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INTRODUCTION |
A major phosphorylation event that occurs with cyclic
nucleotide-dependent relaxation of vascular smooth muscle is an
increase in the phosphorylation of two 20-kDa proteins (isoform 3, pI
5.9; and 8, pI 5.7) (1-3). We recently identified these 20-kDa
phosphoproteins as different phosphorylated forms of a small heat
shock-related protein, HSP201
(4). In addition, HSP20 can be phosphorylated in vitro by both cAMP-dependent protein kinase (PKA) and
cGMP-dependent protein kinase (PKG) (4). HSP20 is also
phosphorylated during endothelial-dependent vasorelaxation
of isolated segments of bovine carotid artery smooth muscle (5).
In a vascular smooth muscle, umbilical artery smooth muscle, that is
refractory to cyclic nucleotide-dependent vasorelaxation, there is no significant increase in the phosphorylation of HSP20 in
response to activation of PKA or PKG (2). HSP20 is present in umbilical
artery smooth muscle and can be phosphorylated by PKA in
vitro using homogenates of umbilical smooth muscle (6). Taken
together, these data support a role for phosphorylated HSP20 in
mediating cyclic nucleotide-dependent vasorelaxation.
Histamine and phorbol ester-induced contractions of bovine carotid
artery smooth muscle are associated with an increase in the
phosphorylation of another 20-kDa protein (isoform 4, pI 6.0) (1). The
subsequent activation of cyclic nucleotide-dependent signaling pathways leads to a decrease in the phosphorylation of
isoform 4. This 20-kDa protein is immunoreactive with specific polyclonal antibodies raised against HSP20 (4). Thus, increases in the
phosphorylation of this isoform of HSP20 are associated with smooth
muscle contraction and decreases are associated with activation of
cyclic nucleotide-dependent signaling pathways.
The purpose of this investigation was to determine the specific site on
the HSP20 molecule that is phosphorylated during cyclic nucleotide-dependent vasorelaxation. We demonstrated that
Ser16 is the site phosphorylated on HSP20 by PKA or PKG. We
then prepared peptides in which the Ser16 site was altered
and determined whether these peptides altered the contractile responses
of transiently permeabilized vascular smooth muscle.
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EXPERIMENTAL PROCEDURES |
Materials--
Human skeletal muscle HSP20 was purified as
described previously (7). The catalytic subunit of
cAMP-dependent protein kinase (PKA) and modified trypsin,
sequence grade, was purchased from Promega (Madison, WI). Hepes was
obtained from American Bioanalytical (Natick, MA). Urea, SDS, glycine,
and Tris were from Research Organics (Cleveland, OH). Coomassie
Brilliant Blue was from ICN Biomedicals Inc (Aurora, OH).
[
-32P]ATP and [32P]orthophosphate were
from Amersham Pharmacia Biotech. Forskolin and
3-isobutyl-1-methylanthine (IBMX) were from Calbiochem. The inhibitor
of PKA, PKI was purchased from Peninsula (Belmont, CA). Piperazine
diacrylamide and other electrophoresis reagents were from Bio-Rad.
CHAPS, EGTA, EDTA, polyoxyethylene-sorbitan monolaurate (Tween 20) and
all other reagent grade chemicals were from Sigma. Purified
cGMP-dependent protein kinase (PKG) was obtained from Dr.
Tom Lincoln (University of Alabama, Birmingham, AL). Polyclonal antibodies against HSP20 were produced as described previously (7),
against
B crystallin were from Upstate Biotechnology (Lake Placid,
NY), and antibodies against myotonic kinase binding protein (MKBP) were
from Dr. Atsushi Suzuki (Yokohama, Japan). Goat anti-rabbit secondary
antibodies were from Jackson ImmunocResearch (West Grove, PA). Protein
concentrations were determined using the Coomassie Plus Protein Assay
Reagent (Pierce).
