Phosphorylation of the 27-kDa heat shock protein via p38 MAP
kinase and MAPKAP kinase in smooth muscle
Janice K.
Larsen1,
Ilia A.
Yamboliev1,
Lee A.
Weber2, and
William T.
Gerthoffer1
1 Department of Pharmacology,
University of Nevada School of Medicine and
2 Department of Biology,
University of Nevada, Reno, Nevada 89557-0046
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ABSTRACT |
The 27-kDa heat shock protein (HSP27) is
expressed in a variety of tissues in the absence of stress and is
thought to regulate actin filament dynamics, possibly by a
phosphorylation/dephosphorylation mechanism. HSP27 has also been
suggested to be involved in contraction of intestinal smooth muscle. We
have investigated phosphorylation of HSP27 in airway smooth muscle in
response to the muscarinic agonist carbachol. Carbachol increased
32P incorporation into canine
tracheal HSP27 and induced a shift in the distribution of charge
isoforms on two-dimensional gels to more acidic, phosphorylated forms.
The canine HSP27 amino acid sequence includes three serine residues
corresponding to sites in human HSP27 known to be phosphorylated by
mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2.
To determine whether muscarinic receptors are coupled to a "stress
response" pathway in smooth muscle culminating in phosphorylation of
HSP27, we assayed MAPKAP kinase-2 activity and tyrosine phosphorylation of p38 mitogen-activated protein (MAP) kinase, the enzyme thought to
activate MAPKAP kinase-2. Recombinant canine HSP27 expressed in
Escherichia coli was a substrate for
MAPKAP kinase-2 in vitro as well as a substrate for endogenous smooth
muscle HSP27 kinase, which was activated by carbachol. Carbachol also
increased tyrosine phosphorylation of p38 MAP kinase. SB-203580, an
inhibitor of p38 MAP kinases, reduced activation of endogenous HSP27
kinase activity and blocked the shift in HSP27 charge isoforms to
acidic forms. We suggest that HSP27 in airway smooth muscle, in
addition to being a stress response protein, is phosphorylated by a
receptor-initiated signaling cascade involving muscarinic receptors,
tyrosine phosphorylation of p38 MAP kinase, and activation of MAPKAP
kinase-2.
carbachol; mitogen-activated protein kinase; p38 mitogen-activated
protein kinase-activated protein; trachea
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INTRODUCTION |
MITOGEN-ACTIVATED PROTEIN (MAP) kinases play a central
role in intracellular signal transduction pathways initiated by a
variety of cellular stimuli. Although activation of MAP kinases often leads to a mitogenic response, there is evidence to support the involvement of MAP kinases in cellular functions in addition to proliferation. For example, in neutrophils, interleukin-8 activation of
the p38 MAP kinase pathway may lead to respiratory burst and granule
secretion (17). In intact, differentiated smooth muscles, extracellular
signal-regulated kinase (ERK)-1 and ERK2 MAP kinases are activated by a
variety of stimuli, including neurotransmitters and phorbol esters (1,
10, 16). The actin-binding protein caldesmon is thought to be an
important downstream target for the ERK MAP kinases in intact smooth
muscle (2). Another downstream target of MAP kinases is
mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2
(29). MAPKAP kinase-2 is a Ser/Thr kinase shown to catalyze
phosphorylation of 27-kDa heat shock protein (HSP) in nonmuscle cells
(3, 19, 29, 32). Although many investigators have demonstrated
activation of MAPKAP kinase-2 via MAP kinase pathways, the cellular
role of MAPKAP kinase-2 and phosphorylation of HSP27 in smooth muscle
has not been described.
The mammalian HSP27 is a member of the highly conserved family of HSPs
(14) and is expressed in a variety of tissues in the presence and
absence of stress. Although HSP27 has been shown to exhibit chaperone
activities in vitro (15) and to modulate actin microfilament dynamics
(4, 22) and although overexpression of the protein confers increased
resistance to heat killing (21), the physiological function of HSP27 in
unstressed cells remains unclear. HSP27 becomes phosphorylated in
response to heat shock and to a variety of cytokines and growth factors
in cultured endothelial cells (28), fibroblasts (32), and monocytic
cells (3). Landry et al. (19) mapped the phosphorylation sites in human HSP27 and showed that MAPKAP kinase-2 phosphorylates human HSP27 protein on Ser-15, Ser-78, and Ser-82. Ser-82 appears to be the major
site of in vivo phosphorylation followed by Ser-78 and Ser-15, the
minor sites (19, 29).
Recent studies suggest that p38 MAP kinases are immediately upstream of
MAPKAP kinase-2 in the stress response pathway leading to
phosphorylation of HSP27. The p38 MAP kinases are homologs of yeast
HOG1 MAP kinase, which is important for growth of yeast in high
osmolarity media as well as for the formation of buds (13). It has been
shown that both ERK and p38 MAP kinases can activate MAPKAP kinase-2 in
vitro; however, only activation of p38 correlates with MAPKAP
kinase-2 activation in vivo (8, 12, 27, 33). Less is known about the
molecular details of the p38 signaling pathway compared with the ERK
MAP kinases. Many investigators have demonstrated that environmental
stresses and proinflammatory cytokines can activate this pathway,
possibly via activation of upstream kinases p21-activated kinase 1 (PAK1) and MAP kinase/ERK kinase (MKK3; see Refs. 8, 9,
27, and 33). However, relatively little is known about the activation of p38 MAP kinases through G protein-linked,
seven-transmembrane-spanning (STM) receptors. To address this issue, we
examined the ability of carbachol, a muscarinic agonist, to increase
phosphorylation of HSP27, activate MAPKAP kinase-2, and increase
tyrosine phosphorylation of p38 MAP kinase. The results suggest that
muscarinic receptors are coupled to the stress response pathway leading
to phosphorylation of HSP27 in intact airway smooth muscle.
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MATERIALS AND METHODS |
Materials. Adult mongrel dogs of
either sex were killed by barbiturate overdose. The trachea was removed
and was placed in cold physiological salt solution (PSS) composed of
(in mM) 2 3-(N-morpholino)propanesulfonic acid (MOPS), pH
7.4, 140 NaCl, 4.7 KCl, 1.2 Mg2SO4,
2.5 CaCl2, 1.2 Na2HPO4,
0.02 EDTA, and 5.6 D-glucose. Tracheal smooth muscle was
dissected free of connective tissue and epithelium. Expression vectors
pET3a and pET24a as well as Escherichia
coli BL21(DE3)pLysS were purchased from Novagen.
