Am J Physiol Cell Physiol 286: C1290–C1301, 2004.
First published January 28, 2004; 10.1152/ajpcell.00458.2003.
Tropomyosin interacts with phosphorylated HSP27 in agonist-induced
contraction of smooth muscle
Sita Somara and Khalil N. Bitar
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109
Submitted 22 October 2003; accepted in final form 27 January 2004
adequately describes the basic
mechanism of contraction in all muscle types, but there are
significant differences in the pathways leading to contraction.
When the muscle is at rest, the tropomyosin molecule covers
the myosin-binding site of the actin molecule, preventing
actin-myosin interaction (22, 36, 41, 44). Upon increase in
intracellular calcium, tropomyosin is displaced off the actin,
exposing the myosin-binding sites for actin-myosin interaction
and leading to contraction (41). In skeletal and cardiac muscle,
upon elevation of intracellular free Ca2⫹ concentration, a
calcium-sensitive multiprotein complex, troponin, binds calcium and initiates the displacement of tropomyosin off the
actin filament. In smooth muscle, troponin is absent and the
mechanism that initiates displacement of tropomyosin off the
actin is not clearly understood.
Tropomyosin is an actin-binding protein widely distributed
in virtually all eukaryotic cells. It is a crucial part of the
contractile apparatus and of thin filament assemblies of both
muscle and nonmuscle cells. Tropomyosin assembles into an
␣-helical coiled-coil dimer with each molecule interacting with
six or seven monomers of actin (21, 38). It also binds to itself
and helps wrap around the actin molecule to stabilize thin
filament assembly (52). The crucial role played by tropomyosin in regulation of contraction and the mechanism of its action
have been studied extensively in cardiac and skeletal muscle,
but its mode of action in smooth muscle is not clearly defined.
Understanding the interaction of tropomyosin with other thin
filament-binding contractile proteins may provide valuable
information regarding the role of tropomyosin in smooth muscle contraction.
Contraction in smooth muscle is initiated by several signaling pathways that lead to the activation of myosin light chain
kinase (MLCK) (23). MLCK phosphorylates the regulatory
chain of myosin, which activates actomyosin adenophosphatase activity of smooth muscle, leading to contraction.
Smooth muscle can maintain contraction even in the absence of
myosin light chain phosphorylation, implicating the involvement of other regulatory pathways in smooth muscle contraction (7, 47). Protein kinase C (PKC) functions in an alternative
signaling pathway leading to sustained smooth muscle contraction (42, 45, 49). PKC-mediated contraction results from PKC
activation and translocation to the membrane, where it may
activate kinases such as mitogen-activated protein (MAP) kinase, leading to the phosphorylation of several contractile
proteins (9, 19, 27). Agonists that induce PKC-mediated contraction have been shown to be involved in the phosphorylation
of heat shock protein (HSP)27, a thin filament-binding contractile protein involved in smooth muscle contraction (6).
HSP27 is an actin-associated protein that modulates actin
filament dynamics (29, 33, 39). HSP27 undergoes rapid phosphorylation in response to a number of extracellular factors (3)
such as heat shock, growth factors, phorbol esters, calcium
ionophores, interleukin (IL)-1, and tumor necrosis factor
(TNF)-␣ (1, 53). In smooth muscle, during agonist-induced
contraction p38 MAP kinase is activated, which in turn phosphorylates HSP27 (17, 31). HSP27 is phosphorylated rapidly at
30 s after stimulation, and the phosphorylation remains sustained at 4 min after stimulation. In smooth muscle, HSP27 has
significant effects on the actin cytoskeleton and these effects
are regulated by phosphorylation and dephosphorylation (17,
31). Phosphorylated HSP27 plays a crucial role in cytoskeletal
reorganization by increasing cytoskeletal stability. HSP27 also
appears to play an important role in smooth muscle contraction
by forming a link between signaling cascade and contractile
machinery as it colocalizes with the contractile proteins actin,
myosin, caldesmon, and tropomyosin and also associates with
the signaling proteins PKC-␣ and RhoA (25, 51). The phosphorylation sites in human HSP27 have been mapped to be
Ser15, Ser78, and Ser82 (30), of which Ser82 appears to be the
major site of in vivo phosphorylation, followed by Ser78 and
Ser15, the minor sites (30, 48). Phosphorylation is accompanied by a decrease in the size of HSP27 oligomers (26). Small
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: [email protected]).
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.
acetylcholine; fusion proteins; serine
THE SLIDING FILAMENT MODEL
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0363-6143/04 $5.00 Copyright © 2004 the American Physiological Society
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Somara, Sita, and Khalil N. Bitar. Tropomyosin interacts with
phosphorylated HSP27 in agonist-induced contraction of smooth
muscle. Am J Physiol Cell Physiol 286: C1290–C1301, 2004. First
published January 28, 2004; 10.1152/ajpcell.00458.2003.—Displacement of the contractile protein tropomyosin from actin filament
exposes the myosin-binding sites on actin, resulting in actin-myosin
interaction and muscle contraction. The objective of the present study
was to better understand the interaction of tropomyosin with heat
shock protein (HSP)27 in contraction of smooth muscle cells of the
colon. We investigated the possibility of a direct protein-protein
interaction of tropomyosin with HSP27 and the role of phosphorylated
HSP27 in this interaction. Immunoprecipitation studies on rabbit
smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with
HSP27 phosphorylation mutants indicated that the association of
tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)tagged HSP27 mutant proteins show that tropomyosin has greater
direct interaction to phosphomimic HSP27 mutant compared with
wild-type and nonphosphomimic HSP27. Our data suggest that, in
response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin.
SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
MATERIALS AND METHODS
Materials
The following reagents were purchased: monoclonal ␣-smooth
muscle actin antibody and monoclonal mouse anti-tropomyosin antibody (developed against chicken gizzard tropomyosin) and tropomyosin from chicken gizzard from Sigma (St. Louis, MO); monoclonal
F-actin antibody from Abcam (Cambridge, MA); monoclonal mouse
anti-myosin antibody (MAB1670) from Chemicon International (Temecula, CA); monoclonal mouse anti-human HSP27 antibody (2B4 –
123) as previously described (4); polyclonal rabbit p-HSP27 (Ser82),
polyclonal rabbit p-HSP27 (Ser78), and polyclonal goat p-HSP27
(Ser15) from Santa Cruz Biotechnology (Santa Cruz, CA); protein G
Sepharose from Amersham Biosciences Sweden; Silver-Plus staining
kit and polyvinylidene fluoride (PVDF) membranes from Bio-Rad
(Hercules, CA); QiaGen Effectene transfection kit from QiaGen
(Valencia, CA); enhanced chemiluminescence (ECL) detection reagents from Amersham Biosciences UK; G-418, penicillin/streptomycin, fetal bovine serum (FBS), collagen IV, and Dulbecco’s (DMEM)
from GIBCO-BRL (Grand Island, NY); actin-binding protein spindown kit from Cytoskeleton (Denver, CO); all other reagents from
Sigma.