Preparation of Vascular Smooth Muscle Strips--
Intact bovine
carotid arteries were obtained from an abattoir (Shapiro's
meatpackers, Augusta, GA). Human aortic tissues were obtained from
organ donors with approval from the Medical College of Georgia
Institutional Review Board. The adventitia was dissected from the
arteries, and the endothelial lining was denuded with a cotton-tipped
applicator. The arteries were opened longitudinally, and thin
transverse strips were cut. Vessel viability was determined by
concurrent muscle bath experiments as described previously (2).
In Vitro Phosphorylation of HSP20--
HSP20 was phosphorylated
in a reaction mixture containing 20 mM Tris (pH 7.4), 10 mM magnesium acetate, 100 nM of the catalytic subunit of cAMP-dependent protein kinase or 100 nM of cGMP-dependent protein kinase. For
experiments using PKG, the peptide inhibitor of PKA (PKI, 1 µM, final concentration), was added. The reaction was
initiated with the addition of 200 µM
[
-32P]ATP (800 cpm/pmol) and incubated for 30 min at
30 °C. The reaction was stopped by the addition (1:1, v:v) of 6.25 mM Tris (pH 6.8), 2% SDS, 5%
-mercaptoethanol, 10%
glycerol, 0.025% bromphenol blue and boiled for 5 min. The proteins
were separated on 15% polyacrylamide/SDS gels, fixed in 10%
trichloroacetic acid, and stained with Neuhoff's Coomassie stain (10%
ammonium sulfate, 2.4% phosphoric acid, 0.1% Coomassie Brilliant Blue
G-250, 20% methanol) (8).
In Situ Phosphorylation of HSP20--
Strips of bovine carotid
artery smooth muscle were incubated in 150 µCi/ml
[32P]orthophosphate in 10 mM Hepes (pH 7.4),
150 mM NaCl, 4.7 mM KCl, 1.0 mM
MgSO4, 1.0 mM NaH2PO4,
10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3 and oxygenated with with
95% O2, 5% CO2 at 37 °C for 4 h. The
muscle strips were then stimulated with serotonin (1 µM)
for 10 min and then IBMX (1 mM) and forskolin (10 µM) for 10 min, phorbol dibutyrate (1 nM-1 µM) for 45 min, or phorbol dibutyrate
(1 µM) for 45 min followed by forskolin (10 µM) for 10 min. The vessels were snap-frozen in liquid
nitrogen, ground to a fine powder with a mortar and pestle, and
resuspended in 90% acetone, 10% trichloroacetic acid, 10 mM dithiothreitol. The suspension was quickly frozen in
liquid nitrogen and then allowed to return to room temperature. The
suspension was centrifuged (10,000 × g) and washed
three times in acetone. The pellets were dried under a stream of
nitrogen and solubilized in 9 M urea, 2% CHAPS, and 100 mM dithiothreitol overnight at room temperature.
Two-dimensional Gel Electrophoresis--
The isolated
phosphoproteins were separated by two-dimensional gel electrophoresis
using the method of O'Farrell (9) modified by Hoschstrasser et
al. (10). In brief, 5 mg of protein was loaded onto 12 × 15-cm slab isofocusing gels consisting of 4% acrylamide, 0.1%
piperazine diacrylamide, 9 M urea, 5% ampholines (5 parts
6-8, 3 parts 5-7, and 2 parts 3-10), and 2% CHAPS. Temed (0.04%)
and ammonium persulfate (0.1%) were used to initiate polymerization. The cathode buffer consisted of 20 mM sodium hydroxide and
the anode buffer 10 mM phosphoric acid. The proteins were
focused for 10,000 V h. The gels were fixed in 10% trichloroacetic
acid and stained with Neuhoff's Coomassie stain (8), and the lanes of
stained proteins were cut from the isofocusing slab gels and equilibrated in 10 mM Tris (pH 6.8), 3% SDS, 19% ethanol,
4%
-mercaptoethanol, and 0.004% bromphenol blue for 10 min. The
proteins were then separated on 12% acrylamide SDS gels (14). The gels
were fixed in 10% trichloroacetic acid, stained with Neuhoff's
Coomassie stain, and the spots corresponding to the specific isoforms
of HSP20 were excised from the gels.