32P was purchased from ICN
Biomedicals. MAPKAP kinase-2 and anti-ERK1-CT antibody was
purchased from Upstate Biotechnology (Lake Placid, NY). Goat
anti-chicken immunoglobulin (Ig) G was purchased from Southern
Biotechnology Associates (Birmingham, AL). Anti-p38 antibody (C-20) was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Phospho-specific p38 MAP kinase antibody was purchased from New England
Biolabs (Beverly, MA). Anti-rabbit IgG alkaline phosphatase conjugate
antibody was purchased from Promega (Madison, WI).
Recombinant HSP27. A full-length cDNA
encoding the canine HSP27 protein was isolated from a canine colonic
smooth muscle library in lambda Zap II by hybridization with a
full-length human HSP27 cDNA probe provided by Drs. Eileen Hickey and
Lee Weber (14, 20). Full-length human and canine recombinant proteins
were created by fusing human and canine cDNA with pET24a, whereas
truncated canine recombinant protein containing the majority of the
canine HSP27s sequence was created by fusing a cDNA fragment encoding amino acids 33-209 with the T7 promoter in pET3a. When expressed in BL21(DE3)pLysS cells, all recombinant HSP27 (rHSP27s) comprised >40% of the total cell protein 1.5 h after induction. Total soluble protein from each preparation was applied to a 1 × 13-cm DEAE Sephacel Column equilibrated with 20 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 10 mM NaCl, 0.1 M EDTA, 1.0 mM dithiothreitol (DTT),
and the rHSP27 eluted at 10 ml/h with a linear NaCl gradient (0.01-0.4 M). Fractions highly enriched in rHSP27 were collected and pooled, and purity was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Purity was >85%.
Preparation of anti-HSP27 antibodies and immunoblot
analysis. DEAE column fractions enriched in rHSP27 were
further purified by SDS-PAGE (12% acrylamide), and the gel-purified
rHSP27 (~100 µg) was then injected into the pectoral muscles and
thighs of laying hens. Booster injections were administered every 2 wk, and eggs were collected. Antibodies were isolated from egg yolks by
polyethylene glycol precipitation. Polyclonal HSP27 antibodies were
affinity purified with a canine rHSP27-Sepharose CL4B column, and
specificity was tested on Western blots of homogenates of canine colon
and tracheal smooth muscle. The polyclonal antibody was found to react
against truncated canine rHSP27 and a protein of 27 kDa in both canine
colon and tracheal smooth muscle extracts. The antibody reacted only
very weakly to human rHSP27.
Immunoblots were performed by transferring proteins from
SDS-polyacrylamide gels to pure nitrocellulose paper using either a
Hoefer TE Transfor electrophoresis unit (90 V, 4-14 h, 15°C) or a Bio-Rad (Hercules, CA) Mini Trans-Blot unit (120 V, 1-2 h, 4°C). Nitrocellulose was blocked with 5% powdered milk in TBS (10 mM Tris · HCl, pH 7.4, and 150 mM NaCl) and was
probed with chicken anti-canine HSP27 antibodies (1:150 dilution), and
bands were visualized with goat anti-chicken IgG alkaline phosphatase conjugate (1:1,000 dilution).
For detection of p38 tyrosine phosphorylation, tracheal smooth muscle
strips were treated with 1 µM carbachol and were frozen by immersion
in liquid nitrogen, and proteins were extracted in MAP kinase
extraction buffer containing 20 mM Tris, pH 7.5, 5 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM
Na3VO4,
20 mM
-glycerophosphate, 10 mM NaF, 1 mM DTT, 1 µg/µl aprotinin,
and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and were clarified by
centrifugation at 10,000 g for 10 min
at 4°C. The protein extract was separated on a 12%
SDS-polyacrylamide gel and was transferred to nitrocellulose paper.
Immunodetection was performed using anti-phosphotyrosine p38 antibody
(1:3,000) followed by goat anti-rabbit alkaline phosphatase secondary
antibody (1:5,000).
For identification of contractile proteins on two-dimensional gels,
Western blots were probed with anti-calponin polyclonal antibodies (a
gift of Dr. Michael Walsh, University of Calgary), anti-actin (A-2547)
monoclonal antibody from Sigma (St. Louis, MO), anti-tropomyosin
(T-2780) monoclonal antibody from Sigma, and anti-22-kDa smooth muscle
protein (SM22) polyclonal antibodies (a gift of Dr. L. B. Smillie,
University of Alberta). Affinity-purified anti-myosin light chain (MLC)
antibodies were prepared by immunizing rabbits with chicken gizzard
20-kDa MLC. Proteins on Western blots were detected using goat
anti-chicken IgG alkaline phosphatase (Southern Biotechnology
Associates) or goat anti-rabbit or goat anti-mouse alkaline
phosphatase-conjugated secondary antibodies (Promega).
HSP27 phosphorylation. Canine tracheal
smooth muscle strips were mounted on stainless steel hooks and were
incubated for 3 h at 37°C in oxygenated phosphate-free PSS
containing 0.2 mCi/ml 32P. After
32P labeling, tissue strips were
allowed to equilibrate in PSS for 30 min. The strips were stimulated
two times with 60 mM K+ for 5 min,
each followed by a 15-min wash. Tissues were then stimulated with 1 µM carbachol for specified times as described below and were frozen
in acetone-5% trichloroacetic acid over dry ice. After warming to room
temperature in pure acetone, the muscle strips were vacuum dried and
weighed. The tissues were then homogenized in 50 µl of lysis buffer
per milligram dry weight. Lysis buffer was composed of 9.5 M urea,
0.5% SDS, 5.0%
-mercaptoethanol, and 0.16%
Ampholine, pH 3-10. Proteins were resolved by
two-dimensional nonequilibrium pH gel electrophoresis (NEpHGE)-SDS-PAGE
(24). The tissue extract was first subjected to NEpHGE in 0.1 × 13-cm tube gels for 4,800 V · h (pH 3-10)
followed by separation in the second dimension by SDS-PAGE (12%
acrylamide). The slab gels were then stained overnight with 0.4%
Coomassie brilliant blue R-250 in 25% isopropanol and 10% acetic
acid. Gels were completely destained in 25% isopropanol and 10%
acetic acid, and the protein bands were quantified by densitometry. The
mass of HSP27 was calculated from densitometric scans using 1-8
µg of purified rHSP27 as dye binding standards. HSP27 bands were then
cut from the slab gels, and the amount of radioactivity was measured by
scintillation counting. Protein content and radioactivity were
calculated for each HSP27 isoform (a, b, and c). Phosphorylation was
expressed as counts per minute of each isoform per total micrograms of
HSP27.