Methods
Preparation of smooth muscle cells from rabbit rectosigmoid. All
procedures were performed according to the “Guiding Principles for
Research Involving Animals and Human Beings” of the American
Physiological Society. Smooth muscle cells of rabbit rectosigmoid
were isolated as described previously (4). Briefly, the internal anal
sphincter (IAS) from anesthetized New Zealand White rabbits, conAJP-Cell Physiol • VOL
sisting of the distalmost 3 mm of the circular muscle layer, ending at
the junction of skin and mucosa, was removed by sharp dissection. A
5-cm length of the rectosigmoid orad to the junction was dissected and
digested to yield isolated smooth muscle cells. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml of HEPES
buffer (pH 7.4). The composition of the buffer was (in mM) 115 NaCl,
5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, 5.6
glucose with 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS
type II), 0.01% (wt/vol) soybean trypsin inhibitor, and 0.184% (wt/
vol) in DMEM. At the end of the second enzymatic incubation period,
the medium was filtered through 500-␮m Nitex mesh. The partially
digested tissue left on the filter was washed four times with 10 ml of
collagenase-free buffer solution. Tissue was then transferred into 15
ml of fresh collagenase-free buffer solution, and cells were gently
dispersed. After a hemocytometric cell count, the harvested cells were
resuspended in collagenase-free HEPES buffer (pH 7.4). Each
rectosigmoid yielded 10 –20 ⫻ 106 cells.
Transfection of smooth muscle cells with HSP27 mutants. Smooth
muscle cells were cultured in DMEM with 10% FBS and 3% penicillin/streptomycin on collagen IV-coated dishes. Cells were passed
on the day before transfection and allowed to reach 70% confluence
on the day of transfection. Cells were washed twice with PBS. 3D or
3G human HSP27 mutant cDNA was transfected into the cells with a
QiaGen Effectene transfection kit. Briefly, the cDNA was diluted with
buffer EC (QiaGen) and mixed with enhancer followed by incubation
at room temperature for 5 min. The DNA-enhancer mixture was well
mixed with Effectene transfection reagent followed by incubation at
room temperature for 10 min to allow complex formation. The
transfection complex was then mixed with cell culture medium and
overlaid on the cells. After 2 days of transfection, the cells were
selected with G-418 (3 mg/ml) for 1–2 days. The estimated transfection efficiency was ⬃40% as measured by the number of cells
remaining attached to the plate upon G-418 treatment. These cells
were maintained as a stable transfection and were grown to confluence
for further studies. Expressions of mutant HSP27 proteins were
confirmed by immunoblotting of the whole cell lysates with human
HSP27-specific antibody (Stress Gen) (5). The protein has been
shown not to cross-react with rabbit and mouse antibody. Overexpression of HSP27 in transfected cells was confirmed by immunoblot
with HSP27 antibody (2B4 –123). Furthermore, phenotypic characterization of the stable HSP27-expressing cell lines was done by studying the expression of ␣-actin and smooth muscle-myosin heavy chain
by Western blot. All the cells used for the experiments were from
passage 1.
Immunoprecipitation and immunoblotting. Smooth muscle cells
isolated from rabbit colon were diluted in HEPES buffer as needed.
Rabbit colon smooth muscle cells and confluent smooth muscle cells
in culture were separately divided into three sets for stimulation: the
first set was treated with 0.1 ␮M acetylcholine for 30 s; the second set
was treated with 0.1 ␮M acetylcholine for 4 min; and the third set was
untreated and served as control. After treatment, the cells were
washed with PBS (in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4; pH
7.4). The cells were then suspended in lysis buffer [in mM: 20
Tris䡠HCl, pH 7.4, 150 NaCl, 2 phenylmethylsulfonyl (PMSF), 1
Na3VO4, 1 NaF, 1 Na4MoO4, 1 dithiothreitol (DTT), 20 Na2HPO4, 20
NaH2PO4, 20 Na4P2O7䡠10H2O, and 50 EDTA with 5 ␮g/ml DNase/
RNase, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin
A, and 10 ␮g/ml antipain]. The suspended cells were lysed by
sonication for 30 s followed by vortexing for 30 s and incubated on ice
for 30 min. The cells were centrifuged for 15 min at 14,000 g, and the
supernatant was collected. Protein content was estimated by Bio-Rad
protein assay solution. Antibody (1–2 ␮g) was added to 500 ␮g of cell
lysate protein in a total of 500 ␮l of lysis buffer and rocked overnight
at 4°C. Fifty microliters of 50% protein G Sepharose bead slurry were
added to the overnight mixture and rocked at 4°C for 2 h. The beads
bound with proteins were then collected by centrifuging at 14,000 g
for 3 min at 4°C. Supernatant was discarded, and the bead pellet was
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oligomers of HSP27 regulate and stabilize the cytoskeleton as
phosphorylation of HSP27 leads to changes in the actin cytoskeleton and actin-dependent events (32, 33, 54).
The colocalization of tropomyosin with HSP27 and their
individual roles in triggering and maintaining smooth muscle
contraction led us to study the direct interaction of tropomyosin
with HSP27 (25, 51). We previously proposed a model (5)
whereby phosphorylated HSP27 could lead to actin-myosin
interaction, possibly by interaction of HSP27 with tropomyosin. The objective of the present study was to investigate the
possibility of direct interaction of HSP27 with tropomyosin
and to study how phosphorylation of HSP27 modulates this
interaction in/during agonist-induced sustained smooth muscle
contraction. Immunoprecipitation studies on rabbit smooth
muscle cells indicate that, upon agonist-induced contraction,
tropomyosin shows increased association to HSP27 phosphorylated at Ser78 and Ser82. Transfection of smooth muscle cells
with the HSP27 phosphorylation mutants 3D-HSP27 mutant
(Ser15, Ser78, and Ser82 mutated to aspartate to mimic the
phosphorylated HSP27) and 3G-HSP27 mutant (Ser15, Ser78,
and Ser82 phosphorylation sites mutated to nonphosphorylatable glycine) indicated that association of tropomyosin with
HSP27 is affected by HSP27 phosphorylation. In vitro binding
studies with glutathione S-transferase (GST)-tagged HSP27
mutant proteins show that tropomyosin has greater direct
interaction to the phosphomimic HSP27 mutant compared with
nonphosphomimic and wild-type HSP27. These data confirm
our previously proposed model that phosphorylation of HSP27
may result in a conformational change, leading to greater direct
interaction of phosphorylated HSP27 with tropomyosin. This
interaction may help pull the tropomyosin away from thin
filament, leading to a sustained actin-myosin interaction.