In Gel Tryptic Digests and Amino Acid Sequencing--
The gel
pieces containing the specific isoforms of HSP20 were digested by the
in gel tryptic digest method of Hellman et al. (11). In
brief, the gel pieces were destained in 40% methanol and washed in 0.2 M ammonium bicarbonate (pH 8.9), 50% acetonitrile. The gel
pieces were then dried under a stream of nitrogen and reconstituted in
0.2 M ammonium bicarbonate (pH 8.9), 50% acetonitrile, 0.02% Tween 20. The proteins were digested with the addition of 0.5 µg of trypsin for 12 h. The proteolytic fragments were extracted with 60% acetonitrile, 0.1% trifluoroacetic acid. The peptides were
separated with reverse phase narrow-bore liquid chromatography on a
C-18 column using a 260-min gradient of 0-40% acetonitrile in 0.065 to 0.05% trifluoroacetic acid at a flow rate of 100 µl/min. with the
Smart System (Pharmacia Biotech, Uppsala, Sweden). 50-µl fractions
were collected and counted in a scintillation counter.
Peptide Sequencing--
Peptides from the Smart system were
applied to a ProSorb membrane (Perkin-Elmer and Applied Biosystems) as
per the manufacturers' directions. The peptides were sequenced on a
Procise (Applied Biosystems, model 492) instrument using standard protocols.
Phosphopeptide Mapping--
Peptide mapping was performed
according to the method of Cleveland et al. (12). A strip of
a two-dimensional gel containing isoforms 8, 4, and 3 was rehydrated
with 125 mM Tris, 1% SDS (pH 6.8) for 1 h. The
rehydrated gel piece was placed on top of a 15% SDS-PAGE gel and
overlaid with 10 µg of Staphylococcus aureus V8 protease
in 125 mM Tris, 1% SDS, 15% glycerol (pH 6.8). The gel
was run at 150 V until the dye front reached the end of the gel. The
gels were stained, dried, and exposed to Kodak XAR-5 film.
Peptide Synthesis--
Peptide synthesis was conducted on an
Applied Biosystems model 433A peptide synthesizer using standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. The synthesis of
the phosphopeptides involved an increase in coupling times of 20 min.
The phosphorylated amino acid derivatives were purchased from
Calbiochem. Peptide purity was determined by high pressure liquid
chromatography. Phosphopeptides were analyzed by mass spectrometry.
Production and Affinity Purification of Phosphorylation
State-specific Antibodies--
The synthetic peptide, WLRRASPLPGLK (S
denotes phosphoserine), conjugated to keyhole limpet hemocyanin (0.5 mg) was injected subcutaneously into two rabbits in collaboration with
Babco (Berkeley, CA). The rabbit serum was tested with an ELISA using
the synthetic peptide. A titer that extinguished at a dilution of
1:10,000 was obtained, and this serum was affinity purified using
AminoLink® Plus Immobilization Kit (Pierce). The serum was first
applied to a column linked with 5 mg of the non-phosphorylated peptide (EIPVPVPQPSW LRRASAPLPGLK). The eluted serum was then applied to a
column linked with 5 mg of the phosphorylated peptide (WLRRASPLPGLK, where S indicates phosphorylated serine). Fractions (1 ml) were collected and neutralized with 50 µl of sodium phosphate buffer (pH
8.0). The fractions were analyzed in a spectrophotometer, and the
280-nm peak fractions were combined and used for Western blots at a
dilution of 1:100.