In vitro phosphorylation of HSP27.
Canine rHSP27 (3.7 µM) was added to rabbit skeletal muscle MAPKAP
kinase-2 (1 U/80 µl) in a kinase reaction buffer (in mM: 10 MOPS, pH
7.5, 10
-glycerophosphate, 6 MgCl2, 0.4 EGTA, 0.04 NaF, 0.4 Na3VO4,
and 1.6 DTT). A mixture of MgATP (0.25 mM) plus
[
-32P]ATP (2 µCi)
was then added to the kinase reaction to total 120 µl. After
incubation at 30°C for 0, 5, 10, 30, 60, 90, and 120 min, 15-µl
samples were removed. Concentrated SDS-PAGE sample buffer was added to
each sample to yield 60 mM Tris · HCl, pH 6.8, 2%
SDS, 10% glycerol, 0.025% bromphenol blue, and 1 mM DTT. HSP27 was
separated from MAPKAP kinase-2 by one-dimensional SDS-PAGE (12%
acrylamide), and phosphorylation of canine rHSP27 was visualized using
a Bio-Rad model 525 Molecular Imager.
Activation of in vivo HSP27 kinase.
Muscle strips (30-40 mg) were stimulated with 1 µM carbachol for
0.5, 1, 2, 5, 15, 30, and 60 min and then were frozen by immersion in
liquid nitrogen. Frozen muscle strips were pulverized and homogenized
in MAP kinase extraction buffer (see above). The extracts were
clarified by centrifugation at 10,000 g for 10 min at 4°C. The kinase
reaction mixture contained (in 40 µl) 25 mM Tris, pH 7.0, 0.1 mM
EGTA, 0.2 mM
Na3VO4,
10 mM magnesium acetate, 1 mM DTT, and 3.3 µM human rHSP27 plus
clarified tissue extract. The reaction was started by the addition of
Na2ATP to give a 0.25 mM final
concentration containing 2 µCi
[
-32P]ATP. After 15 min, the reaction was terminated by addition of SDS sample buffer (see
above). Proteins were separated by SDS-PAGE and were stained with
Coomassie brilliant blue. Phosphorylation of rHSP27 was detected with a
Bio-Rad model 525 Molecular Imager. Background signal due to the
phosphorylation of endogenous HSP27 comprised <9% of the total
kinase activity and was corrected by subtracting signal from reactions
lacking rHSP27 from reactions containing rHSP27.
Mono-Q chromatography. Tracheal tissue
strips (50 mg) were frozen by immersion in acetone chilled with crushed
dry ice (
80°C). The proteins were extracted in MAP kinase
extraction buffer. The extract was clarified by centrifugation at
100,000 g for 10 min at 4°C and
then was applied to a Mono-Q HR 5/5 column (Pharmacia Biotech,
Piscataway, NJ) equilibrated with 20 mM Tris, pH 7.5, 2 mM EGTA, 1 mM
Na3VO4,
10 mM
-glycerophosphate, 1 mM DTT, 1 µg/µl aprotinin, and 0.1 mM
PMSF. The column was washed with 10 column volumes of equilibration
buffer and was developed with a 60-ml linear NaCl gradient (0-0.4
M) at a flow rate of 0.5 ml/min. One-milliliter fractions were
collected and concentrated, and every other fraction was analyzed by
12% SDS-PAGE. The proteins were transferred to
nitrocellulose paper and were probed separately with either
anti-ERK1-CT antibody (1:2,000 dilution) or anti-p38 antibody (1:1,000
dilution) as described above.
Treatment of tracheal smooth muscle with
SB-203580. Canine tracheal smooth muscle strips (~2 × 10 mm) were mounted on stainless steel hooks and were incubated
in oxygenated PSS at 37°C. Muscle strips were stimulated three
times for 5 min with 70 mM K+ to
produce stable, reproducible contractions. The muscles were then
stimulated with 1 µM carbachol for 10 min to serve as a
control response before the addition of SB-203580. Muscle strips were then incubated with either 0.1% dimethyl sulfoxide (DMSO; vehicle) or
25 µM SB-203580 dissolved in 0.1% DMSO for 1 h. They were then stimulated with 1 µM carbachol, frozen in liquid nitrogen, and homogenized in MAP kinase extraction buffer. The extracts were clarified by centrifugation at 10,000 g for 10 min. The soluble proteins
were added to equal volumes of lysis buffer composed of 9.5 M urea,
2.5% Triton X-100, 5%
-mercaptoethanol, and 0.16% Ampholine, pH
3-10 (Pharmacia LKB Biotechnology). Charge isoforms of HSP27 were
then resolved by NEpHGE-SDS-PAGE as described above, and proteins were
stained with Coomassie brilliant blue as described above. Images of
gels scanned with a UMAX Powerlook flatbed scanner were analyzed using
the Volume Analyze feature of Molecular Analyst software (Bio-Rad) to
determine the mass of HSP27 in each charge isoform. Dye binding
standard curves were constructed by densitometry of one-dimensional
SDS-polyacrylamide gels containing 1-8 µg rHSP27.
Statistical methods. Results are
presented as means ± SE. Hypothesis testing was performed using
SigmaStat, version 1.0 (Jandel Scientific, San Rafael, CA). Differences
between treatment means were evaluated by Student's
t-test for unpaired data as
appropriate. Multiple comparisons among mean protein densities of HSP27
charge isoforms were made using the Bonferroni
t-test. A probability of
P < 0.05 was accepted as a
significant difference.
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RESULTS |
Identification of canine smooth muscle HSP27 by
Western blotting. To study HSP27 in canine airway
smooth muscle, we developed a canine specific antibody. Despite high
homology among eucaryotic HSPs, antibodies raised against human and
rodent HSP27 do not cross-react with canine HSP27. Therefore, we
isolated, cloned, and sequenced cDNAs encoding canine HSP27 from a
canine colonic smooth muscle cDNA library using a human HSP27 cDNA
probe (20). The deduced amino acid sequence showed a high degree of
identity to other mammalian species (Fig.
