Agonist-induced association of tropomyosin with protein kinase C{alpha} in colonic smooth muscle

Sita Somara, Haiyan Pang, and Khalil N. Bitar

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

Submitted 27 July 2004 ; accepted in final form 7 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKC{alpha} in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKC{alpha} in the particulate fraction may be due to acetylcholine-induced translocation of PKC{alpha} to the particulate fraction. To investigate whether the association of PKC{alpha} with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKC{alpha} indicated direct interaction of tropomyosin with PKC{alpha}. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKC{alpha} with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

acetylcholine; fusion proteins


THE CLASSIC PATHWAY OF SMOOTH muscle contraction indicates that contraction occurs when myosin regulatory light chain (MLC20) is phosphorylated by activated calcium/calmodulin-dependent myosin light chain kinase (MLCK). Although myosin phosphorylation is undoubtedly crucial for initiation and maintenance of contractility, the modulation of contraction at the level of thin filament also plays an equally important role. The alternative system regulating contraction at the thin-filament level involves PKC, which plays an important role in controlling sustained smooth muscle contraction (26, 32). PKC-mediated contraction results from PKC activation and translocation to the membrane. PKC plays a role in the regulation of myosin phosphorylation via phosphorylation of CPI17, resulting in inhibition of myosin phosphatase (MLCP) (5, 15, 17, 20). Phosphorylation of MLC20 by MLCK is counterbalanced by its dephosphorylation by MLCP. Therefore, inhibition of MLCP by PKC via CPI17 maintains phosphorylation of MLC20. In vascular smooth muscle, agonist stimulation has been shown to increase the phosphorylation of CPI17 (16). Preincubation of smooth muscle cells with calphostin C, a PKC inhibitor, results in inhibition of contraction (14), inhibition of MAP kinase activation (25), inhibition of heat shock protein (HSP)27 phosphorylation (13) and inhibition of PKC translocation (3). Although the role of PKC in MLC phosphorylation is well understood, the precise role of PKC-mediated phosphorylation of other contractile proteins involved in smooth muscle contraction has not been well established (28). PKC has been known to interact with other contractile proteins such as calponin and HSP27 (2, 18).

The major regulatory protein components of smooth muscle thin filaments are caldesmon, calponin, and tropomyosin (TM) (19). Caldesmon and calponin regulate smooth muscle contraction by inhibiting activation of myosin Mg2+-ATPase. Phosphorylation of caldesmon and calponin reverses the inhibition leading to contraction. TM also averts the activation of myosin Mg2+-ATPase, due to its inhibitory position on actin, by covering myosin-binding sites (4). TM forms a continuous strand along each actin helix in thin filaments, which is built up from a double helix of actin monomers. TM assembles into an {alpha}-helical coiled-coil dimer, with each molecule interacting with six or seven monomers of actin (10, 21). It also binds to itself and helps wrap around the actin molecule to stabilize the thin-filament assembly (36). TMs, present in virtually all eukaryotic cells, are known to be expressed in developmental and tissue specific patterns. Although the crucial role played by TM in regulation of contraction has been extensively studied in cardiac and skeletal muscle (7), the interactive, functional, and biological role of TM during smooth muscle contraction is only poorly understood. The understanding of the interaction of TM with signaling molecules may provide a better insight into the involvement of TM in signal transduction and thin-filament regulation during smooth muscle contraction.

Phosphorylation of {alpha}{alpha}-TM has been associated in vitro with increased myosin Mg2+/ATPase activity in rabbit striated muscle (9). Phosphorylation of TM downstream of ERK has been reported to modulate its interaction with actin (12). ML-7, an MLCK inhibitor, has been reported to inhibit TM phosphorylation in endothelial cells (12). Our data indicate that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of TM in colonic smooth muscle cells. Acetylcholine also induces a significant and sustained increase in PKC-mediated association of TM with PKC{alpha} in the particulate fraction of rabbit colonic smooth muscle cells. Increased association of TM with PKC{alpha} in the particulate fraction of rabbit colonic smooth muscle cells may be due to acetylcholine-induced translocation of PKC{alpha} to the particulate fraction, because immunoblotting studies showed that the amount of actin and TM in the particulate fraction in response to acetylcholine is constant. We have investigated the possibility of a direct protein-protein interaction of TM with the signaling molecule PKC{alpha} TM cDNA of ~1 kb was RT-PCR amplified from RNA extracted from rabbit colon smooth muscle. The PCR amplified TM cDNA was cloned and expressed as GST fusion protein in the pGEX-KT vector. Coelution of PKC{alpha} with GST-TM during in vitro studies indicates a direct association of GST-TM with PKC{alpha}. Direct interaction of TM with PKC{alpha} and PKC-mediated phosphorylation of TM suggests that TM could be a substrate for PKC{alpha}.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

The following reagents were purchased: monoclonal mouse anti-TM antibody (developed against chicken gizzard TM); polyclonal anti-PKC{alpha} antibody from Panvera (Madison, WI); recombinant human PKC{alpha} from Cytoskeleton (Denver, CO); polyvinylidene fluoride (PVDF) membranes from Bio-Rad (Hercules, CA); protein G sepharose and enhanced chemiluminescence (ECL) detection reagents from Amersham Biosciences (Buckinghamshire, England); 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 from Sigma (St. Louis, MO).