Cloning and Expression of HSP20--
The rat cDNA for HSP20
(13) was polymerase chain reaction-amplified using sense (GAA TTC ATA
TGG AGA TCC GGG TGC CTG TGC) and antisense (CGT ACT CGA GCT ACT TGG CAG
CAG GTG GTG ACT) primers (synthesized by Life Technologies, Inc.). The
polymerase chain reaction products were ligated into pCR-script SK(+)
cloning vector and transformed into Escherichia coli
supercompetent cells according to the manufacturer's directions
(pCR-Script SK(+) Cloning Kit, Stratagene, La Jolla, CA). Plasmid
minipreps were performed with the Wizard miniprep DNA purification
system (Promega, Madison, WI) using appropriate colonies from the
transformed E. coli. The isolated DNA was then sequenced
using an Applied Biosystems Prism automatic DNA sequencer. The plasmid
was then cut with Xho-1, isolated on a 1% agarose gel, and inserted
into a pET-19b plasmid and transformed into E. coli JM109
cells (Promega, Madison, WI), and plasmid preparations were performed
as above. The plasmids were then transformed into BL21(DE3)pLysS cells,
and appropriate colonies were then inoculated into LB broth (containing
carbenicillin and chloramphenicol) and grown for approximately 2.5 h at 37 °C. The bacteria were harvested by centrifugation 2500 × g for 10 min, and the HSP20 was affinity purified using a
HIS-bind resin column (Novagen, Madison, WI). Proteins from the
fractions were separated on SDS-PAGE gels (14), and fractions
containing a single band at 20 kDa were dialyzed against decreasing
concentrations of urea (6 M urea, 1% Triton to 0 M urea, 1% Triton) in phosphate-buffered saline. Finally,
the HSP20 was dialyzed against phosphate-buffered saline, 1%
CHAPS.
Immunoblotting--
The proteins were separated on 15% SDS-PAGE
gels (14) and transferred to Immobilon for 210 V h. The blots were
air-dried and subsequently blocked with Tris-buffered saline (TBS: 10 mM Tris, 150 mM NaCl (pH 7.4)), 5% milk for
1 h. The blots were then incubated with anti-HSP20 antibodies in
TBS/milk for 1 h at room temperature. The blots were washed 3 times (5 min each) in TBS/Tween 20 (0.5%). Immunoreactive protein was
determined using 125I-protein A. The blots were again
washed 6 times (5 min each) in TBS/Tween 20. The blots were then
exposed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) screen
for 18 h. In other experiments the amount of immunoreactive
protein was determined using enhanced chemiluminescence (NEN Life
Science Products).
Enzyme-linked Immunosorbent Assay (ELISA)--
Phosphorylated
and non-phosphorylated recombinant HSP20 were diluted in borate buffer
(100 mM boric acid, 25 mM
Na2B4O7, 75 mM NaCl (pH
8.5)), and 100 µl was added to each well of Dynatec Immulon 2 plates
(Fisher). The plates were incubated overnight at 4 °C and washed 3 times with wash buffer (10 mM Tris, 0.05% Tween 20 (pH
8.0)). The plates were blocked with borate buffer, 1% bovine serum
albumin for 30 min at room temperature and again washed three times.
The plates were incubated with the affinity purified phosphorylation
state-specific antibodies (1:1000 dilution) and incubated for 2 h
at room temperature. The plates were again washed three times and
subsequently incubated with anti-rabbit antibodies conjugated to
alkaline phosphatase (Promega, Madison, WI) for 2 h at room
temperature. The plates were washed three times and developed with
alkaline phosphatase substrate (Sigma 104 phosphatase substrate). The
optical density was read at 405 nM.
Transient Permeabilization of Isolated Strips of Vascular Smooth
Muscle--
Fine strips of bovine carotid artery smooth muscle (0.1 mm
wide × 8 mm long) were cut with a razor blade under a dissecting microscope and permeabilized using a protocol that has been modified to
introduce the 21-kDa photoprotein, aequorin (15-18). The strips were
washed 3 times in stripping solution, 25 mM Hepes, 120 mM KCl, 5.6 mM glucose, 0.2% bovine serum
albumin, and 3 mM EGTA and then incubated in stripping
solution for 30 min at room temperature while gently shaken. The strips
were then incubated in the stripping solution with the specific
peptides for another 30 min on ice. Calcium was added directly to the
solution in three increments, 5 min apart to a final concentration of 1 mM. It has been determined that a 13-kDa molecule with a
radioactive tag is not released after this protocol (18) suggesting
that the cell membranes regain their integrity.