1). The canine HSP27 sequence also has
three potential MAPKAP kinase-2 phosphorylation sites at
Ser-15, Ser-82, and Ser-86 that correspond to known phosphorylation sites in human HSP27 (boxed sequences in Fig. 1). The canine HSP27 cDNA
was then subcloned into an expression vector and rHSP27 expressed in
E. coli. rHSP27 was purified by DEAE
Sephacel chromatography and SDS-PAGE. We then immunized chickens with a
truncated form of canine rHSP27 (amino acids 33-209) and prepared
affinity-purified polyclonal anti-HSP antibodies that were used to
identify HSP27 in homogenates of canine tracheal smooth muscle.

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Fig. 1.
Heat shock protein (HSP) 27 sequence comparison. Alignment of the
209-amino acid canine HSP27 sequence shows high degree of identity with
rat (86.41%), mouse (87.56%), hamster (89.47%), and human (86.83%)
HSP27 proteins. Sequences within the boxes represent mitogen-activated
protein kinase-activated protein (MAPKAP) kinase-2 recognition motif
HXRXXS (H = hydrophobic amino acid, X = any amino acid). Bold
letter S, phosphorylated serines. GenBank accession numbers are M86389,
L11609, X51747, and X54079, respectively. Alignment determined by the
CLUSTAL program. * Amino acid identity; periods (.),
similarity.
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SDS extracts of canine tracheal smooth muscle were subjected to
NEpHGE-SDS-PAGE as shown in Fig. 2.
Proteins identified by Western blotting of similar gels include actins,
tropomyosins, calponin, SM22, and the 20-kDa MLC. The identity of HSP27
was determined by Western blotting after NEpHGE-SDS-PAGE (Fig.
3). The charge isoforms shown in Fig. 2 and
Fig. 3A were identified with
anti-HSP27 antibodies (Fig. 3A).
Canine HSP27 resolved into three spots referred to as charge isoforms
"a," "b," and "c."

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Fig. 2.
Separation of HSP27 charge isoforms by 2-dimensional nonequilibrium pH
gel electrophoresis (NEpHGE)-SDS-polyacrylamide gel electrophoresis
(PAGE) of canine tracheal protein extracts. Tissue protein extracts
were subjected to NEpHGE in the first dimension and then were separated
by molecular weight (MW) by SDS-PAGE in the second dimension. + and
, polarity of pH gradient in the first dimension of the separation.
Known proteins clearly separated by this process are indicated in the
image of a Coomassie brilliant blue-stained second-dimension gel and
include actin, tropomyosin (TM), myosin light chain (MLC), 22-kDa
smooth muscle protein (SM22), calponin (CP), and HSP27. HSP27 resolved
into a, b, and c spots as identified by Western blotting (Fig. 3).
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Fig. 3.
A: section of a NEpHGE-SDS-PAGE
Coomassie brilliant blue-stained gel as shown in Fig. 2 demonstrating
HSP27 spots a, b, and c. B:
corresponding Western blot of gel in A
probed with affinity-purified chicken polyclonal anti-HSP27. Anti-HSP27
antibodies recognize all 3 HSP27 charge isoforms.
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Phosphorylation of canine HSP27 in
tissue. To investigate the function of HSP27 in
mammalian smooth muscle, we tested the notion that it is phosphorylated
in intact smooth muscle in response to stimulation with a muscarinic
agonist. Carbachol was used to stimulate strips of canine tracheal
smooth muscle that had been metabolically labeled with
32P. Muscle strips were frozen 0, 1, 2, and 60 min after treatment with carbachol, and proteins were
resolved by NEpHGE-SDS-PAGE. Protein bands were visualized in slab gels
stained with Coomassie brilliant blue. In a single representative
experiment, phosphoproteins were visualized with a Phosphorimager as
shown in Fig. 4. The phosphorimages show
that the a spot does not contain any radioactivity. Both b and c spots
contain measurable radioactivity in the unstimulated muscle and
incorporate increased 32P after
stimulation (Fig. 4, bottom).

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Fig. 4.
Top: HSP27 isolated from canine
tracheal smooth muscle tissue strips under control conditions (0 min)
and after stimulation with carbachol for 2 and 60 min. Extracted
proteins were resolved by NEpHGE-SDS-PAGE and were stained with
Coomassie brilliant blue. Bottom:
corresponding gels from top imaged
with a Bio-Rad Molecular Imager to detect changes in radioisotope
labeling. HSP27 b and c spots incorporated additional
32P within 2 min after stimulation
with carbachol. In addition, phosphorylation of the b and c spots was
maintained for 60 min. MLC, used as an internal control, was also
phosphorylated at 2 min but decreased somewhat at later time points.
HSP27 a spot did not contain any radioactivity.
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We measured the effect of carbachol stimulation on the level of
radioactivity in each isoform and on the distribution of protein in
each isoform. Gels were scanned, and protein content was determined by
densitometry using purified rHSP27 as a dye binding standard. Radioactivity incorporated in each spot was determined by scintillation counting. Figure
5A shows
that stimulation with 1 µM carbachol caused the tissue to contract
tonically. Figure 5, B and
C, shows the relative increase in
radioactivity in isoforms b and c after 1, 2, and 60 min normalized to
basal levels (0 min). We observed a 1.6-fold increase in
phosphorylation of the b isoform and a 1.7- to 1.8-fold increase in
phosphorylation of the c isoform. Both increases were statistically
significant at all time points compared with 0 min. The distribution of
protein among the charge isoforms also changes significantly upon
stimulation. The unphosphorylated a form decreased significantly from
41 ± 1% of total HSP27 to 27 ± 5% after 60 min
(P < 0.05, Bonferroni
t-test). This was accompanied by a
corresponding increase in the protein content of the more acidic,
phosphorylated b and c isoforms. The b isoform increased from 36 ± 4% of total HSP27 to 39 ± 6%. The c isoform increased from 23 ± 5 to 34 ± 3% of total HSP27 after 60 min. This suggests that
the charge isoforms are produced by changes in the incorporation of
phosphorus where the a spot is unphosphorylated and both b and c spots
are phosphorylated.

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Fig. 5.
Changes in phosphorylation of HSP27 charge isoforms in trachea muscle
strips stimulated with 1 µM carbachol.
A: force developed by an intact strip
of tracheal smooth muscle stimulated with 1 µM carbachol (Carb).