Methods

Preparation of smooth muscle cells from the rabbit rectosigmoid. Smooth muscle cells of rabbit rectosigmoid were isolated as described (1). Briefly, the internal anal sphincter 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 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) DMEM. At the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 10 ml 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 the freshly isolated smooth muscle cells were prepared as described previously (23). 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 followed by immediate freezing in acetone/dry ice slurry. 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 Na4P2O7·10H2O 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, and 5 mg/ml Pefbloc]. The sonicated cells were centrifuged at 100,000 g for 60 min. The supernatant material was collected as cytosolic fraction, whereas the pellet material was resuspended in the lysis buffer with 1% Triton X-100 followed by sonicating twice for 30 s each time and collected as particulate fraction. The protein content was determined by using Bio-Rad protein assay reagent.

Immunoprecipitation and immunoblotting. Antibody (1–2 µg) was added to 500 µg of sample protein in 500 µl of lysis buffer and rocked overnight at 4°C. Fifty percent protein G-Sepharose bead slurry (50 µl) was added to the overnight mixture and rocked at 4°C for 2 h. The beads bound with proteins were then collected by centrifuging at 14,000 g for 3 min at 4°C. Supernatant was discarded, and the bead pellet was washed three times at room temperature with Tris-buffered saline (TBS) bead wash buffer (20 mM Tris·HCl, 150 mM NaCl, pH 7.6). The beads were then resuspended in 25 µl of 2x sample buffer and boiled for 5 min. Proteins from the immunoprecipitates were separated on SDS-PAGE and transferred to PVDF membrane. The membrane was immunoblotted with the desired antibodies. Replicates of experiments were performed using completely separate sets of cells.

Western blot analysis. The proteins were separated on SDS-PAGE and electrophoretically transferred to PVDF membrane. The PVDF membrane was then blocked with 5% nonfat dry milk for 1 h. After being blocked, the membrane was incubated in an appropriate dilution of primary antibody in 5% nonfat dry milk in TBS with Tween 20 (TBST) for 1 h. The membrane was washed three times with TBS 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.

RNA preparation. Total RNA was extracted from fresh tissue using TRIzol following manufacturers' protocol. Briefly, 1 ml TRIzol reagent was added to ~50–100 mg tissue and incubated for 5 min at 30°C. After incubation, 0.2 ml of chloroform were added and shaken vigorously by hand for 15 s, followed by incubation at 30°C for 2–3 min. The sample was then centrifuged at 12,000 g for 15 min at 2–8°C. Upper colorless aqueous layer was transferred to fresh tube. To this, 0.5 ml of isopropyl alcohol was added and incubated at 30°C for 10 min. The sample was centrifuged again at 12,000 g for 10 min at 2–8°C. The supernatant was discarded, and the RNA pellet was washed with 1 ml of 75% ethanol. The pellet was air dried and dissolved in minimum volume of DEPC water.

RT-PCR. One microgram of total RNA was taken in 10 µl of double-distilled RNAse/DNAse-free water. RNA was then denatured by heating at 75°C for 5 min, followed by rapid chilling. To denatured RNA was added 2.0 µl of 10x amplification buffer, 1.0 µl of 20 mM dNTPs, 1.0 µl of oligo(dT) primers, 1.0 µl of ~20 U/µl RNase, 1.0 µl of 50 mM MgCl2, and 1.0 µl of RT 100–200 U/µl, and total volume was made up to 20.0 µl with nuclease-free water. The mixture was incubated at 37°C for 60 min followed by heat inactivation of RT and denaturation of template cDNA complexes at 95°C for 5 min. The cDNA formed was then subjected to polymerase chain reaction by adding 20 pmol of sense and antisense primers followed by the addition of 77 µl of 1x amplification buffer, 0.5 U of Taq polymerase. The cDNA underwent 35 cycles of PCR (94°C for 45 s; 55°C for 45 s; 72°C for 1 min 15 s) followed by one cycle of 94°C for 1 min; 55°C for 45 s; 72°C for 1 min 15 s. The sense and antisense primers used were 5'-TTT GGA TCC ATG GAT TCC ATC AAG AAG AAG ATG CAG ATG-3' (restriction site BamHI) and reverse primer 5'-TTT GAA TTC TCA CAG GTA GTT GAG TTC CAG CAG GGT CTG-3' (restriction site EcoRI), respectively. After PCR, 5 µl of sample were run on the gel and a PCR amplified product of ~900 bp was observed under an ultraviolet illuminator. The PCR product was purified using Qiagen gel purification kit and sent for sequencing at the University of Michigan Sequencing Core facility. On sequencing, the PCR product was confirmed to be TM cDNA.

Cloning of amplified cDNA in GST-tagged expression vector. The RT-PCR amplified TM cDNA was double digested with the restriction enzymes BamH1 and EcoR1. The digested PCR product was then cloned in frame with the NH2-terminal GST tag of the vector pGEX-KT (Dr. Jack Dixon, University of Michigan) (8). Recombinant pGEX-KT was transformed into E. coli DH5{alpha}. After confirmation of the clones by double-digest release of the inserts, the plasmid was purified and sent in for sequencing at the University of Michigan Sequencing Core facility. Recombinant pGEX-KT was then transformed into an expression host BL21 (DE3) pLysS cells.

Protein expression and purification of GST-TM fusion protein in E. coli. The cloned TM gene was expressed as a GST fusion protein in E. coli (BL21 strain) by isopropyl-D-thiogalactopyranoside induction as described previously (29). Purification of GST-TM was performed by affinity chromatography using the glutathione Sepharose column (Sigma) according to the protocol described in the GST purification module (Pharmacia Biotech, Buckinghamshire, UK). Purified protein samples were analyzed by SDS-PAGE according to the method of Laemmli and by Western blot analysis. The purified protein was quantified by protein assay kit (Bio-Rad).