Physiologic Contractile Responses--
The strips were tied
under magnification at each end with a 7-0 prolene suture (Johnson and
Johnson, Cincinatti, OH) and suspended in a muscle bath in Hepes
solution (10 mM Hepes, 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 1.0 mM
CaCl2, 10 mM glucose (pH 7.4)) at room
temperature. The muscle strips were placed under 0.5 g of tension
and allowed to relax to a basal tension over 15 min. The strips were
fixed at one end to a stainless steel wire and attached to a Kent
Scientific (Litchfield, CT) force transducer (TRN001) interfaced with a
Data Translation A-D board, DT2801 (Data Translation, Inc., Marlboro,
MA). Data were acquired with Lab Tech Notebook software (Laboratory
Technologies Corp., Wilmington, MA). Agonists were added directly to
the bath.
In Vitro Phosphorylation of Synthetic Peptides--
The peptides
(200 µg) were phosphorylated in a 50-µl reaction mixture containing
20 mM Tris (pH 7.4), 10 mM magnesium acetate, 5 mM K2PO4, 5 mM EDTA, 2 mM 2-mercaptoethanol, 6 units (15 nM) of the
catalytic subunit of PKA. The reactions were initiated with the
addition of 200 µM [
-32P]ATP (800 cpm/pmol) and incubated for 15 min at 30 °C, terminated by spotting
20-µl aliquots onto phosphocellulose papers (Whatman P81). The papers
were washed three times in ice-cold 75 mM phosphoric acid,
rinced in acetone, and allowed to air dry. The papers were counted in a
scintillation counter (Beckman, Irving, CA).
Data Analysis--
Values are reported as mean ± S.E., and
n refers to the number of animals examined. The statistical
difference between the two groups was determined with Student's
t test and between multiple groups with one-way repeated
measures analysis of variance using Sigma Stat software (Jandel
Scientific, San Rafeal, CA). A p value less than 0.05 was
considered significant. Densitometric analysis was performed with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
Identification of the Phosphorylation Sites on the HSP20
Molecule--
Phosphorylation of purified rat skeletal muscle HSP20
with the catalytic subunit of PKA resulted in two proteolytic fractions that contained radioactive counts (Fig.
1). The amino acid sequence obtained from
the major proteolytic fragment (peak 1, fraction 19) was
XAXXPLPGLSAPGGRRQ and that from the minor
fragment (peak 2, fractions 27 and 28) was APSVALPVAQVPTDPG. These
peptides displayed significant homology with the known amino acid
sequence of human HSP20 (fraction 19, 70% homology, and fraction
27/28, 100% homology) (7). The fragment from peak 1 had a PKA
consensus sequence (RRAS) corresponding to Ser16 on the
HSP20 molecule. The fragment from peak 2 had a less suitable PKA
consensus sequence (RAPS) corresponding to Ser59 on the
HSP20 molecule. Phosphorylation of purified rat skeletal muscle HSP20
with PKG resulted in one fraction that contained the majority of the
radioactive counts. This fraction had a similar mobility on the column
as the major peak obtained after phosphorylation with PKA (fraction
19). Peptide analysis was not performed on this fraction.

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Fig. 1.
In vitro phosphorylation of HSP20
by PKA and PKG. Purified HSP20 was phosphorylated in
vitro by cAMP-dependent protein kinase (A,
PKA) or cGMP-dependent protein kinase (B,
PKG). The HSP20 was separated by SDS-PAGE (gel inserts), and after
in gel tryptic digestion, the proteolytic fragments were separated by
reversed phase fast protein liquid chromatography, and the fractions
were counted in a scintillation counter (cpm × 10 3). Phosphorylation of HSP20 by PKA led to two
fractions containing radioactivity, fraction 19 (A, peak 1)
and fractions 27 and 28 (A, peak 2). The amino acid sequence
of the peptide from fraction 19 was
XAXXPLPGLSAPGGRRQ and that from fraction 27/28
was APSVALPVAQVPTDPG. Phosphorylation of HSP20 by PKG led to one major
fraction containing radioactivity, fraction 19 (B, peak
1).