B: relative phosphorylation of the
HSP27 b isoform was determined by scintillation counting of the b spots
cut from 2-dimensional gels prepared as in Fig. 3. Radioactivity
[counts/min (cpm)] was corrected for protein content to
give cpm/total µg HSP27 protein, which was then normalized to
phosphorylation at 0 min. Relative phosphorylation of the b
spot at 1, 2, and 60 min was significantly greater than at 0 min.
C: phosphorylated c isoform of HSP27
showed a >1.7-fold increase over basal levels at 1, 2, and 60 min
after carbachol treatment. * Significant difference from 0 min,
P < 0.05, Student's
t-test;
n = 4 (1 min) and 5 (2 and 60 min).
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Phosphorylation of canine rHSP27 by MAPKAP
kinase-2. Stokoe et al. (29) showed that
small-molecular-weight HSPs are phosphorylated by MAPKAP kinase-2. To
determine if canine HSP27 is a substrate for MAPKAP kinase-2, we
incubated full-length canine rHSP27 with purified skeletal muscle
MAPKAP kinase-2 in vitro. Phosphorylation of canine rHSP27 by MAPKAP
kinase-2 was initiated by the addition of MgATP (2 µCi
[
-32P]ATP) and was
allowed to proceed for 120 min. Aliquots were removed at the times
shown in Fig. 6, and rHSP27 was isolated by
SDS-PAGE. Phosphorylation of rHSP27 occurred within 5 min and reached a maximum in this reaction by 120 min. This result clearly demonstrates that canine rHSP27 is a substrate for MAPKAP kinase-2.

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Fig. 6.
In vitro phosphorylation of canine recombinant HSP27 (rHSP27) by MAPKAP
kinase-2. Kinase reaction, in 120-µl total volume, was started with
[ -32P]ATP and was
stopped at the times indicated by removing 15-µl samples and mixing
with concentrated SDS-PAGE sample buffer (see MATERIALS AND
METHODS). Phosphorylated HSP27 was isolated by SDS-PAGE (12%
acrylamide). Phosphorimage of 1-dimensional polyacrylamide gel
illustrates that canine rHSP27 (27 kDa) is a substrate phosphorylated
by MAPKAP kinase-2.
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HSP27 kinase activity in canine
trachea. Because canine rHSP27 was a good substrate for
MAPKAP kinase-2 in vitro, we tested for endogenous HSP27 kinase
activity in smooth muscle tissue. Muscle strips were stimulated with
carbachol for times varying between 30 s and 1 h and then were frozen
in liquid nitrogen. The homogenates were assayed for in vivo HSP27
kinase activity using human rHSP27 as a substrate. As shown in Fig.
7, rHSP27 was rapidly phosphorylated by an
in vivo kinase. This HSP27 kinase activity reached maximum by 5 min and
was sustained for 60 min. The in vivo HSP27 kinase activity increased
1.1-fold over basal levels at 30 s, 1.4-fold at 1 min, and 2-fold at 5 min. The time course of activation of endogenous HSP27 kinase activity
(Fig. 7) correlated well with phosphorylation of HSP27 observed in
tissue strips after carbachol treatment (Fig. 5); that is, we observed a 1.6-fold increase in HSP27 phosphorylation at 1 and 2 min (Fig. 5)
and a 1.1- to 1.4-fold increase in the in vivo kinase at 30 s and 2 min
(Fig. 7) after carbachol treatment. Moreover, both HSP27
phosphorylation and HSP27 kinase activity remain elevated at 60 min
after stimulation.

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Fig. 7.
Activation of HSP27 kinase activity in vivo. Tissue strips were
stimulated with 1 µM carbachol and were assayed for in vivo kinase
activity using human rHSP27 as a substrate. Phosphorylated rHSP27 was
isolated by SDS-PAGE, and relative phosphorylation was measured with a
Bio-Rad Molecular Imager. Phosphorylation was corrected for total
protein and background signal due to phosphorylation of endogenous
HSP27. Results are normalized to kinase activity at 0 min.
* Significant difference from 0 min,
P < 0.05, Student's
t-test;
n = 6.
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From the deduced amino acid sequence, we know that HSP27 contains four
different kinase recognition motifs. These include consensus
phosphorylation sites for MAPKAP kinase-2 (HXRXXS, where H is
hydrophobic amino acid and X is any amino acid), protein kinase C
(S/TXR/K), protein kinase A (R/KXXS/T), and tyrosine kinase
(R/KXXD/EXXY). Therefore, a crude tissue homogenate could contain
multiple kinase activities that might phosphorylate HSP727 in our
assay. To further define the endogenous HSP27 kinase, we added
chelerythrine to the kinase extract to inhibit any protein kinase C
activity. We observed no changes in the in vivo kinase activity with
the addition of 10 mM chelerythrine (data not shown).
Expression of MAP kinases in canine
trachea. MAPKAP kinase-2 is known to be a
substrate for ERK1 and ERK2 MAP kinases in vitro (29) and p38 MAP
kinase in vivo (8). To investigate the role of these MAP kinase
signaling pathways in phosphorylation of HSP27, we tested for the
expression of these MAP kinases in tracheal tissue. Tracheal protein
extracts were separated by Mono-Q chromatography and were analyzed for
expression of ERK1, ERK2, and p38 MAP kinases with isoform-selective
antibodies. As shown in Fig. 8, all three MAP kinases are expressed in canine tracheal smooth muscle. These MAP
kinases eluted at different salt concentrations, with p38 MAP kinase
eluting later (0.32-0.35 M NaCl) than the ERK MAP kinases (0.24-0.29 M NaCl). The order of elution is consistent with
differences in isoelectric points (pI) calculated from deduced amino
acid sequences of human MAP kinases (ERK2, pI 6.72; ERK1, pI 6.29; p38,
pI 5.41).

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Fig. 8.
Separation of extracellular signal-regulated kinase (ERK) and p38
mitogen-activated protein (MAP) kinases. Soluble protein extracts from
canine trachea were applied to Mono-Q HR 5/5 column at a flow rate of
0.5 ml/min. Column was developed with a 60-ml linear gradient from 0 to
0.4 M NaCl (dotted line). Absorbance at 218 nm, shown as the solid
line, is plotted as arbitrary units. Fractions (1 ml) were collected,
concentrated, and subjected to SDS-PAGE and Western blotting. Specific
anti-ERK1 MAP kinase and anti-p38 MAP kinase antibodies were used to
probe separate Western blots of fractions containing MAP kinases as
shown in inset. ERK1 and ERK2 MAP
kinases eluted from the column at 0.24-0.29 M NaCl, corresponding
to fractions
36-44, whereas
p38 MAP kinase eluted later at 0.32 M NaCl
(fraction 48).