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reduced glutathione). The fusion proteins were analyzed by immunoblotting with anti-GST and anti-HSP27 antibody.
In vitro binding of different GST-HSP27 fusion proteins isoforms
with tropomyosin. Different isoforms of GST-HSP27 (50 ␮g each)
were mixed with 200 ␮l of glutathione-agarose beads separately and
rocked for 2 h at 4°C. Unbound GST-HSP27 fusion proteins were
removed by washing five times with MTPBS. All the washes were
retained for further analysis. Twenty-five micrograms of tropomyosin
were added to the different GST-HSP27 isoform-bound beads and
rocked for 1 h at 4°C. Unbound tropomyosin was removed by washing
five times with MTPBS. All the washes were retained for further
analysis. The bound proteins were eluted twice with 1 bed volume of
elution buffer (50 mM Tris·HCl, pH 8.0, containing 10 mM reduced
glutathione). Ten microliters each of all the washes and the eluates
were spotted on a PVDF membrane. The membrane was blocked with
5% nonfat skim milk in TBS and immunoblotted with anti-GST
antibody, anti-tropomyosin antibody, and anti-HSP27 antibody. The
spots were detected by chemiluminescence.
Dot blot. Dry blotting paper and the PVDF membrane were cut
according to the dot blot apparatus size. The cut PVDF membrane was
soaked in 100% methanol for 2–3 min followed by equilibration in
1⫻ PBS for 5 min at room temperature. The cut dry blotting paper
was incubated in 1⫻ PBS for 5 min. The wet blotting paper was
placed on the dot blot apparatus, followed by the equilibrated PVDF
membrane onto the blotting paper. The dot blot apparatus was clasped
with a clamp on the sides and was attached to the vacuum pipe. The
vacuum was passed at the rate of 1 ml/min. Ten microliters of the
sample were added into the each slot. The slots were then washed with
100 ␮l of 1⫻ PBS. The clamps were opened to remove the membrane.
The membrane was incubated in 5% nonfat dry milk in TBST with
rocking for 1 h at room temperature. The membrane was washed twice
for 10 min each with TBST. The membrane was then incubated in the
appropriate dilution of primary antibody for 1 h with rocking at room
temperature. The membrane was washed three times for 15 min each
with TBST. The membrane was then incubated in the appropriate
dilution of secondary antibody for 1 h with rocking at room temperature followed by washing three times for 15 min each with TBST.
The membrane was then incubated with Amersham’s ECL reagent for
1 min. The proteins were detected on the membrane by immediately
exposing the membrane to the film.
F-actin cosedimentation assay. F-actin cosedimentation assays
were performed as described by the actin manufacturer (Cytoskeleton)
with slight modification. Briefly, whole cell lysates were incubated in
F-actin buffer (in mM: 20 Tris䡠HCl, pH 7.5, 75 KCl, 10 NaCl, 2 DTT,
and 2.5 MgCl2) for 30 min at room temperature and subjected to
ultracentrifugation at 110,000 g for 1 h at 24°C to pellet out the
F-actin from the cell lysate. The supernatant was collected, and an
equal amount of F-actin prepared per the manufacturer’s protocol was
added to each sample, followed by incubation at room temperature for
1 h. After 1 h, the samples were subjected to ultracentrifugation at
110,000 g to pellet F-actin and proteins bound to F-actin. After
solubilization of the pellet fraction in a volume equal to the initial
incubation volume, 20 ␮l of the pellet fractions was loaded onto
SDS-PAGE.
Data analysis. Western blot bands were quantitated with a densitometer (model GS-700, Bio-Rad Laboratories), and band volumes
(absorbance units ⫻ mm2) were calculated and expressed as a percentage of the total volume. Band data are within the linear range of
detection for each antibody used. The control band intensities were
standardized to 100%. The band intensities of samples from treated
cells were compared with the control and expressed as percent change
from the control. Blotting data are within the linear range of detection
for each antibody used. All the means were compared and analyzed
with Student’s t-test.
Dot blot spots were quantitated with a densitometer (model GS700, Bio-Rad Laboratories), and spot volumes (absorbance units ⫻
mm2) were calculated and expressed as a percentage of the total
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washed three times at room temperature with Tris-buffered saline
(TBS) bead wash buffer (20 mM Tris䡠HCl, 150 mM NaCl, pH 7.6).
The beads were then resuspended in 25 ␮l of 2⫻ sample buffer
and boiled for 5 min. Immunoprecipitates were immunoblotted with
polyclonal anti-HSP27s78 (1:500), anti-HSP27s82 (1:500), antiHSP27s15 (1:500), and anti-tropomyosin antibody (1:500). Replicates
of experiments were performed with completely separate sets of cells.
Silver staining. The proteins were separated on SDS-PAGE, and
the gel was silver stained following the manufacturer’s protocol.
Western blot. The proteins were separated on SDS-PAGE and
electrophoretically transferred to PVDF membrane. The PVDF membrane was then blocked with 5% nonfat dry milk for 1 h. After
blocking, the membrane was incubated in an appropriate dilution of
primary antibody in 5% nonfat dry milk in TBS-Tween 20 (TBST) for
1 h. The membrane was washed three times for 15 min each with TBS
at room temperature to remove unbound primary antibody. The
membrane was then incubated in an appropriate dilution of secondary
antibody in 5% nonfat dry milk in TBST for 1 h at room temperature.
The membrane was washed three times with TBST for 15 min each at
room temperature to remove unbound secondary antibody. The membrane was then incubated with ECL reagent for 1 min. The proteins
were detected on the membrane by immediately exposing the membrane to the film for 30 s and 1 min.
Cloning of HSP27 mutants in GST-tagged expression vector. Three
isoforms of human HSP27 were used in these studies: 1) wild-type
(wt)HSP27, which has three phosphorylation sites, Ser15, Ser78, and
Ser82; 2) 3G-HSP27 mutant, in which all three phosphorylation sites
were mutated to glycine to mimic nonphosphorylatable HSP27; and 3)
3D-HSP27 mutant, in which all three phosphorylation sites were
mutated to aspartate to mimic constitutively phosphorylated HSP27.