In vitro binding of GST-TM protein with recombinant PKC{alpha}. In vitro binding assay was done as described previously (29). Briefly, 25 µg of GST-TM fusion protein were mixed with 200 µl glutathione agarose beads (50% slurry) separately and rocked for 2 h at 4°C. GST alone was used as control. Unbound GST-TM fusion protein was removed by washing five times with PBS. All the washes were retained for further analysis. Ten micrograms of recombinant PKC{alpha} were added to the GST-TM bound to agarose beads and rocked for 1 h at 4°C. Unbound PKC{alpha} was removed by washing five times with MT-PBS. All of the washes were retained for further analysis. The bound proteins were eluted twice with one bed volume of elution buffer (50 mM Tris·HCl, pH 8.0, containing 10 mM reduced glutathione). Ten microliters of all of the washes and the eluates were spotted on a PVDF membrane. The membrane was blocked with 5% nonfat skim milk in TBS and immunoblotted with anti-GST antibody, anti-TM antibody, and anti-PKC{alpha} antibody. The spots were detected by chemiluminescence.

Dot blot. Dot blot of the washes and eluates of in vitro binding assay was done as described previously (23). Briefly, the methanol soaked and PBS-equilibrated PVDF was placed in the dot-blot apparatus and was clasped with clamps on the sides. The vacuum was passed at the rate of 1 ml/min. Ten microliters of the sample were added to each slot in an order. The slots were then washed with 100 µl of 1x PBS. The membrane was incubated in 5% nonfat dry milk in TBST rocking for 1 h at room temperature and washed twice with TBST. The membrane was then incubated in appropriate dilution of primary antibody (anti-TM, 1:500; anti-PKC{alpha}, 1:200; anti-GST, 1:4,000) for 1 h with rocking at room temperature and washed three times with TBST. The membrane was then incubated in appropriate dilution of secondary antibody for 1 h with rocking at room temperature followed by washing three times with TBST. The membrane was then incubated with Amersham's ECL reagent for 1 min. The proteins were detected on the membrane by immediately exposing the membrane to the film.

Data Analysis

Data analysis was done as described previously (29). 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 intensities were standardized to 100%. The band intensities of samples from treated cells were compared with the control and expressed as percent change from the control. All the means were compared and analyzed using Student's t-test. Blotting data are within the linear range of detection for each antibody used as described previously (22, 29).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acetylcholine-Induced Phosphorylation of TM in Rabbit Colon Smooth Muscle

TM is an actin binding protein that plays a key role in regulation of contraction by controlling the actomyosin interaction and activation of myosin Mg2+-ATPase. Phosphorylation of TM has been known to activate myosin Mg2+-ATPase by facilitating actomyosin interaction (9). We investigated the phosphorylation status of TM on acetylcholine stimulation of colon smooth muscle cells. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Immunoprecipitation of whole cell lysates with anti-phosphoserine antibody followed by immunoblotting with anti-TM antibody resulted in a significant and sustained increase in the serine phosphorylation of TM [411.13 ± 12.37 and 401.29 ± 15.89% compared with control (100) at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 1A]. Similarly, immunoprecipitation of whole cell lysates with anti-TM antibody followed by immunoblotting with anti-phosphoserine antibody resulted in a significant and sustained increase in the serine phosphorylation of TM [414.14 ± 7.85 and 407.90 ± 13.02% compared with control (100) at 30 s and 4 min, respectively, P ≤ 0.001, n = 3; Fig. 1B].



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Fig. 1. ACh-induced phosphorylation of tropomyosin in rabbit colon smooth muscle cells. Freshly isolated rabbit colon smooth muscle cells were stimulated with 0.1 µM ACh for 30 s and 4 min. A: 500 µg of whole cell lysates were immunoprecipitated (IP) with anti-phosphoserine antibody followed by immunoblotting (IB) with anti-tropomyosin antibody (1:500). Whole cell lysates of unstimulated rabbit colon smooth muscle cells were used as control. Representative blot is presented to show a sustained increase in phosphorylation of tropomyosin after stimulation with ACh for 30 s and 4 min compared with control. A graph shows ACh-induced significant and sustained increase in the phosphorylated tropomyosin at 30 s and at 4 min (411.13 ± 12.37 and 401.29 ± 15.89% at 30 s and 4 min, respectively; P ≤ 0.001, n = 3) compared with control (100%) in colonic smooth muscle cells. B: 500 µg of whole cell lysates were immunoprecipitated with anti-tropomyosin antibody followed by immunoblotting with anti-phosphoserine antibody (1:200). Whole cells lysates of unstimulated rabbit colon smooth muscle cells were used as control. Representative blot is presented to show a sustained increase in phosphorylation of tropomyosin after stimulation with ACh for 30 s and 4 min compared with control. A graph shows ACh-induced significant and sustained increase in the phosphorylated tropomyosin at 30 s and at 4 min (414.14 ± 7.85 and 407.90 ± 13.02% at 30 s and 4 min, respectively; P ≤ 0.001, n = 3) compared with control in colonic smooth muscle cells.