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Activation of p38 MAP kinase in tracheal smooth
muscle. ERK MAP kinases as well as p38 MAP kinases are
potential upstream activators of MAPKAP kinase-2, and, as shown in Fig.
8, canine tracheal smooth muscle express ERK1, ERK2, and p38 MAP
kinases. Cuenda et al. (8) suggest that p38 MAP kinase is upstream of MAPKAP kinase-2 in PC-12 and KB cells because the p38 MAP kinase inhibitor SB-203580 blocks MAPKAP kinase-2 activity and HSP27 phosphorylation, whereas ERK MAP kinase activity remains unaffected. We
examined the question of p38 MAP kinase activation by assaying tyrosine
phosphorylation of p38 MAP kinase in response to carbachol stimulation.
Tissue strips were stimulated with carbachol for 10 min and then were
frozen in liquid nitrogen. Protein extracts from unstimulated and
stimulated tissue were processed by SDS-PAGE, transferred to
nitrocellulose, and probed with anti-phospho-p38 MAP kinase antibodies
that recognize only tyrosine-phosphorylated p38 MAP kinase (Fig.
9). Figure 9 shows that tyrosine
phosphorylation of p38 is increased after 10 min of carbachol
treatment. These results show that tyrosine phosphorylation of p38 MAP
kinase is an early event after muscarinic receptor activation.

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Fig. 9.
Tyrosine phosphorylation of p38 MAP kinase in canine tracheal smooth
muscle. Two muscle strips from a single animal were isolated, and 1 muscle strip was stimulated with 1 µM carbachol for 10 min. Total
proteins were extracted in SDS-PAGE sample buffer (see MATERIALS
AND METHODS) and were resolved by SDS-PAGE. Tyrosine
phosphorylation of p38 MAP kinase was detected by Western blotting with
anti-phospho-p38 MAP kinase antibody. Tyrosine phosphorylation
increased by ~2-fold after 10 min of stimulation with carbachol.
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Phosphorylation of HSP27 through p38 MAP kinase
pathway. Because p38 MAP kinase is expressed in canine
tracheal smooth muscle and it becomes tyrosine phosphorylated upon
carbachol stimulation, we used SB-203580 to test the notion that p38
MAP kinase is upstream of MAPKAP kinase and phosphorylation of HSP27.
Tracheal tissue strips were stimulated with 1 µM carbachol
for 10 min in the absence or presence of 25 µM SB-203580, frozen in
liquid nitrogen, and homogenized. HSP27 kinase activity was assayed as
described above (Fig. 7). Stimulation with carbachol in the absence of
SB-203580 induced a 3.6-fold increase in kinase activity (Fig.
10). Preincubation of muscle strips for
60 min with 25 µM SB-20580 reduced carbachol-stimulated kinase
activity to a 1.9-fold increase above basal activity. SB-203580 alone
had no significant effect on basal kinase activity. The selectivity of
SB-203580 was confirmed by assaying activation of ERK MAP kinases using
the same homogenates that were assayed for HSP27 kinase activity. An
in-gel kinase assay was used as described previously (10, 11). Figure
11A
shows phosphorylation of myelin basic protein by ERK1 and ERK2 in
tracheal smooth muscle homogenates. Figure
11B shows that there was no effect of
pretreatment with SB-203580 on the mean activation of ERK1 or ERK2
produced by 1 µM carbachol. We also assayed tyrosine phosphorylation
of p38 MAP kinase in the absence and presence of SB-203580 to determine whether upstream steps in activation of p38 MAP kinase were sensitive to the antagonist. Figure 11C shows an
example of a Western blot probed with antibody selective for
tyrosine-phosphorylated p38 MAP kinase. Densitometry of blots from five
experiments (Fig. 11D) demonstrated
that phosphorylation of p38 MAP kinase was not affected by SB-203580.
The results suggest that treatment of intact tracheal smooth muscle
with SB-203580 inhibits activation of MAPKAP kinase with no effect on
activation of the ERK MAP kinases or tyrosine phosphorylation of p38
MAP kinase.

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Fig. 10.
Inhibition of in vivo activation of HSP27 kinase by SB-203580. Tracheal
smooth muscle strips were treated with 25 µM SB-203580-0.1%
dimethyl sulfoxide (DMSO; first 2 bars) or with 0.1% DMSO (last 2 bars). Two muscle strips were frozen before stimulation, and two were
stimulated with 1 µM carbachol for 10 min. Strips were frozen, and
homogenates were assayed for HSP27 kinase activity using human rHSP27
as substrate. Inset shows a
phosphorimage of 32P incorporation
into HSP27 under basal (open bars) and stimulated (filled bar and gray
bar) conditions. Kinase activities were determined by densitometry of
the phosphorylated HSP27 bands corrected for total protein loaded in
each lane and normalized to basal kinase activity in the absence of
carbachol (n = 7).
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Fig. 11.
Lack of effect of SB-203580 on in vivo activation of ERK1, ERK2 MAP
kinases, and tyrosine phosphorylation of p38 MAP kinase. Activation of
ERK and p38 MAP kinases was assayed using the same homogenates assayed
for HSP27 kinase activity in Fig. 10. ERK kinase activation was
measured using an in-gel kinase assay with myelin basic protein as the
substrate (A and
B).
A: phosphorimage of carbachol
stimulation of ERK1 and ERK2 MAP kinases in the absence and presence of
25 µM SB-203580. Bar graph in B
shows kinase activities determined by densitometry of the
phosphorylated myelin basic protein bands corrected for total protein
loaded in each lane and normalized to basal kinase activity in the
absence of carbachol (open bar; n = 7). Filled bars in B are relative ERK1
activities in the absence and presence of SB-203580. Hatched bars in
B are relative ERK2 activities in the
absence and presence of SB-203580. C:
Western blot showing tyrosine phosphorylation of p38 MAP kinase in
basal and stimulated muscles in the absence and presence of 25 µM
SB-203580. D: mean data from
densitometry of phosphotyrosine bands as in
C. Volume of each band was corrected
for total protein in each lane and was normalized to band volume of
unstimulated tissues in the absence of SB-203580
(n = 7).