The cDNAs encoding the human wtHSP27, 3G-HSP27, and 3DHSP27 cloned in vectors pcDNA3.1 (Dr. R. Benndorf, University of
Michigan) were used as template for PCR amplification of mutant
HSP27 cDNAs. The sense and antisense primers used were 5⬘-TTT
GGA TCC ATG ACC GAG CGC CGC GT-3⬘ (restriction site
BamHI) and reverse primer 5⬘-TTT GAA TTC GCT AAG GCT TTA
CTT G-3⬘ (restriction site EcoRI), respectively. The amplified PCR
products of ⬃700 bp were digested with the restriction enzymes
BamHI and EcoRI. The digested PCR products were cloned in frame
with the NH2-terminal GST tag of the vector pGEX-KT (Dr. J. Dixon,
University of Michigan) (18). Recombinant pGEX KT was transformed into Escherichia coli DH5␣. After confirmation of the clones
by double digest release of the inserts, recombinant pGEX KT-HSP27
isoforms were transformed into expression host BL21 (DE3) pLysS
cells. All plasmid constructs were further verified by sequencing.
Purification of recombinant GST-HSP27 fusion protein isoforms.
The GST-HSP27 fusion proteins were expressed and purified with
glutathione-agarose beads as described by Hakes and Dixon (18) with
slight modification. Five milliliters of overnight culture were inoculated in five hundred milliliters of fresh LB medium with 100 ␮g/ml
ampicillin. The culture was grown with vigorous shaking at 37°C until
the culture reached 0.4 – 0.5 optical density at 600 nm (⬃3 h).
Isopropyl-␤-d-1-thiogalactopyranoside (IPTG) was then added to the
culture at 1 mM final concentration, and the cultures were grown for
another 2–3 h with vigorous shaking at 37°C. The cells were then
collected by centrifugation at 6,000 rpm for 5 min. The cell pellet was
resuspended in 1/10th volume of ice-cold mercaptoethanol-Triton
X-100-PBS (MTPBS; 150 mM NaCl, 16 mM Na2HPO4, 4 mM
NaH2PO4, pH 7.3, and 1% Triton X-100 plus 0.1% ␤-mercaptoethanol), and lysozyme at a final concentration of 100 ␮g/ml (freshly
prepared from 10 mg/ml stock) was added, followed by an incubation
on ice for 30 min. The cells were lysed by mild sonication and
centrifuged at 10,000 rpm for 15 min. The supernatant was mixed
with glutathione-agarose beads (50 ␮l of each 500-␮l supernatant)
and rocked at 4°C for 2 h. The beads were washed three times with 5
bead volumes of MTPBS. The fusion protein was eluted with 1 bed
volume of elution buffer (50 mM Tris䡠HCl, pH 8.0 containing 10 mM
SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
volume. Spot data are within the linear range of detection for each
antibody used. The control spot intensity was standardized to 100%.
The spot intensities of eluted fractions of the tropomyosin were
compared with the control and expressed as percent change from the
control. All the means were compared and analyzed with Student’s
t-test.
RESULTS
Acetylcholine-Induced Coimmunoprecipitation of
Tropomyosin with Phosphorylated HSP27 in Whole Cell
Lysate of Rabbit Colon Smooth Muscle Cells
unstimulated control. The data thus suggest that in rabbit colon
smooth muscle cells acetylcholine-induced contraction is associated with an increase in the association of tropomyosin
with HSP27 phosphorylated at Ser78 and Ser82.
Characterization of Stable HSP27 Mutant-Expressing Cells
Phenotypic characterization of the stably maintained HSP27
mutant-transfected smooth muscle cells was carried out by
immunoblotting the whole cell lysate with anti-␣-smooth muscle actin and smooth muscle-myosin heavy chain. No difference in expression of smooth muscle-specific actin and myosin
between normal and transfected cells was observed (Fig. 2A).
Overexpression of HSP27 in stably maintained HSP27 mutant-transfected smooth muscle cells was carried out by immunoblotting whole cell lysates of nontransfected and mutant
HSP27-transfected cells with anti-HSP27 antibody (2B4 –123).
Figure 2B shows that HSP27 was overexpressed in mutant
HSP27-transfected cells.
To check whether there were proteins other than actin and
HSP27 coimmunoprecipitating with tropomyosin, proteins
separated on SDS-PAGE were silver stained. The silverstained gel showed that, in addition to actin and HSP27, there
were other proteins coimmunoprecipitating with tropomyosin
(Fig. 3).
Acetylcholine-Induced Coimmunoprecipitation of
Tropomyosin with Phosphorylated HSP27 in Whole Cell
Lysates of Smooth Muscle Cells Transfected with
HSP27 Mutants
To study the gain of function and loss of function of
phosphorylated HSP27, cultured smooth muscle cells were
transfected with HSP27 mutants. Cells were transfected with
3G-HSP27, in which all the potential serine phosphorylation
sites, i.e., Ser15, Ser78, and Ser82, were replaced with glycine
Fig. 1. Acetylcholine-induced coimmunoprecipitation of tropomyosin with phosphorylated heat shock protein (HSP)27 in the
whole cell lysates of rabbit colon smooth muscle cells (SMC).
Freshly isolated rabbit colon smooth muscle cells were divided
into 3 sets: 1) unstimulated control cells (Ctr), 2) acetylcholine
(ACh; 0.1 ␮M) stimulated for 30 s, and 3) ACh (0.1 ␮M)
stimulated for 4 min. Whole cell lysates (500 ␮g) were immunoprecipitated (IP) with 2 ␮g of anti-tropomyosin antibody
followed by immunoblotting (IB) with different phosphorylated
HSP27 antibody (1:200). A: representative blot showing association of tropomyosin with HSP27 probed with phosphoSer15-HSP27 antibody, phospho-Ser78-HSP27 antibody, and
phospho-Ser82-HSP27 antibody after stimulation with ACh for
30 s and 4 min. No bands were observed when probed with
phospho-Ser15-HSP27 antibody. B: association of tropomyosin
with HSP27 phosphorylated at Ser78 when stimulated with
ACh. Stimulation with ACh resulted in a significant increase in
association of tropomyosin with HSP27 phosphorylated at
Ser78, with 182 ⫾ 4 and 146 ⫾ 19% of control at 30 s and 4
min, respectively, after stimulation (P ⱕ 0.001, n ⫽ 4). Unstimulated cells were used as control. C: association of tropomyosin with HSP27 phosphorylated at Ser82 when stimulated
with ACh. Stimulation with ACh resulted in a significant
increase in association of tropomyosin with HSP27 phosphorylated at Ser82, with 185 ⫾ 7 and 158 ⫾ 28% of control at 30 s
and 4 min, respectively, after stimulation (P ⱕ 0.001, n ⫽ 4).
Unstimulated cells were used as control.
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Tropomyosin was previously reported by our laboratory (25)
to colocalize with HSP27 in colonic smooth muscle cells. In
the present study, we characterized the form of HSP27 associated with tropomyosin upon acetylcholine stimulation.