 
Effect of Calphostin C on Acetylcholine-Induced Phosphorylation of TM in Rabbit Colon Smooth Muscle Cells

We investigated the effect of calphostin C, a PKC inhibitor, on acetylcholine-induced phosphorylation of TM. Freshly isolated rabbit colon smooth muscle cells were preincubated with calphostin C (10 µM) for 20 min before stimulation with 0.1 µM acetylcholine. Preincubation with calphostin C inhibited acetylcholine-induced increase in serine phosphorylation of TM (112 ± 14 and 135 ± 20% decrease at 30 s and 4 min, respectively P ≤ 0.001, n = 3) in freshly isolated rabbit colon smooth muscle cells (Fig. 2A).



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Fig. 2. A: effect of calphostin C (Calp C) on ACh-induced phosphorylation of tropomyosin in rabbit colon smooth muscle cells. Freshly isolated rabbit colon smooth muscle cells were incubated with 10 µM Calp C, PKC{alpha} inhibitor for 20 min before stimulation with 0.1 µM ACh for 30 s and 4 min. Whole cell lysates of Calp C-incubated unstimulated rabbit colon smooth muscle cells were used as control. Whole cell lysates (500 µg) were immunoprecipitated with anti-phosphoserine antibody followed by immunoblotting with anti-tropomyosin antibody (1:500). Preincubation with Calp C inhibited ACh-induced increase in tropomyosin phosphorylation (112 ± 14 and 135 ± 20% at 30 s and 4 min, respectively; P ≤ 0.001, n = 3) in freshly isolated rabbit colon smooth muscle cells. B: effect of calcium on ACh-induced phosphorylation of tropomyosin in colonic smooth muscle cells. Rabbit colon smooth muscle cells were isolated in calcium-free buffer, i.e., 0 Ca2+/2 mM EGTA, and stimulated with 0.1 µM ACh for 30 s and 4 min. Whole cells lysates of unstimulated rabbit colon smooth muscle cells were used as control. Whole cell lysates (500 µg) were immunoprecipitated with anti-phosphoserine antibody followed by immunoblotting with anti-tropomyosin antibody (1:500). Isolation of colonic smooth muscle cells in the absence of calcium resulted in reduced ACh-induced serine phosphorylation of tropomyosin (144 ± 5 and 133 ± 6% at 30 s and 4 min, respectively; P ≤ 0.001; n = 3).

 
Effect of Calcium on Acetylcholine-Induced Phosphorylation of TM in Colonic Smooth Muscle Cells

We further investigated whether the acetylcholine-induced phosphorylation of TM was calcium dependent. Rabbit colon smooth muscle cells were isolated in calcium-free buffer, i.e., 0 Ca2+/2 mM EGTA, and stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Acetylcholine-induced serine phosphorylation was greatly reduced in rabbit colon smooth muscle cells isolated in the absence of calcium (144 ± 5 at 30 s and 133 ± 65 at 30 s and 4 min, respectively; P ≤ 0.001, n = 3; Fig. 2B).

Acetylcholine-Induced Coimmunoprecipitation of TM with PKC{alpha} in Rabbit Colon Smooth Muscle Cells

PKC-mediated phosphorylation of TM led us to investigate the association of TM with the signaling molecule PKC{alpha} on acetylcholine stimulation of colon smooth muscle cells. Acetylcholine stimulation leads to activation and translocation of PKC{alpha} to particulate fraction (14). Therefore, we investigated the association of TM with PKC{alpha} in both particulate and cytosolic fraction. Smooth muscle cells isolated from rabbit colon were stimulated with 0.1 µM acetylcholine for 30 s and 4 min. Whole cell lysates were then subjected to high-speed centrifugation to separate particulate and cytosolic fractions. Particulate fractions were immunoprecipitated with anti-TM antibody. The proteins in the immunoprecipitates were separated on SDS-PAGE and were transferred onto PVDF membrane. The membrane was then immunoblotted with anti-PKC{alpha} antibody. Stimulation with the contractile agonist acetylcholine resulted in a significant and sustained increase in the association of TM with PKC{alpha} in the particulate fractions (227 ± 52 and 173 ± 51% compared with control at 30 s and 4 min, respectively, n = 4, p ≤ 0.001; Fig. 3A). The observed increase in association of TM with PKC{alpha} in the particulate fraction suggests that TM is associated with translocated PKC{alpha} in the particulate fraction.



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Fig. 3. ACh-induced coimmunoprecipitation of tropomyosin with PKC{alpha} in rabbit colon smooth muscle cells. Freshly isolated rabbit colon smooth muscle cells were stimulated with 0.1 µM ACh for 30 s and 4 min. Particulate and cytosolic fractions were separated as described in METHODS. A: particulate fraction: 500 µg of particulate fractions were immunoprecipitated with anti-tropomyosin antibody followed by immunoblotting with anti-PKC{alpha} antibody (1:200). Representative blot is presented to show a sustained increase in association of tropomyosin with PKC{alpha} after stimulation with ACh for 30 s and 4 min compared with control. A graph shows sustained association of tropomyosin with PKC{alpha} in the particulate fraction when stimulated with ACh (227 ± 52 and 173 ± 51% compared with control at 30 s and 4 min, respectively; P ≤ 0.001, n = 4). B: effect of Calp C on ACh-induced association of tropomyosin with PKC{alpha} in the particulate fraction. freshly isolated rabbit colon smooth muscle cells were incubated with 10–6 M Calp C for 20 min before stimulation with 0.1 µM ACh for 30 s and 4 min. Particulate fractions were separated from cytosolic fraction as described in METHODS. Particulate fractions (500 µg) were immunoprecipitated with anti-tropomyosin antibody followed by immunoblotting with anti-PKC{alpha} antibody (1:200). Treatment of smooth muscle cells with calphostin C before stimulation with ACh resulted in an inhibition of the association of tropomyosin with PKC{alpha} (97 ± 1 and 116 ± 5% compared with control at 30 s and 4 min, respectively; P ≤ 0.001, n = 4).