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Inhibition of MAPKAP kinase activity by SB-203580 also resulted in
inhibition of HSP27 phosphorylation, as demonstrated by separation of
charge isoforms of HSP27 by NEpHGE-SDS-PAGE. Figure 12A
shows a cropped image of a Coomassie blue-stained gel and a bar graph
illustrating the distribution of HSP27 charge isoforms in unstimulated
muscle (basal). About one-half of the HSP27 is in the unphosphorylated
a spot. As shown in Fig. 12B, 10 min
of stimulation with 1 µM carbachol induces a redistribution of charge isoforms with a significant reduction in the unphosphorylated a spot
and a significant increase in the phosphorylated c spot. Figure 12, C and
D, shows that pretreatment for 60 min
with 25 µM SB-203580 blocks the agonist-induced shift in isoform
distribution. In unstimulated muscles treated with SB-203580, 78 ± 10% of HSP27 was in the unphosphorylated a isoform (Fig.
12C), and there was no significant
change in isoform distribution in response to carbachol (Fig.
12D). The results show that the p38
MAP kinase antagonist SB-203580 inhibits the shift of HSP27 to more
acidic, phosphorylated isoforms. This is consistent with the hypothesis
that muscarinic receptors are coupled to the activation of p38 MAP
kinase and MAPKAP kinase-2 that ultimately leads to phosphorylation of
HSP27.

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Fig. 12.
Inhibition of HSP27 phosphorylation by SB-203580. Muscle strips were
incubated for 60 min with 0.1% DMSO
(A and
B) or 25 µM SB-203580 and 0.1%
DMSO (C and
D). Muscle strips were stimulated
with 1 µM carbachol for 10 min and were frozen in liquid nitrogen.
Homogenates were subjected to NEpHGE-SDS-PAGE, and the gel was then
stained with Coomassie blue. Protein content of the 3 charge
isoforms of HSP27, labeled a, b, and c, was determined by scanning
densitometry. Protein content of each spot was expressed as percentage
of the total HSP27 and is presented as means ± SE in the bar graphs
below each gel image. A: unstimulated
muscles (basal) pretreated with 0.1% DMSO.
B: muscles pretreated with 0.1% DMSO,
then stimulated with 1 µM carbachol for 10 min.
C: unstimulated muscles (basal)
pretreated with 25 µM SB-203580-0.1% DMSO.
D: muscles pretreated with
SB-203580-0.1% DMSO and then stimulated with 1 µM carbachol for
10 min. * and ** Significant differences from basal,
DMSO-treated samples, P < 0.05, Student's t-test;
n = 5.
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 |
DISCUSSION |
We have evaluated the effects of carbachol stimulation on the
phosphorylation of HSP27 in isolated canine tracheal smooth muscle
strips. In response to stimulation with carbachol, the distribution of
HSP27 charge isoforms was shifted to more acidic, phosphorylated forms
(Figs. 4 and 5), which is consistent with previous studies of HSP27
phosphorylation in response to heat stress and chemical stressors (3,
4, 19, 28, 32) in nonmuscle cells. Studies of nonmuscle cells have
identified MAPKAP kinase-2 as the enzyme most likely to phosphorylate
HSP27 in vivo (19, 29). The canine HSP27 sequence contains
phosphorylation sites identical to known MAPKAP kinase-2
phosphorylation sites in the human HSP27 protein (Fig. 1). In vitro
phosphorylation of canine rHSP27 by purified skeletal muscle MAPKAP
kinase-2 (Fig. 6) confirmed that HSP27 is a substrate for this kinase.
In addition, activation of endogenous HSP27 kinase by carbachol
stimulation is rapid, as is HSP27 phosphorylation, and both are
sustained for at least 60 min (Fig. 7). Finally, canine rHSP27 is a
substrate for endogenous HSP27 kinase, which was not inhibited by
chelerythrine. Moreover, studies by Stokoe et al. (29) demonstrated
that MAPKAP kinase-1, calmodulin-dependent protein kinase-II,
adenosine 3',5'-cyclic monophosphate-dependent protein
kinase, protein kinase C, and ribosomal protein S6 kinase II are not
directly involved in the phosphorylation of HSP27. Therefore, the in
vivo HSP27 kinase activity stimulated by carbachol in tracheal smooth
muscle is most likely MAPKAP kinase-2, or possibly MAPKAP kinase-3,
which has similar substrate selectivity and sensitivity to inhibition by SB-203580 (7).
MAPKAP kinase-2 can be activated in vitro by both ERK MAP kinases (3,
29) and p38 MAP kinase (8). Activation of either pathway in vivo might
lead to phosphorylation of HSP27. We have shown that the ERK MAP
kinases are activated after muscarinic stimulation of canine colonic
and airway smooth muscle (10, 11), raising the formal possibility that
activation of ERK MAP kinases leads to activation of MAPKAP kinase-2
and phosphorylation of HSP27. However, the alternative hypothesis that
phosphorylation of HSP27 occurs via a stress response pathway involving
the p38 MAP kinases seems more likely. Cuenda et al. (8) showed that the p38 MAP kinase inhibitor SB-203580 completely blocked HSP27 phosphorylation in KB cells treated with arsenite, sorbitol, and interleukin-1. The question of whether muscarinic receptors in smooth
muscle are coupled to a stress response pathway was addressed by
demonstrating that the p38 MAP kinase is expressed in tracheal smooth
muscle (Fig. 8) and that stimulation with carbachol increased tyrosine
phosphorylation of the p38 MAP kinase (Fig. 9). Consistent with our
results, Kramer et al. (18) showed that p38 MAP kinase is activated in
platelets by thrombin, which acts via G protein-coupled STM receptors.
We also found that the agonist-induced shift in HSP27 charge isoforms
to the more acidic, phosphorylated forms was blocked by SB-203580 (Fig.
12). As shown in Figs. 4 and 10, muscarinic activation results in HSP27
shifting from the unphosphorylated state to more acidic forms, which
probably represent the mono- and diphosphorylated states (Fig. 12).
These results suggest a signaling cascade in which HSP27 is
phosphorylated by activation of the p38 MAP kinase and activation of
MAPKAP kinase-2. More direct support of this hypothesis was provided by
the partial inhibition of HSP27 kinase activity by SB-203580 and no
inhibition of ERK MAP kinases or tyrosine phosphorylation of p38 MAP
kinase (Fig. 11).