HSP27 has been reported to be phosphorylated at three sites,
Ser15, Ser78, and Ser82. We have thus studied the phosphorylation state of HSP27 that is associated with tropomyosin, in
response to acetylcholine stimulation. Smooth muscle cells
isolated from rabbit colon were stimulated with 0.1 ␮M acetylcholine for 30 s and 4 min. Whole cell lysates were immunoprecipitated with anti-tropomyosin antibody. The proteins in
the immunoprecipitates were separated on SDS-PAGE and
were transferred onto PVDF membrane. The membrane was
then immunoblotted with different phospho-HSP27 antibodies,
i.e., anti-HSP27s15, anti-HSP27s78, and anti-HSP27s82 antibodies separately. No bands were observed when the membrane was probed with anti-HSP27s15 antibody, whereas
bands were observed with anti-HSP27s78 and anti-HSP27s82
antibodies (Fig. 1A). Acetylcholine induced a significant and
sustained increase in the association of tropomyosin with
HSP27s78 (182 ⫾ 4 and 146 ⫾ 19% of control at 30 s and 4
min, respectively; P ⱕ 0.001, n ⫽ 4; Fig. 1B) and HSP27s82
(185 ⫾ 7 and 158 ⫾ 28% of control at 30 s and 4 min,
respectively; P ⱕ 0.001, n ⫽ 4; Fig. 1C) compared with the
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Fig. 2. A: phenotypic characterization of stable HSP27-expressing cell lines.
Whole cell lysate from normal and 3D-HSP27 mutant- and 3G-HSP27 mutanttransfected stable smooth muscle cell lines were probed for the expression of
␣-actin and myosin heavy chain for phenotypic characterization of the stable
cell lines. B: immunoblotting of human HSP27 mutant-transfected cells showing overexpression of HSP27. The whole cell lysates of nontransfected (N) and
HSP27 mutant-transfected cells were subjected to SDS-PAGE and immunoblotted with anti-HSP27 (2B4 –123) antibody. Mutant HSP27-transfected cells
showed overexpression of HSP27.
to mimic the nonphosphorylatable HSP27, and with 3DHSP27, in which all the potential serine phosphorylation sites,
i.e., Ser 15, Ser78, and Ser82, were replaced with aspartate to
mimic the constitutively phosphorylated HSP27. These mutant
constructs were expressed in transfected smooth muscle cells,
and the expressions of mutant HSP27 proteins were confirmed
by immunoblotting the whole cell lysates. Confluent transfected and nontransfected rabbit colon smooth muscle cells
were stimulated with 0.1 ␮M acetylcholine for 30 s and 4 min.
Whole cell lysates from these cells were immunoprecipitated
with anti-tropomyosin antibody. The immunoprecipitates were
AJP-Cell Physiol • VOL
Fig. 3. Silver staining of IP-tropomyosin. Whole cell lysates of normal and
3D-HSP27 mutant- and 3G-HSP27 mutant-transfected cells were immunoprecipitated with tropomyosin antibody and subjected to SDS-PAGE electrophoresis. The gel was further silver stained to show that there are other proteins
involved in addition to actin, tropomyosin, and HSP27.
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then subjected to SDS-PAGE, and the separated proteins were
transferred onto PVDF membrane. The membrane was immunoblotted with anti-HSP27s78 and anti-HSP27s82 antibodies.
HSP27s78. An increase in band intensity was observed in
phosphomimic-transfected cells upon stimulation with acetylcholine when probed with anti-HSP27s78 compared with nonphosphomimic-transfected cells and nontransfected cells (Fig.
4A). The densitometric analysis of these bands showed a
significant increase in association of tropomyosin with phospho-Ser78-HSP27 in response to stimulation with acetylcholine at 30 s in the cells transfected with 3D-HSP27 mutant
(287 ⫾ 11% of control; P ⱕ 0.01, n ⫽ 3) compared with cells
transfected with 3G-HSP27 mutant (174 ⫾ 25% of control;
P ⱕ 0.01, n ⫽ 3) and nontransfected cells (172 ⫾ 4% of
control; P ⱕ 0.01, n ⫽ 3) above unstimulated cells (Fig. 4B).
The increased association was observed to be sustained at 4
min after acetylcholine stimulation in cells transfected with
3D-HSP27 mutants (172 ⫾ 7% of control; P ⱕ 0.01, n ⫽ 3)
compared with cells transfected with 3G-HSP27 mutant
(145 ⫾ 21% of control; P ⱕ 0.01, n ⫽ 3) and nontransfected
cells (140 ⫾ 7% of control; P ⱕ 0.01, n ⫽ 3) above unstimulated cells (Fig. 4B).
HSP27s82. A similar pattern of increased association of
tropomyosin with HSP27 was observed when the lysates of
phosphomimic-transfected cells compared with nonphosphomimic-transfected cells and nontransfected cells were probed
with anti-HSP27s82 antibody (Fig. 4C). Densitometric analysis showed a significant increase in association of tropomyosin
SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
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with phospho-Ser82-HSP27 in response to stimulation with
acetylcholine at 30 s in the cells transfected with 3D-HSP27
mutant (289 ⫾ 23% of control; P ⱕ 0.01, n ⫽ 3) compared
with cells transfected with 3G-HSP27 mutant (175 ⫾ 28% of
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control; P ⱕ 0.01, n ⫽ 3) and nontransfected cells (185 ⫾ 8%
of control; P ⱕ 0.01, n ⫽ 3) above unstimulated cells (Fig.
4D). The increased association was observed to be sustained at
4 min after acetylcholine stimulation in cells transfected with
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Fig. 4. ACh-induced coimmunoprecipitation of tropomyosin with phosphorylated HSP27 in the whole cell lysates of cultured
smooth muscle cells transfected with HSP27 mutant. Normal nontransfected cells from rabbit colon, cells transfected with the
phosphomimic HSP27 (3D) constructs, and cells transfected with the nonphosphomimic (3G) constructs were maintained in culture
until confluence. The cells were serum starved for 24 h before stimulation with and without 0.1 ␮M ACh for 30 s and 4 min
separately. The whole cell lysates were immunoprecipitated with anti-tropomyosin, followed by immunoblotting with different
anti-phospho-HSP27 antibodies. A: representative blots showing association of tropomyosin with HSP27 phosphorylated at Ser78
in nontransfected, 3G-HSP27-transfected, and 3D-HSP27-transfected cells after stimulation with 0.1 ␮M ACh for 30 s and 4 min.