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To determine the sites on the HSP20 molecule that are phosphorylated
when intact strips of muscle are stimulated with substances that
activate cyclic nucleotide-dependent signaling pathways, strips of bovine carotid artery smooth muscle were incubated with [32P]orthophosphate and stimulated with forskolin (10 µM) and isobutylmethylxanthine (IBMX, 1 mM)
for 10 min. This combination of an adenylate cyclase activator,
forskolin, and a phosphodiesterase inhibitor, IBMX, led to the maximal
phosphorylation of HSP20 (data not shown). The proteins were separated
with two-dimensional gel electrophoresis. Autoradiography revealed two
20-kDa spots that were immunoreactive with antibodies against HSP20
(Fig. 2, immunoblots not shown). The
protein that had been previously described as isoform "3" with a pI
of 5.9 was digested, and the proteolytic fragments were separated by
reversed phase fast protein liquid chromatography. The peak of
radioactivity was again in fraction 19, with a minor component of
counts in fractions 27 and 28 (Fig. 2B). The proteolytic fragment from fraction 19 contained the amino acid sequence
RAXXXLPGLSAPGX. This sequence had 100% homology
to the known sequence of human HSP20 and with the peptide isolated from
fraction 19 after the in vitro phosphorylation of the
purified HSP20. The RAXX likely represents RAS corresponding
to Ser16 on the HSP20 molecule that was phosphorylated by
PKA. We were unable to resolve the proteolytic fragment from fractions
27/28 for sequence analysis. The proteolytic fragment with the peak amount of radioactivity from isoform "8" (pI of 5.7) was again in
fraction 19 (Fig. 2C). The proteolytic fragment from
fraction 19 contained the amino acid sequence RASAPLPGLSAPGR (100%
homology with human HSP20). This peptide again contained the consensus sequence for PKA phosphorylation: RRAS with Ser16
representing the phosphorylation site. The proteolytic fragment from
fractions 27/28 had a sequence of LPPGVDPAAVTSALSPEG (100% homology
with human HSP20), corresponding to the carboxyl terminus of the HSP20
molecule, and contained no consensus sites for PKA or PKG
(Fig. 3).

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Fig. 2.
In situ phosphorylation of HSP20
by forskolin. Radiolabeled strips of vascular smooth muscle were
treated with the adenylate cyclase activator, forskolin (10 µM), and the phosphodiesterase inhibitor,
isobutylmethylxanthine (1 mM, for 10 min) (A),
or with phorbol dibutyrate (1 µM, for 45 min)
(D) and homogenized, and the proteins were separated by
two-dimensional gel electrophoresis, and autoradiographs were obtained
(m refers to the myosin light chains, and 8, 3, 4 refer to the specific isoforms of HSP20). The spots corresponding to
isoforms 3, 4, and 8 of HSP20 were digested with trypsin, and the
proteolytic fragments were separated by reversed phase fast protein
liquid chromatography, and the fractions were counted in a
scintillation counter (cpm × 10 3). The fraction
with the peak amount of radioactivity from isoform 3 was fraction 19 (B, peak 1) and contained the amino acid sequence,
RAXXXLPGLSAPGX. There were two fractions with
radioactivity from isoform 8, fraction 19 (C, peak 1), and
fractions 27/28 (C, peak 2). The amino acid sequence of peak
1 was RASAPLPGLSAPGR and peak 2 was LPPGVDPAAVTSALSPEG. The fraction
with the peak amount of radioactivity from isoform 4 was fractions
27/28 (E, peak 2) and contained the amino acid sequence
RYRLPPGVPPAAVTSAL.
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Fig. 3.
. Location of the HSP20 phosphorylation sites
within the aligned amino acid sequences of human and rat HSP20.
The sequences of human and rat HSP20 from Kato et al. (7)
were aligned, and the tryptic digest sites are marked with a
slash (/). The amino acid sequences of proteolytic fragments
from specific column fractions are underlined. The in
situ cAMP-dependent phosphorylation site is marked
with a dashed box.
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To determine the phosphorylation site on the isoform with a pI of 6.0 (isoform 4), strips of bovine carotid artery smooth muscle were
incubated in the presence of [32P]orthophosphate and
stimulated with the phorbol ester, phorbol dibutyrate (PDBu, 1 µM, 45 min), and the proteins were separated by
two-dimensional electrophoresis. The spot corresponding to isoform 4 was digested, and the proteolytic fragments were separated. The peak of
radioactivity was in fractions 27 and 28 (Fig. 2E), and the
peptide sequence from this fraction was RYRLPPGVPPAAVTSAL (94%
homology with human HSP20). This sequence is found at the carboxyl
terminus of the HSP20 molecule (amino acids 120-137).