Activation of muscarinic receptors in airway smooth muscle is a normal
physiological event in vivo and would not ordinarily be considered a
"stress" stimulus in contrast to common experimental stressors
such as heat stress, ultraviolet radiation, arsenite, or anisomycin.
This raises the interesting question of what the role of the p38 MAP
kinase is in the normal physiology of airway smooth muscle and how the
extracellular signals are transduced to kinase activation and substrate
phosphorylation. A model based on what is known about MAP kinase
signaling in both muscle and nonmuscle cells is illustrated in Fig.
13. Agonists, including carbachol and
interleukin-8, activate ERK1 and ERK2 via an STM receptor (17, 30),
whereas stress stimuli, such as sorbitol and anisomycin or ultraviolet
light, activate two other parallel MAP kinase stress pathways: MKK3
leading to p38 activation and MKK4 activating
jun-NH2-terminal
kinase (JNK)/stress-activated protein kinase (SAPK; see Ref. 6). It is
known that there is cross talk among MAP kinase homologs. For example,
an osmotic stressor, sorbitol, activates three MAP kinase homologs,
ERK1/ERK2 (6), p38 (8), and JNK (6). The cross talk between MAP kinase
pathways can occur at several levels, including the dual- specificity
protein kinases that activate the MAP kinases. A relevant example is
MKK4, which activates JNK/SAPK as well as the p38 MAP kinase in
cultured fibroblasts (23).

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Fig. 13.
Possible parallel arrangement of MAP kinase cascades in mammalian
cells. Carbachol and interleukin-8 activate ERK1/ERK2 pathway via
7-transmembrane receptor (STMR) while cellular stresses activate MKK3
and MKK4 pathways. In addition, MAP kinase pathways converge and
activate parallel cascades. For example, sorbitol activates MAP
kinase/ERK (MKK) 3 as well as ERK1/ERK2 and
jun-NH2-terminal
kinase (JNK)/stress-activated protein kinase (SAPK). MKK4 can activate
both JNK and p38 MAP kinases. Activation of ERK1/ERK2 by carbachol and
interleukin-8 normally leads to nonproliferative events, whereas
activation of the JNK/SAPK pathway leads to proliferation. MEK, MAP
kinase; MEKK, MAP kinase kinase; UV, ultraviolet.
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Our studies suggest that similar activation of parallel MAP kinase
pathways occurs in airway smooth muscle. Several agonists acting via
STM receptors activate ERK MAP kinases in canine smooth muscles (10,
11). These include carbachol, neurokinin A, and histamine. In the
present study, we have shown that HSP27 is phosphorylated after
activation of muscarinic receptors, and the signaling pathway probably
includes p38 MAP kinase. Until recently, phosphorylation of HSP27 has
been thought to be coupled primarily to the stress response pathway
(Fig. 11), and there have been few reports that link activation of p38
MAP kinase with STM receptors. Our results suggest that G
protein-linked muscarinic receptors activate the p38 MAP kinase pathway
in airway smooth muscle and that one result of activating the p38 MAP
kinase pathway is phosphorylation of HSP27.
Although the physiological function of HSP27 is unknown, several
reports suggest that HSP27 may modulate actin filament dynamics in
vivo. This modulation of actin dynamics may be regulated by phosphorylation, since rHSP27 mutated at the phosphorylation sites is
unable to promote F-actin polymerization normally seen in control cells
transfected with wild-type rHSP27 (22). Moreover, Benndorf et al. (4)
observed that nonphosphorylated HSP25 isolated from Ehrlich ascites
tumor cells inhibited actin polymerization in vitro, whereas
phosphorylated HSP25 failed to exhibit any inhibiting activity. Using
isolated, permeabilized rectosigmoid smooth muscle cells, Bitar et al.
(5) found that contraction induced by bombesin was blocked by a
monoclonal antibody against HSP27. Therefore, phosphorylation and
activation of HSP27 may be linked to several cellular functions,
including proliferation, locomotion, and contraction via actin
remodeling. Control of these cellular responses in rectosigmoid smooth
muscle was suggested to be through MAP kinase cascades in part because
ERK MAP kinases and HSP27 codistribute in resting and stimulated cells
(31), and HSP27 phosphorylation is coupled to MAP kinase cascades in
nonmuscle cells (3, 8, 19, 31).
Activation of MAP kinases occurs in response to stimuli that promote
actin remodeling in other nonproliferative cells. For example,
interleukin-8 activates ERK MAP kinases (17) and induces migration of
neutrophils, which is a process involving changes in filamentous actin
content. However, the identity and regulation of proteins upstream of
p38 MAP kinases in regulating cytoskeletal and contractile proteins is
unclear. The protein kinases PAK1 and MKK3 are thought to be upstream
of p38 MAP kinase, but details of coupling to STM receptors are not yet
defined. In addition, activation of
rho and
rac are also thought to be important
for formation of actin stress fibers, focal contacts, and membrane ruffling in nonmuscle cells (25, 26). If actin filament structure is
regulated by MAP kinases in smooth muscle cells, then it may be that
HSP27 modulates actin filament dynamics after activation of G
protein-coupled STM receptors linked sequentially to
ras, rho/rac,
p38 MAP kinase, and MAPKAP kinase-2, leading to the phosphorylation of
HSP27 and regulation of the actin cytoskeleton.
 |
ACKNOWLEDGEMENTS |
We acknowledge Dr. Burton Horowitz for providing the colonic cDNA
smooth muscle library. We thank Dr. Eileen Hickey for assistance in the
cloning, sequencing, and construction of the canine rHSP27. A special
acknowledgment is extended to Dr. Jennifer Pohl for canine HSP27
polyclonal antibody production and isolation.
 |
FOOTNOTES |
This study was supported by Grants HL-48183 and DK-41315 (to W. T. Gerthoffer) from the National Institutes of Health.
Present address of J. K. Larsen: Dept. of Molecular and Integrative
Physiology, University of Illinois, 524 Burill Hall, 407 S. Goodwin
Ave., Urbana, IL 61801.
Address for reprint requests: W. T. Gerthoffer, Dept. of
Pharmacology/318, University of Nevada School of Medicine, Reno, NV
89557-0046.
Received 29 August 1996; accepted in final form 29 July 1997.
 |
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