 
Effect of Calphostin C on Acetylcholine-Induced Association of TM with PKC{alpha} in the Particulate Fraction

We investigated the effect of calphostin C, a PKC inhibitor, on the association of TM with PKC{alpha} during acetylcholine-induced contraction in smooth muscle cells. Freshly isolated smooth muscle cells from rabbit colon were incubated with 10 µM calphostin C for 20 min before stimulation with 0.1 µM acetylcholine for 30 s and 4 min. Particulate fractions were separated from cytosolic fraction. Particulate fractions (500 µg) were immunoprecipitated with anti-TM antibody followed by immunoblotting with anti-PKC{alpha} antibody (1:200). Treatment of smooth muscle cells with calphostin C before stimulation with acetylcholine resulted in an inhibition of the acetylcholine-induced association of TM with PKC{alpha} (97 ± 1 and 116 ± 5% compared with control at 30 s and 4 min, respectively; n = 4, P ≤ 0.001; Fig. 3B).

Effect of Acetylcholine on the Amount of Actin and TM

To study the changes in the amount of actin and TM in colonic smooth muscle cells in response to acetylcholine, immunoblotting with anti-TM antibody and anti-actin antibody was performed. Colonic smooth muscle cells were stimulated with acetylcholine (0.1 µM) for 4 min, and equal amounts (50 µg) of fractions were separated on SDS-PAGE. Separated proteins were probed against anti-TM antibody and anti-actin antibody. Immunoblotting of particulate and cytosolic fractions shows that all of the TM (90.5%) is present in the particulate fraction with only trace amounts present in cytosolic fraction (9.5%). Furthermore, our data show that acetylcholine induces no change in amount of TM in the particulate fraction (91.06 ± 0.30% at 4 min; P ≤ 0.05, n = 3) compared with control (100%; Fig. 4A). Similarly, immunoblotting with anti-actin antibody indicates that acetylcholine induced no change in the amount of smooth muscle thin filament actin in the particulate fraction (101.99 ± 1.28% at 4 min; P ≤ 0.002, n = 4) compared with control (100%; Fig. 4B). Our data thus show that increased association of TM with PKC{alpha} is due to agonist-induced activation and translocation of PKC{alpha} per se to the particulate fraction.



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Fig. 4. Effect of ACh on amount of tropomyosin and actin in rabbit colon smooth muscle cells. Freshly isolated rabbit colon smooth muscle cells were stimulated with 0.1 µM ACh for 4 min. Equal amounts (50 µg) of particulate fractions of ACh stimulated and unstimulated colon smooth muscle cells were separated on SDS-PAGE. Separated proteins were probed against anti-tropomyosin antibody and anti-actin antibody. A: immunoblotting with anti-tropomyosin antibody indicated that a significant amount of tropomyosin is present in the particulate fraction, whereas only traces of tropomyosin are present in the cytosolic fraction {9.53 ± 0.19% in unstimulated and 9.78 ± 0.26% at 4 min after stimulation; P ≤ 0.44 [not significant (NS)], n = 3}. Furthermore, ACh induces no significant change in the amount of tropomyosin in the particulate fraction [90.47 ± 0.19% in unstimulated and 91.06 ± 0.30% at 4 min; P ≤ 0.38 (NS), n = 3] compared with control tropomyosin in whole cell lysates (100). B: similarly, immunoblotting with anti-actin antibody indicates that ACh induced no change in the amount of smooth muscle thin filament actin in the particulate fraction [101.99 ± 1.28% at 4 min; P ≤ 0.29 (NS), n = 4] compared with control unstimulated (100).

 
RT-PCR Amplification of Rabbit Colon Smooth Muscle TM

To examine whether the observed in vivo interaction of TM with PKC{alpha} is due to a direct protein-protein interaction of TM with the signaling molecule PKC{alpha}, in vitro binding studies were carried out by constructing GST-TM. For constructing GST-TM, TM cDNA was RT-PCR amplified from the rabbit colon smooth muscle RNA as described in METHODS. Amplified RT-PCR product of ~890 bp (Fig. 5) was sequenced and confirmed to be TM by sequence analysis using BLAST tool.



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Fig. 5. RT-PCR amplification of tropomyosin cDNA from rabbit colon smooth muscle RNA. Approximately 890-bp cDNA product was amplified by RT-PCR from the rabbit colon smooth muscle RNA using the primers designed based on smooth muscle cell-specific tropomyosin. Amplified RT-PCR product of ~890 bp was confirmed to be tropomyosin after the analysis of the sequence obtained by sequencing of RT-PCR amplified cDNA.

 
Expression and Purification of GST-Fused TM

GST-fused TM was constructed to carry out in vitro binding assay to investigate the possibility of direct protein-protein interaction between TM and PKC{alpha}. RT-PCR-amplified TM cDNA of ~890 bp was cloned into GST tag containing vector, pGEX-KT at BamH1 and EcoR1 sites to express TM as NH2-terminal GST-fused proteins. The clone was then confirmed to be in the correct open reading frame by sequencing. The GST-fused TM protein was expressed by induction with 1 mM IPTG.

The expressed fusion protein was purified using the glutathione agarose beads as described in the METHODS and analyzed by staining with Coomassie blue stain (Fig. 6A). Fusion protein was also confirmed to be GST-TM protein by immunoblotting using anti-TM (Fig. 6B) and anti-GST antibodies (Fig. 6C). A band of ~64-kDa molecular mass was detected on the blot both by anti-TM antibody and anti-GST antibody.