The chromatographs of the peptides from in vitro
phosphorylation of HSP20 with PKA (Fig.
4A) were similar to the
chromatographs of isoform 3 after IBMX/FSK treatment (Fig.
4B). The peptide patterns on the chromatographs from
isoforms 8 and 4 were also similar (Fig. 4, C and
D). However, there were differences between the peptide
patterns from isoform 3 and isoforms 4 and 8. Finally, phosphopeptide
mapping of a strip of gel containing all three isoforms demonstrated
that there were phosphorylated peptides unique to isoforms 4 and 8 (Fig. 4, E and F).

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Fig. 4.
Phosphopeptide mapping of isoforms 3, 4, and
8 of HSP20. Chromatograms from the tryptic digests of HSP20
phosphorylated with PKA (A), isoform 3 (B), and
isoform 8 (C) after in situ phosphorylation with
3-isobutyl-1-methylxanthine and forskolin and isoform 4 after in
situ phosphorylation with phorbol dibutyrate (D) reveal
the relative mobility of the peptide fragments. The fractions
containing the peak amount of radioactivity are indicated in
Panel A (Peak 1 and Peak 2). A strip
of the two-dimensional gel containing isoforms 3, 4, and 8 of HSP20
(E) was digested with staphylococcal V8 protease, and the
phosphopeptides generated are depicted in F. There were
peptides unique to isoforms 8 and 4 (arrows).
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Characterization of Phosphorylation State-specific Polyclonal
Antibodies--
Affinity purified phosphorylation state-specific
antibodies for phosphorylated HSP20 recognized only isoforms 3 and 8 of
HSP20 that were phosphorylated by PKA
(Fig. 5). On the other hand, a purified
polyclonal antibody (7) recognized all isoforms of HSP20 including
non-phosphorylated HSP20.

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Fig. 5.
Characterization of phosphorylation
state-specific antibodies. HSP20 was purified from human skeletal
muscle, and some of the protein was phosphorylated in vitro
using PKA. The proteins (1 µg for each gel) were then separated by
two-dimensional electrophoresis and transferred to Immobilon.
Non-phosphorylated HSP20 is shown in A and C.
HSP20 phosphorylated by PKA is shown in B and D.
A and B were probed with the phosphorylation
state-specific antibody and C and D with the
antibody that recognizes all isoforms of HSP20. The * denotes a
hyperphosphorylated form of HSP20 that is present only when HSP20 is
phosphorylated by PKA in vitro (4). The relative mobility of
molecular weight markers is indicated on the left of each
panel and the mobility of isoelectric focusing markers on the
top of A.
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To determine the sensitivity of the phosphorylation state-specific
antibodies, recombinant HSP20 was phosphorylated in vitro by
the catalytic subunit of PKA. The phosphorylation state-specific antibodies recognized 3-100 ng of phosphorylated HSP20 in an ELISA (Fig. 6). By Western blotting, the
antibodies recognized 1-10 µg of phosphorylated HSP20 in a linear
fashion (Fig. 6).

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Fig. 6.
. Sensitivity of the phosphorylation
state-specific antibodies. The sensitivity of the phosphorylation
state-specific antibodies was determined using recombinant HSP20 that
was phosphorylated in vitro with PKA. An ELISA was performed
using phosphorylated HSP20 (A, closed circles) and
non-phosphorylated HSP20 (A, open circles). Immunoblots were
performed using 1-10 µg of phosphorylated HSP20 (B) or
non-phosphorylated HSP20 (C). Molecular weight markers are
on the left, and the amount of recombinant protein in
micrograms is on the bottom of the blot. D,
densitometric analysis of the immunoblot from B is
shown.