B: association of tropomyosin with HSP27 phosphorylated at Ser78 in nontransfected, 3G-HSP27-transfected, and 3D-HSP27transfected cells upon stimulation with 0.1 ␮M ACh. The graph shows an increase in association of tropomyosin with HSP27
phosphorylated at Ser78 after stimulation with ACh for 30 s in the cells transfected with 3D-HSP27 mutant (287 ⫾ 11% of control;
P ⱕ 0.01, n ⫽ 3) compared with cells transfected with 3G-HSP27 mutant (174 ⫾ 25% of control; P ⱕ 0.01, n ⫽ 3) and control
nontransfected cells (172 ⫾ 4% of control; P ⱕ 0.01, n ⫽ 3). Similar increase in association was observed at 4 min in the cells
transfected with 3D-HSP27 mutant (172 ⫾ 7% of control; P ⱕ 0.01, n ⫽ 3) compared with cells transfected with 3G-HSP27 mutant
(145 ⫾ 21% of control; P ⱕ 0.01, n ⫽ 3) or control nontransfected cells (140 ⫾ 7% of control; P ⱕ 0.01, n ⫽ 3). C: representative
blots showing association of tropomyosin with HSP27 phosphorylated at Ser82 in nontransfected, 3G-HSP27-transfected, and
3D-HSP27-transfected cells upon stimulation with 0.1 ␮M ACh. D: association of tropomyosin with HSP27 phosphorylated at
Ser82 in nontransfected, 3G-HSP27-transfected, and 3D-HSP27-transfected cells on stimulation with 0.1 ␮M ACh. The graph
shows an increase in association of tropomyosin with HSP27 phosphorylated at Ser82 after stimulation with ACh for 30 s in cells
transfected with 3D-HSP27 mutant (289 ⫾ 23% of control; P ⱕ 0.01, n ⫽ 3) compared with cells transfected with 3G-HSP27
mutant (175 ⫾ 28% of control; P ⱕ 0.01, n ⫽ 3) and control nontransfected cells (185 ⫾ 8% of control; P ⱕ 0.01, n ⫽ 3). Similar
increases were observed at 4 min after stimulation with ACh in cells transfected with 3D-HSP27 mutant (174 ⫾ 20% of control;
P ⱕ 0.01, n ⫽ 3) compared with cells transfected with 3G HSP27 mutant (152 ⫾ 18% of control; P ⱕ 0.01, n ⫽ 3) and control
nontransfected cells (147 ⫾ 19% of control; P ⱕ 0.01, n ⫽ 3).
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SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
3D-HSP27 mutant (174 ⫾ 20% of control; P ⱕ 0.01, n ⫽ 3)
compared with cells transfected with 3G-HSP27 mutant
(152 ⫾ 18% of control; P ⱕ 0.01, n ⫽ 3) and nontransfected
cells (147 ⫾ 19% of control; P ⱕ 0.01, n ⫽ 3) above
unstimulated cells (Fig. 4D).
The data suggest that acetylcholine induced an increased
association of tropomyosin with HSP27 in phosphomimic
(3D-HSP27)-transfected smooth muscle cells and this increased association remained sustained at 4 min after stimulation, whereas in nonphosphomimic (3G-HSP27)-transfected
smooth muscle cells and nontransfected cells the association
was relatively lower.
Interaction of tropomyosin with F-actin in the presence or
absence of phosphorylated HSP27 was studied in smooth
muscle cells transfected with different HSP27 mutants. Whole
cell lysates from acetylcholine-stimulated transfected and nontransfected cells were immunoprecipitated with anti-tropomyosin antibody, and the immunoprecipitates were subjected to
SDS-PAGE. The separated proteins were then immunoblotted
with F-actin-specific antibody. The data indicate that transfection of smooth muscle cells with the nonphosphomimic mutant
of HSP27 resulted in an increase in the association of tropomyosin with actin in the relaxed cells (137 ⫾ 1% of control;
P ⱕ 0.01, n ⫽ 3) compared with phosphomimic mutanttransfected cells (84 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 3). The
levels of interaction of tropomyosin with actin remained significantly higher in the nonphosphomimic-transfected cells in
response to stimulation with acetylcholine at 30 s (121 ⫾ 1%
of control; P ⱕ 0.001, n ⫽ 3) or 4 min (120 ⫾ 1% of control;
P ⱕ 0.001, n ⫽ 3). The cells transfected with the phosphomimic mutant of HSP27 maintained the same lower level of
interaction of actin with tropomyosin after stimulation with
acetylcholine at 30 s (82 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 3)
and at 4 min (80 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 3) (Fig. 5).
The data suggest that in the presence of phosphomimic HSP27
less actin is associated with tropomyosin compared with the
amount of actin associated with tropomyosin in the presence of
nonphosphomimic HSP27. The decreased association of tropomyosin with actin in phosphomimic-transfected cells correlated with the concomitant increased association of tropomyosin with HSP27 in these cells. This suggested a possibility of
direct interaction of tropomyosin with HSP27.
Expression and Purification of GST-Fused Mutant
HSP27 Protein
To study whether the association of tropomyosin with
HSP27 was direct or indirect, in vitro binding studies were
carried out with GST-tagged HSP27 mutant fusion proteins.
Fragments (652 bp) containing mutant HSP27 cDNAs were
amplified from the respective HSP27 mutant pcDNA3.1
clones. The PCR-amplified fragments from each mutant were
inserted into pGEX-KT at BamH1 and EcoRI sites to express
an NH2-terminal GST-fused protein. The clones were confirmed to be in the correct open reading frames by sequencing.
The GST-fused mutant HSP27 proteins were expressed in
response to induction with 1 mM IPTG.
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Fig. 5. ACh-induced coimmunoprecipitation of tropomyosin with actin in the
whole cell lysates of smooth muscle cells transfected with HSP27 mutants.
Freshly isolated rabbit colon smooth muscle cells were divided into 3 sets: 1)
unstimulated control cells, 2) ACh (0.1 ␮M) stimulated for 30 s, 3) ACh (0.1
␮M) stimulated for 4 min. Whole cell lysates (500 ␮g) were immunoprecipitated with 2 ␮g of anti-tropomyosin antibody, followed by immunoblotting
with actin antibody (1:200). The graph shows association of tropomyosin with
actin upon stimulation with ACh. In the relaxed noncontracted cells, the
association of actin with tropomyosin was higher in nonphosphomimic mutanttransfected cells (137 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 3) than phosphomimic
mutant-transfected cells (84 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 3). In the
phosphomimic mutant-transfected cells, upon ACh stimulation the association
of tropomyosin with actin remained at the same lower levels: 82 ⫾ 1% (P ⱕ
0.01, n ⫽ 3) and 82 ⫾ 1% (P ⱕ 0.01, n ⫽ 3) of control at 30 s and 4 min,
respectively, after stimulation. The association of tropomyosin with actin was
found to be sustained at higher levels, after stimulation with ACh, in the
nonphosphomimic mutant: 121 ⫾ 1% (P ⱕ 0.001, n ⫽ 3) and 120 ⫾ 1% (P ⱕ
0.001, n ⫽ 3) of control at 30 s and 4 min, respectively.