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Fig. 6. Expression and purification of GST-tropomyosin (TM) protein. The confirmed clone of Escherichia coli BL21 containing recombinant TM cDNA was used for expression and purification of GST-tagged fusion protein as described in Methods. The purified fusion proteins were subjected to SDS-PAGE followed by Coomassie blue staining and immunoblotting using anti-TM and anti-GST antibodies. A: Coomassie blue staining of the purified protein: GST-TM (lane 1) and GST alone (lane 2); B: immunoblotting of purified protein with anti-TM antibody: GST-TM (lane 1) and GST alone (lane 2); C: immunoblotting of purified protein with anti-TM antibody: GST-TM (lane 1) and GST alone (lane 2). Molecular weight (MW) marker is marked on the left.

 
Direct Association of Recombinant PKC{alpha} with GST-TM Fusion Protein

The purified GST-TM conjugated to glutathione agarose beads was incubated with commercially available recombinant PKC{alpha} protein (Cytoskeleton). The GST-TM fusion proteins bound with PKC{alpha} were eluted as described in the METHODS. Analysis of all the unbound fraction washes and eluted fractions on dot blot showed that PKC{alpha} coeluted with GST-TM (Fig. 7, lanes 11 and 12, GST-TM). Glutathione agarose beads conjugated to GST alone were used as controls and did not show coelution of PKC{alpha} with GST alone (Fig. 7, lanes 11 and 12, GST). Our results suggest that acetylcholine-induced association of TM with PKC{alpha} was due to a direct protein-protein interaction. This is the first evidence of direct protein-protein interaction of TM with PKC{alpha}.



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Fig. 7. Direct interaction of GST-TM with recombinant PKC{alpha}. GST-TM (25 µg) was incubated with 200 µl of 50% suspension of glutathione agarose beads in PBS/0.1% {beta}-mercaptoethanol at 4°C for 30 min. Fractions 1–5 are washings of unbound GST-TM. Recombinant human PKC{alpha} (10 µg) was added to the agarose-bound GST-TM beads and rocked for 1 h. Fractions 6–10 are washings of unbound PKC{alpha}. Protein bound to the glutathione-agarose beads was eluted with 10 mM reduced glutathione in fractions 11 and 12. Dot blots of all the fractions (1–12) were probed with antibodies specific for PKC{alpha}, TM, and GST. In fractions 11–12, coelution of PKC{alpha} with GST-TM was observed. This indicates that there is a direct association of TM with PKC{alpha}. Experiments were done in triplicate, and GST alone bound to glutathione beads was used as control to show that binding of PKC{alpha} to TM was specific.

 

    DISCUSSION
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Contraction of smooth muscle occurs when myosin S1 heads of the thick filament interacts with the actin molecules in the thin filament (6) forming a cross bridge. After the formation of the cross bridges, filaments slide together as the myosin heads bend and pull on the actin filaments causing them to slide. Regulation of contraction in smooth muscle cells at the thin filament level is not clearly understood. Three different contractile regulatory proteins, calponin, caldesmon, and TM have been implicated in the regulation of the smooth muscle contraction at the thin-filament level. In striated muscles (skeletal and cardiac muscle), the calcium-sensitizing contractile protein complex, troponin with the help of TM, initiates the interaction of myosin with actin. In the absence of calcium, the troponin complex binds to actin and positions the TM, thus blocking the myosin binding sites on actin (27). Binding of calcium to troponin causes detachment of troponin from actin, allowing TM to move over the surface of thin filaments, thereby exposing the myosin binding sites on actin, and this allows myosin to bind to actin leading to contraction (37). However, in smooth muscle, the troponin complex is absent, and the mode of regulation of thin filament contraction by TM is not clearly understood.

In smooth muscle, contraction evolves into two different phases: initial contraction followed by sustained contraction. Initial contraction is characterized by increase in intracellular calcium that binds to calmodulin, which in turn activates MLCK. MLCK causes phosphorylation of the myosin regulatory chain, leading to actin-activated myosin ATPase activation, actomyosin interaction, and muscle contraction (30, 31). The initial calcium transient is rapidly dissipated, causing rapid decline in MLCK activity, but the phosphorylated state of myosin regulatory chain and muscle contraction are sustained (11, 30, 31). The mechanism proposed to be activated during sustained contraction involves PKC and RhoA. Both PKC and RhoA inhibit MLCP and thus maintain the myosin regulatory chain in a phosphorylated state (30, 33, 35). Our laboratory and others (14) have previously shown that in smooth muscle cells, PKC{alpha} redistributes in response to contractile agonists. We have recently shown that there is a direct association of translocated PKC{alpha} with calponin in the particulate fraction and a role for the complex calponin-PKC{alpha}-HSP27 in contraction of colonic smooth muscle cells (23). We thus suggest that PKC{alpha} is not only involved in maintaining phosphorylation of the myosin regulatory light chain during sustained smooth muscle contraction (24), but it is also involved in regulating contraction at the thin-filament level by modulating the association of the different contractile proteins (22).

