caldesmon phosphorylation. There was a significant increase of PKC{alpha} levels in caldesmon immunoprecipitates, whereas there was a significant decrease of PKC{alpha} levels in phospho-caldesmon immunoprecipitates. Similar results were seen when caldesmon and phospho-caldesmon immunoprecipitates were probed with phospho-PKC{alpha}. Our results are consistent with reports from other laboratories that show direct phosphorylation of caldesmon by PKC{alpha} and direct interaction of caldesmon with PKC{alpha} (58, 59). Previous studies have shown that tropomyosin binds to PKC{alpha} and that tropomyosin is phosphorylated in response to acetylcholine (49). Previous studies have also indicated that acetylcholine induces PKC{alpha} translocation and phosphorylation in colonic smooth muscle cells (28, 42). The present study showed that phosphorylated PKC{alpha} is translocated to the particulate fraction. We thus hypothesize that phospho-PKC{alpha}, on translocation, may associate with caldesmon and tropomyosin resulting in phosphorylation of caldesmon and tropomyosin. On caldesmon phosphorylation, phospho-PKC{alpha} may dissociate from phospho-caldesmon. Dissociated phospho-PKC{alpha} may either continue to phosphorylate other proteins involved in contraction or it may be dephosphorylated to subsequently initiate a relaxation cycle.

PKC-mediated contraction has been suggested to activate two parallel MAPK contractile pathways: an ERK-MAPK pathway that regulates smooth muscle contraction by caldesmon or calponin phosphorylation and a p38 MAPK pathway that regulates smooth muscle contraction by HSP27 phosphorylation (11, 20). HSP27 is a putative contractile protein whose phosphorylation plays crucial role in PKC-mediated smooth muscle contraction (6, 19, 28, 31). Sustained smooth muscle contraction is associated with sequential activation of kinases resulting in activation of MAPKAP2 kinase, which phosphorylates HSP27 (17, 32, 33). Agonist-induced colonic smooth muscle contraction is associated with sustained increase in HSP27 phosphorylation (6, 43, 48). Acetylcholine stimulation of colonic smooth muscle is also accompanied by caldesmon phosphorylation at ser789. We have previously shown that caldesmon associates with HSP27 in colonic smooth muscle cells in response to agonist stimulation (28). We investigated the effect of caldesmon phosphorylation on the association of HSP27 with caldesmon. Our data indicate that acetylcholine induced a significant and sustained association of phosphorylated caldesmon with HSP27 in colonic smooth muscle cells. The function of this association requires further investigation. As mentioned earlier, phosphorylation of caldesmon weakens its affinity for actin leading to actomyosin interaction and contraction (13). Thus we propose that the association of phosphorylated caldesmon with HSP27 may by an as yet unidentified mechanism assist in changing conformation thus weakening the affinity of caldesmon with actin.

The actomyosin ATPase inhibition by caldesmon is enhanced in the presence of tropomyosin on actin (36, 37). Tropomyosin is an actin-binding protein that is positioned in the grooves of actin blocking the myosin binding sites on actin. Binding of caldesmon to actin-tropomyosin enhances the ability of tropomyosin to block the myosin binding sites on actin (24). As mentioned above, phosphorylation of caldesmon has been known to weaken the affinity of caldesmon for actin-tropomyosin (36–38). Present data show that acetylcholine stimulation of colonic smooth muscle cells results in caldesmon phosphorylation. Caldesmon phosphorylation leads to decreased association of caldesmon with tropomyosin resulting in the sliding of tropomyosin on actin, thus exposing the myosin binding sites for actomyosin interaction (Fig. 9 ).


Figure 9
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  		Fig. 9. Association of phospho-caldesmon with HSP27 results in dissociation of phospho-caldesmon with tropomyosin allowing tropomyosin to slide on actin, exposing myosin-binding sites for sustained contraction. During the relaxed state, caldesmon is bound to tropomyosin and actin. On ACh stimulation, HSP27 is phosphorylated. Phospho-HSP27 helps translocation and phosphorylation of PKC{alpha}. Phospho-PKC{alpha} (ser657) may associate with caldesmon and with tropomyosin resulting in PKC-dependent phosphorylation of caldesmon (ser789) and tropomyosin. On phosphorylation of caldesmon, phospho-PKC{alpha} (ser657) dissociates from phospho-caldesmon (ser789). Simultaneous association of phospho-caldesmon and phospho-tropomyosin with phospho-HSP27 results in dissociation of phospho-tropomyosin from phospho-caldesmon. This results in sliding of tropomyosin on actin and thus exposing the myosin-binding sites on actin. The net result is exposing the myosin-binding sites on actin for sustained colonic smooth muscle contraction.