The expressed fusion protein was purified with the glutathione-agarose beads as described in Methods and analyzed by
Western blot with anti-HSP27 antibody. A band of ⬃53-kDa
(27-kDa HSP27 ⫹ 26-kDa GST) molecular mass was detected
on the blot by both anti-HSP27 antibody and anti-GST antibody (Fig. 6).
Direct Association of Recombinant Tropomyosin with
GST-HSP27 Fusion Protein
The purified GST-fused HSP27 mutant proteins were conjugated to glutathione-agarose beads and incubated with commercially available tropomyosin protein. The GST-HSP27 fusion proteins bound with tropomyosin were eluted as described
in Methods. Eluted fractions were analyzed on the Western blot
by probing with anti-tropomyosin and anti-HSP27 antibodies.
Increased band intensity of tropomyosin was observed in
elution from the glutathione agarose-bound GST-3D-HSP27
fraction (Fig. 7B). All the unbound fractions, washes, and
eluted fractions were analyzed on dot blot. Glutathione-agarose
beads conjugated to GST alone were used as controls and
showed no binding of tropomyosin to GST (Fig. 7A). The
binding affinity of tropomyosin with GST-3D HSP27 fusion
protein, which represents a phosphomimic HSP27 protein, was
333 ⫾ 76% of control (P ⱕ 0.001, n ⫽ 6) compared with
nonphosphomimic HSP27 mutant fusion protein, which was
168 ⫾ 17% of control (P ⱕ 0.001, n ⫽ 6), and with wild-type
HSP27 fusion protein, which was 200 ⫾ 44% of control (P ⱕ
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Acetylcholine-Induced Coimmunoprecipitation of
Tropomyosin with F-actin in Whole Cell Lysates of Smooth
Muscle Cells Transfected with HSP27 Mutants
SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
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Fig. 7. Direct association of tropomyosin and HSP27: GST-HSP27 mutant fusion proteins (50 ␮g) conjugated to glutathioneagarose beads were incubated with 25 ␮g of tropomyosin obtained commercially, and direct association was studied as described
in Methods. A: representative dot blot showing the association of tropomyosin with GST-tagged HSP27 mutant fusion proteins.
GST alone conjugated to glutathione-agarose beads was used as control. All the unbound fractions, washes, and eluates were
spotted, and dot blot was carried out as described in Methods. Fractions 1–3 are washes of unbound GST-HSP27 mutant proteins;
fractions 4 –7 are washings of unbound tropomyosin; fractions 8 –10 are elutions of GST-HSP27 mutant proteins bound to the
glutathione-agarose with 10 mM reduced glutathione. Coelution of tropomyosin with GST-HSP27 mutant proteins was observed
in fractions 8 –10, an indication of direct association of recombinant tropomyosin with GST-HSP27(s). B: representative blots of
eluates of GST, GST-wtHSP27, GST-3D-HSP27, and GST-3G-HSP27 were run on SDS-PAGE and probed with anti-HSP27 or
anti-tropomyosin antibodies. Increased affinity of tropomyosin to GST-3D-HSP27 is shown compared with GST-3G-HSP27 and
GST-wt-HSP27. C: percent binding of tropomyosin to GST-tagged HSP27 mutant fusion proteins. Percent binding of tropomyosin
to GST-3D-HSP27 was 333 ⫾ 76% of control (P ⱕ 0.001, n ⫽ 6), compared with GST-wtHSP27 (200 ⫾ 44% of control; P ⱕ
0.001, n ⫽ 6) and GST-3G-HSP27 (168 ⫾ 17% of control; P ⱕ 0.001, n ⫽ 6). GST alone was used as control.
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Fig. 6. Expression and purification of glutathione S-transferase
(GST)-HSP27 mutant fusion protein. The confirmed clone of E.
coli BL21 containing recombinant HSP27 mutant 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 immunoblotting
with anti-HSP27 and anti-GST antibodies. Lane 1, purified
GST-wild-type (wt)HSP27; lane 2, purified GST-3D-HSP27;
lane 3, purified GST-3G-HSP27. A: immunoblotted with antiHSP27 (1:1,000). B: immunoblotted with anti-GST antibody
(1:4,000). A and B, right: molecular mass standards.
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SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
0.001, n ⫽ 6) above the control GST (Fig. 7, A and C). The
data suggest that tropomyosin is associated with HSP27 directly and this direct binding of tropomyosin with HSP27
varies depending on the phosphorylation state of HSP27.
Cosedimentation of Tropomyosin with F-actin in the
Presence of Phosphomutant HSP27
DISCUSSION
The present study describes a model for the displacement of
tropomyosin off the thin filament and the role of HSP27 in this
displacement in smooth muscle cells. Our lab previously re-
Fig. 8. Cosedimentation of tropomyosin with actin from the whole cell lysates
of smooth muscle cells transfected with HSP27 mutants. Whole cell lysates
(500 ␮g) of smooth muscle cells transfected with HSP27 mutants were
incubated in F-actin buffer and subjected to high-speed centrifugation to
sediment F-actin. The supernatants that contained no F-actin (data not shown)
were collected and incubated in equal amounts of F-actin stock, followed by
sedimentation of F-actin and F-actin-bound proteins. The pellet obtained from
centrifugation was subjected to SDS-PAGE followed by immunoblotting with
anti-tropomyosin and F-actin-specific antibody. A: representative immunoblot
showing the cosedimentation of tropomyosin with actin. B: % cosedimentation
of tropomyosin with actin. In the relaxed noncontracted cells, the association
of actin with tropomyosin was reduced in phosphomimic mutant-transfected
cells (86 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 2) compared with nonphosphomimic
mutant-transfected cells (119 ⫾ 4% of control; P ⱕ 0.01, n ⫽ 2).
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Interaction of tropomyosin with F-actin in the presence or
absence of phosphorylated HSP27 was also studied by cosedimentation of tropomyosin with actin in smooth muscle cells
transfected with different HSP27 mutants. Whole cell lysates
from acetylcholine-stimulated transfected and nontransfected
cells were incubated in F-actin polymerization buffer and
subjected to high-speed centrifugation to remove all the Factin. The supernatant collected was incubated with equal
amounts of F-actin stock and centrifuged to sediment F-actin
and F-actin binding proteins. The pellet was then subjected to
SDS-PAGE and immunoblotted with tropomyosin- and Factin-specific antibody. The data indicate that transfection of
smooth muscle cells with the phosphomimic 3D-HSP27 mutant resulted in a decrease in the cosedimentation of tropomyosin with actin (86 ⫾ 1% of control; P ⱕ 0.01, n ⫽ 2)
compared with nonphosphomimic 3G-HSP27 mutant-transfected cells (119 ⫾ 4% of control; P ⱕ 0.01, n ⫽ 2) (Fig. 8).