In smooth muscle, agonist-induced contraction is accompanied by phosphorylation and translocation of HSP27 to the membrane (34, 38). We have shown that phosphorylated HSP27 also plays an important role in smooth muscle contraction by modulating the interaction of contractile proteins. We have proposed that phosphorylated HSP27 may aid in the sliding of the TM on actin to expose myosin-binding sites for actomyosin interaction leading to contraction (29). Our present study indicates that acetylcholine induce a significant and sustained phosphorylation of TM in colonic smooth muscle cells. TM, as mentioned earlier, plays a crucial role in the regulation of contraction by preventing actomyosin interaction leading to inactivation of Mg2+-ATPase. Phosphorylation of TM modulates its interaction with actin (12), and in rabbit striated muscle, it has been associated with increased myosin Mg2+/ATPase activity (9). Our results indicate that acetylcholine-induced phosphorylation of TM is PKC mediated and calcium dependent. Our data from immunoprecipitation studies indicate that in colonic smooth muscle cells, acetylcholine induces a significant and sustained increase in the association of TM with PKC{alpha} in the particulate fraction. Our data further indicates that preincubation of smooth muscle cells with calphostin C, a PKC inhibitor, before acetylcholine stimulation results in inhibition of acetylcholine-induced association of PKC{alpha} with TM. We further investigated the changes in the amount of thin filament actin and TM in the particulate fraction in response to acetylcholine. Our data indicate that there is no significant change in the amount of TM or actin in the particulate fraction in response to acetylcholine. We thus postulate that the observed acetylcholine-induced increased association of TM with PKC{alpha} in the particulate fraction is due to agonist-induced activation and translocation of PKC{alpha} to the particulate fraction.

Studies have suggested that PKC{alpha} regulates contraction at the thin filament level by modulating the associations of contractile proteins (22), but association with TM is novel. PKC interacts with many different proteins in direct protein-protein interaction, and our paper presents the first evidence that the novel association of TM with PKC{alpha} is due to direct protein-protein interaction. In vitro direct binding assay was carried out to assess the possibility of direct protein-protein interaction of TM with PKC{alpha}. TM cDNA was amplified from the rabbit colon smooth muscle extracted RNA by RT-PCR and confirmed by sequencing. A recombinant GST-fused TM was constructed by cloning the RT-PCR amplified TM cDNA into pGEX-KT vector. Purified GST-TM was bound to glutathione agarose beads and incubated with recombinant PKC{alpha}. The analysis of eluted samples on the dot blot indicated that the acetylcholine-induced association of TM with PKC{alpha} was indeed due to a direct interaction between the two proteins. This is the first evidence for direct protein-protein interaction of TM with PKC{alpha}.

PKC-mediated phosphorylation of TM and direct interaction of TM with PKC{alpha} in smooth muscle suggests that TM could be a substrate for PKC{alpha}. Our lab has previously shown that calponin interacts with translocated PKC{alpha}, translocated HSP27, and TM in the particulate fraction of smooth muscle on acetylcholine induction (22). On the basis of our present study, we propose that the contractile apparatus containing thin-filament actin with bound TM is present in the particulate fraction. We thus propose that, on agonist induction, HSP27 is phosphorylated and translocated to the particulate fraction along with PKC{alpha}. The complex of phosphorylated HSP27-PKC{alpha}-calponin associates with TM. Whereas the direct interaction of phosphorylated HSP27 with TM may aid in the sliding of TM on actin, exposing the myosin binding sites for actomyosin interaction, the direct interaction of PKC{alpha} with TM may lead to the phosphorylation of TM. Phosphorylated TM along with association of calponin to TM may contribute to retain the slided TM on the actin filament maintaining the exposed myosin binding sites on actin for actomyosin interaction leading to increased Mg2+-ATPase activity during sustained contraction. Our studies also provide a first evidence of interaction of the contractile protein TM with the signaling molecule PKC{alpha}.

Future studies should concentrate on the specificities of interaction of TM with PKC{alpha}. These studies would provide further insight into the regulation of smooth muscle contraction by TM at the thin-filament level.


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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5 RO1-DK-057020.