 
We have recently shown that tropomyosin binds with increased affinity to phosphorylated HSP27 (48). We have also suggested that agonist-induced phosphorylation of HSP27 may result in binding of tropomyosin to phosphorylated HSP27 and that this binding may aid in the displacement of tropomyosin to expose myosin binding sites during contraction. The sustained interaction of tropomyosin with phosphorylated HSP27 is thus affected by the sustained phosphorylated state of HSP27 (28). Phosphorylated HSP27 is essential for the association of the contractile proteins actin with myosin (6) and tropomyosin with HSP27 (48) leading to sustained contraction. Colonic smooth muscle cells transfected with non-phospho-HSP27 showed decreased acetylcholine-induced association of actin with myosin (28) and of tropomyosin with HSP27 as well as reduced contractile response compared with untransfected cells. Our present data show that in response to acetylcholine induction, colonic smooth muscle cells transfected with non-phospho-HSP27 exhibit decreased caldesmon phosphorylation with a concomitant decrease in the association of phosphorylated caldesmon with HSP27 compared with cells transfected with phospho-HSP27 or to untransfected cells. However, the association of phosphorylated caldesmon with tropomyosin is decreased in cells transfected with the phospho-HSP27 or in untransfected cells, whereas the association remained constant in cells transfected with the non-phospho-HSP27 mutant. This suggests that in colonic smooth muscle cells, phosphorylated HSP27 enhances acetylcholine-induced caldesmon phosphorylation, which in turn increases the acetylcholine-induced association of phosphorylated caldesmon with HSP27 with a concomitant increase in acetylcholine-induced dissociation of phosphorylated caldesmon from tropomyosin. Our laboratory has previously shown that phospho-HSP27 is essential for translocation of PKC{alpha} (43). Present data indicate a decrease in caldesmon phosphorylation in smooth muscle cells transfected with non-phospho-HSP27 cDNA, suggesting that the decrease in translocation of PKC{alpha} (43) could account for the decreased phosphorylation of caldesmon.

Whether the phosphorylation of caldesmon marks the end of the relaxation cycle or the initiation of the contraction cycle is not clear and needs further investigation. We propose a model of thin-filament regulation of sustained smooth muscle cells of colon whereby phospho-HSP27 modulates the dissociation of caldesmon from tropomyosin leading to actomyosin interaction and contraction in colonic smooth muscle. During the relaxed state, caldesmon is bound to tropomyosin and actin. On acetylcholine stimulation, HSP27 is phosphorylated. Phospho-HSP27 helps translocation and phosphorylation of PKC{alpha} to the particulate fraction. Phospho-PKC{alpha} may associate with caldesmon and with tropomyosin resulting in PKC-dependent phosphorylation of caldesmon (ser789) and tropomyosin. On phosphorylation of caldesmon, phospho-PKC{alpha} (ser657) dissociates from phospho-caldesmon (ser789). Simultaneous association of phospho-caldesmon and phospho-tropomyosin with phospho-HSP27 results in dissociation of phospho-tropomyosin from phospho-caldesmon. This results in sliding of tropomyosin on actin and thus exposing the myosin-binding sites on actin. The net result is exposing the myosin-binding sites on actin for sustained colonic smooth muscle contraction (Figs. 9 and 10).


Figure 10
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  		Fig. 10. Sequence of events depicting the role of caldesmon (CD) in thin-filament regulation of smooth muscle contraction. Smooth muscle contraction is regulated at both the thin- and thick-filament levels. ACh-induced rise in intracellular calcium levels results in activation of both the thin- and thick-filament pathways: the proposed role of CD in the thin-filament cascade modulating smooth muscle contraction is as follows: 1) heat-shock protein 27 (HSP27) is phosphorylated and translocates PKC{alpha} to the particulate fraction where PKC{alpha} is phosphorylated. Phospho-PKC{alpha} binds to non-phospho CD. 2) CD is phosphorylated, and phospho-PKC{alpha} dissociates from phospho-CD. Phospho-CD binds to phospho-HSP27 resulting in a conformational change of phospho-CD. 3) The conformational change in phospho-CD results in sliding of tropomyosin (TM) on actin (AC) away from phospho-CD and thus exposes myosin (MY) binding sites for actomyosin interaction. CaM, calmodulin; MLCK, myosin light chain kinase; MLC20, myosin light chain.

 

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This study was supported by National Institutes of Health Grant 5 RO1 DK-057020.


   ACKNOWLEDGMENTS
 
We thank S. Blea and K. Cristian for assistance with technical editing and figure preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., MSRB I, Rm. A520, Ann Arbor, MI 48109-0658 (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.


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