The data suggest that in the presence of phosphomimic HSP27
less actin is associated with tropomyosin.
ported (5) that actin-myosin interaction in smooth muscle
contraction is modulated by HSP27 phosphorylation. We also
reported (25) that during agonist-induced contraction, tropomyosin colocalizes with HSP27. A model was proposed
whereby, upon phosphorylation of HSP27, phosphorylated
HSP27 binds to tropomyosin, pulling it away from the myosin
head binding site into the F-actin groove, thus facilitating the
interaction of actin with myosin (5). We have investigated the
direct association of tropomyosin with HSP27 and the possible
role of HSP27 phosphorylation in this association. We have
investigated the possibility of modulation of actin-myosin
interaction by the association of tropomyosin with phosphorylated HSP27, leading to contraction. Our present data suggest
that tropomyosin does bind directly to HSP27 and that the
phosphorylation of HSP27 brings about a conformational
change allowing for greater binding affinity of tropomyosin
with phosphorylated HSP27. We propose that phospho-HSP27
is involved in the displacement of tropomyosin off the actin
filaments, exposing myosin-binding sites for actin-myosin interaction.
Muscle contraction depends on the sliding of thick filaments
past thin filaments driven by myosin cross bridges. The actomyosin ATPase, which energizes muscle contraction, is
switched “on” and “off” by changes in intracellular free Ca2⫹
concentration (11). However, the mechanism that leads to the
activation of actomyosin ATPase and the sliding of the filaments by intracellular free Ca2⫹ concentration varies in different muscle types (15). In skeletal and cardiac muscle, “on-off”
switching is mediated by the binding of Ca2⫹ to the troponin C
subunit of troponin-tropomyosin complex on thin filaments. In
smooth muscle, troponin is absent and Ca2⫹ regulates contraction by binding to calmodulin, which links the MLCK
phosphorylation cascade to activation of actomyosin ATPase
(46, 50).
Actin-myosin interaction during smooth muscle contraction
is mediated by tropomyosin (10, 35). Tropomyosin is localized
on thin filament, sterically blocking myosin-binding sites on
actin in all types of muscle cells (12, 28). The role of tropomyosin in contraction is well studied and appears to be similar
in all types of muscle cells. Upon elevation of intracellular free
Ca2⫹ concentration in skeletal and cardiac muscle, troponin
aids in the displacement of tropomyosin off the actin filament
and exposes myosin-binding sites on actin (14, 43, 52),
whereas in smooth muscle cells, which lack troponin, the
mechanism that initiates displacement of tropomyosin off the
actin is not clearly understood. Tropomyosin is one of the most
conserved of all the actin-binding proteins identified in organisms ranging from yeast to humans, and it is expressed in
nearly all eukaryotic cells (24). Multiple genes as well as
alternatively spliced RNA transcripts have been implicated in
expressing several different isoforms in a tissue-specific manner (34). Differential expression has been found during cell
transformation and differentiation, suggesting that different
isoforms are required for specific roles in regulating both the
actin filament structure and the interaction of other proteins
with the actin cytoskeleton (13). Tropomyosin is a two-chained
parallel coiled-coil 284-amino acid-containing protein and is
aligned head to tail along the helical actin filament (20).
Head-to-tail alignment of tropomyosin is achieved by nine
amino acids at the NH2 terminal interacting with nine amino
acids at the COOH terminal to form an overlap complex (40).
SMOOTH MUSCLE CONTRACTION: PHOSPHO-HSP27 AND TROPOMYOSIN
AJP-Cell Physiol • VOL
binding affinity with nonphosphorylatable HSP27 is observed
compared with control wild-type HSP27. However, the data
suggest that, in vitro, tropomyosin has greater direct affinity to
phosphorylated HSP27 in the absence of F-actin.
The data suggest that, in smooth muscle cells, HSP27 is
rapidly phosphorylated upon agonist stimulation that may
result in structural change of HSP27. This possible change may
be responsible for the stronger association of tropomyosin to
phosphorylated HSP27, thereby reducing the association of
tropomyosin with actin. Marston and Huber (36) have suggested polymerization-depolymerization of actin as a part of
the contraction-relaxation cycle of smooth muscle. Actin polymerization-depolymerization is regulated by a number of
mechanisms, in which HSP27 is likely to contribute to the
equilibrium between polymerization and depolymerization (2,
17, 32). The reduced association of tropomyosin with actin
may be due to an increased association of tropomyosin with
phosphorylated HSP27 or may be due to the equilibrium
between polymerized and depolymerized actin, i.e., the ratio of
F-actin to G-actin. The increased association of tropomyosin
with phosphorylated HSP27 may result in displacement of the
tropomyosin, thus exposing the myosin-binding sites on actin.
How this phosphorylation is achieved is being investigated.
The serine residues in HSP27 might be phosphorylated in a
certain order, i.e., sequentially: Ser82, being the major phosphorylated site, could be phosphorylated first, leading to phosphorylation of the other two minor sites, Ser78 and Ser15 (25,
48). It is also possible that phosphorylation sites on HSP27 are
phosphorylated simultaneously or that each site is phosphorylated specifically depending on the signal. The data also suggest that upon phosphorylation at Ser82 and Ser78, HSP27
undergoes a conformational change leading to a stronger association of HSP27 with tropomyosin. Conformational change
may be just the structural change, i.e., dissociation of oligomers into monomers and dimers that may expose certain
amino acids that are responsible for greater binding of tropomyosin to phospho-HSP27. Colocalization of tropomyosin
with HSP27 upon agonist-induced contraction is primarily due
to the direct association of tropomyosin with phospho-HSP27
as suggested by in vitro binding experiments. We thus propose
that upon phosphorylation HSP27 undergoes structural
changes resulting in greater binding to tropomyosin. This
strong binding may help in the displacement of tropomyosin
off the F-actin filament, exposing the myosin-binding site for
actin-myosin interaction and leading to contraction. Thus we
propose that agonist-induced phosphorylation of HSP27 is of
physiological relevance and that it is involved in modulation of
actin-myosin interaction in smooth muscle contraction.
ACKNOWLEDGMENTS
We thank Dana Thomas and H. Pang for technical assistance, editing, and
figure preparation.
GRANTS
This study was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant 5-R01-DK-057020.
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