    ACKNOWLEDGMENTS
 
We thank Mercy D. Pawar for technical assistance and Kelly Smid 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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  1. Biancani P, Hartnett KM, Sohn UD, Rhim BY, Behar J, Hillemeier C, and Bitar KN. Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh (Abstract). Am J Physiol Gastrointest Liver Physiol 266: G767.
  2. Bitar KN. HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction. Am J Physiol Gastrointest Liver Physiol 282: G894–G903, 2002.[Abstract/Free Full Text]
  3. Bitar KN, Ibitayo A, and Patil SB. HSP27 modulates agonist-induced association of translocated RhoA and PKC-{alpha} in muscle cells of the colon. J Appl Physiol 92: 41–49, 2002.[Abstract/Free Full Text]
  4. Eaton BL. Tropomyosin binding to F-actin induced by myosin heads. Science 192: 1337–1339, 1976.[ISI][Medline]
  5. Eto M, Ohmori T, Suzuki M, Furuya K, and Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem(Tokyo) 118: 1104–1107, 1995.[Abstract]
  6. Geeves MA and Holmes KC. Structural mechanism of muscle contraction. Annu Rev Biochem 68: 687–728, 1999.[CrossRef][ISI][Medline]
  7. Gordon AM, Regnier M, and Homsher E. Skeletal and cardiac muscle contractile activation: tropomyosin "rocks and rolls." News Physiol Sci 16: 49–55, 2001.[ISI][Medline]
  8. Hakes DJ and Dixon JE. New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202: 293–298, 1992.[ISI][Medline]
  9. Heeley DH. Investigation of the effects of phosphorylation of rabbit striated muscle alpha alpha-tropomyosin and rabbit skeletal muscle troponin-T. Eur J Biochem 221: 129–137, 1994.[Abstract]
  10. Hitchcock-DeGregori SE and Varnell TA. Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities. J Mol Biol 214: 885–896, 1990.[ISI][Medline]
  11. Horowitz A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967–1003, 1996.[Abstract/Free Full Text]
  12. Houle F, Rousseau S, Morrice N, Luc M, Mongrain S, Turner CE, Tana S, Moreau P, and Huot J. Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing. Mol Biol Cell 14: 1418–1432, 2003.[Abstract/Free Full Text]
  13. Ibitayo AI, Groblewski G, and Bitar KN. Ceramide induced phosphorylation of Hsp27 and modulation of its distribution within smooth muscle cells (Abstract). Gastroenterology 114: A769, 1998.
  14. Ibitayo AI, Sladick J, Tuteja S, Louis-Jacques O, Yamada H, Groblewski G, Welsh M, and Bitar KN. Hsp27 in signal transduction and association with contractile proteins in smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 277: G445–G454, 1999.[Abstract/Free Full Text]
  15. Ikebe M and Brozovich FV. Protein kinase C increases force and slows relaxation in smooth muscle: Evidence for regulation of the myosin light chain phosphatase. Biochem Biophys Res Commun 14: 370–376, 1996.
  16. Kitazawa T, Eto M, Woodsome TP, and Brautigan DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 275: 9897–9900, 2000.[Abstract/Free Full Text]
  17. Kitazawa T, Takizawa N, Ikebe M, and Eto M. Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in Triton X-100 demembranated rabbit arterial smooth muscle. J Physiol 520: 139–152, 1999.[Abstract/Free Full Text]
  18. Leinweber B, Parissenti AM, Gallant C, Gangopadhyay SS, Kirwan-Rhude A, Leavis PC, and Morgan KG. Regulation of protein kinase C by the cytoskeletal protein calponin. J Biol Chem 275: 40329–40336, 2000.[Abstract/Free Full Text]
  19. Marston SB and Smith CW. The thin filaments of smooth muscles. J Muscle Res Cell Motil 6: 669–708, 1985.[CrossRef][ISI][Medline]
  20. Masuo M, Reardon S, Ikebe M, and Kitazawa T. A novel mechanism for the Ca2+ sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. J Gen Physiol 104: 265–286, 1994.[Abstract]
  21. McLachlan AD and Stewart M. The troponin binding region of tropomyosin. Evidence for a site near residues 197 to 127. J Mol Biol 106: 1017–1022, 1976.[ISI][Medline]
  22. Patil SB, Pawar MD, and Bitar KN. Direct association and translocation of PKC-{alpha} with calponin. Am J Physiol Gastrointest Liver Physiol 286: G954–G963, 2004.[Abstract/Free Full Text]
  23. Patil SB, Pawar MD, and Bitar KN. Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKC{alpha}. Am J Physiol Gastrointest Liver Physiol 286: G635–G644, 2004.[Abstract/Free Full Text]
  24. Patil SB, Tsunoda Y, Pawar MD, and Bitar KN. Translocation and association of ROCK-II with RhoA and HSP27 during contraction of rabbit colon smooth muscle cells. Biochem Biophys Res Commun 319: 95–102, 2004.[CrossRef][ISI][Medline]
  25. Sbrissa D, Yamada H, Hajra A, and Bitar KN. Bombesin-stimulated ceramide production and MAP kinase activation in rabbit rectosigmoid smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 272: G1615–G1625, 1997.[Abstract/Free Full Text]
  26. Silver PJ. Regulation of contractile activity in vascular smooth muscle by protein kinases. Rev Clin Basic Pharmacol 5: 341–395, 1985.
  27. Smith DA and Geeves MA. Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium. Biophys J 84: 3168–3180, 2003.[Abstract/Free Full Text]
  28. Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CL, Harnett KM, Behar J, and Biancani P. Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001.[Abstract/Free Full Text]
  29. Somara S and Bitar KN. Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle. Am J Physiol Cell Physiol 286: C1290–C1301, 2004.[Abstract/Free Full Text]
  30. Somlyo AP and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231–236, 1994.[CrossRef][ISI][Medline]
  31. Stull JT, Kamm KE, Krueger JK, Lin P, Luby-Phelps K, and Zhi G. Ca2+/calmodulin-dependent myosin light-chain kinases. Adv Second Messenger Phosphoprotein Res 31: 141–150, 1997.[ISI][Medline]
  32. Throckmorton DC, Packer CS, and Brophy CM. Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction. J Surg Res 78: 48–53, 1998.[CrossRef][ISI][Medline]
  33. Ueheta M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997.[CrossRef][ISI][Medline]
  34. Wang P and Bitar KN. Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of Hsp27. Am J Physiol Gastrointest Liver Physiol 275: G1454–G1462, 1998.[Abstract/Free Full Text]
  35. Weber LP, Van Lierop JE, and Walsh MP. Ca2+ independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol 516: 805–824, 1999.[Abstract/Free Full Text]
  36. Weigt C, Wegner A, and Koch MH. Rate and mechanism of the assembly of tropomyosin with actin filaments. Biochemistry 30: 10700–10707, 1991.[ISI][Medline]
  37. Xu C, Craig R, Tobacman L, Horowitz R, and Lehman W. Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. Biophys J 77: 985–992, 1999.[Abstract/Free Full Text]
  38. Yamada H, Strahler J, Welsh MJ, and Bitar KN. Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle. Am J Physiol Gastrointest Liver Physiol 269: G683–G691, 1995.[Abstract/Free Full Text]