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To determine the specificity of the phosphorylation state-specific
antibodies for HSP20 in intact tissues, homogenates of bovine carotid
artery smooth muscles (30 µg of protein) were treated with buffer
alone (control), sodium nitroprusside (10 µM, 10 min), or
forskolin (10 µM, 10 min) and then separated on SDS-PAGE
and transferred to Immobilon. The blots were probed with the
phosphorylation state-specific antibodies and subsequently re-probed
with affinity purified polyclonal antibodies that recognize all
isoforms of HSP20 (7). The affinity purified phosphorylation
state-specific antibodies recognized a band at a relative
mobility of 20 kDa in the sodium nitroprusside- and forskolin-treated
tissues, whereas the affinity purified polyclonal antibody recognized
three forms of HSP20 (Fig. 7).

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Fig. 7.
Specificity of the phosphorylation
state-specific antibodies. Bovine carotid artery smooth muscle was
treated with buffer alone (control, C), sodium nitroprusside
(10 µM, 10 min, S), or forskolin (10 µM, 10 min, F). The strips were homogenized,
and 30 µg of protein was loaded into each lane. The blots were probed
with the phosphorylation state-specific antibodies (A)
followed by the antibodies that recognize all isoforms of HSP20
(B).
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The phosphorylation of an additional isoform of HSP20, pI 6.0, (isoform
4), increases with phorbol ester stimulation of carotid artery smooth
muscle (1). The phosphorylation of isoform 4 decreases with activation
of cyclic nucleotide-dependent signaling pathways.
Radiolabeled strips of carotid artery smooth muscle were treated with
phorbol dibutyrate (PDBu, 100 nM, for 45 min) followed by forskolin (10 µM, for 10 min). The strips
were homogenized and the proteins separated by two-dimensional
electrophoresis and transferred to Immobilon. The blots were exposed to
x-ray film (autoradiographs) and subsequently probed with the
phosphorylation state-specific affinity purified antibodies. The
phosphorylation state-specific antibodies recognized isoforms 3 and 8 but did not recognize isoform 4 (Fig.
8B). The blots were again
probed with antibodies that recognize all isoforms of HSP20. Isoforms 3, 4, 8, as well as a nonphosphorylated pool of HSP20 were identified (Fig. 8C). Finally, the blots were probed with an antibody
against another recently identified small heat shock protein myotonic dystrophy-binding protein (19). This antibody recognized a 20-kDa protein with a pI of 5.3 that was not phosphorylated in response to
PDBu or forskolin treatment (Fig. 8, A and D).
This protein does not contain the RRAS16 site (19).

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Fig. 8.
Immunoreactive small heat shock
proteins. Strips of carotid artery smooth muscle were incubated in
the presence of [32P]orthophosphate and treated with
phorbol dibutyrate (100 nM, for 45 min) followed by
forskolin (10 µM, for 10 min). The strips were
homogenized, and the proteins were separated by two-dimensional
electrophoresis and transferred to Immobilon. The blots were exposed to
x-ray film (A) and subsequently probed with the
phosphorylation state-specific affinity purified antibodies
(B), followed by the antibodies that recognize all isoforms
of HSP20 (C), and antibodies against myotonic dystrophy
kinase-binding protein (D, arrow). The relative
mobility of molecular weight markers is indicated on the
left and the isoelectric focusing gradient on the
top of A. The isoforms of HSP20 are designated
3, 4, 8, and NP (nonphosphorylated).
Immunoreactive MKBP is denoted with an arrow.
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Phosphorylation of Isoform 3 in Human Vascular
Tissue--
Previous reports have suggested that only isoform 3 of
HSP20 was present in human vascular tissue (2). To determine which isoforms in human aortic tissues are phosphorylated during cyclic nucleotide-dependent vasorelaxation, strips of human aortic
smooth muscle were labeled with [32P]orthophosphate and
treated with IBMX (1 mM) and forskolin (10 µM). The strips were separated by two-dimensional
electrophoresis and transferred to Immobilon. Autoradiographs were
developed (Fig. 9A), and the
blots were then probed with the phosphorylation state-specific antibodies (Fig. 9B). Only isoform 3 was phosphorylated
after IBMX and forskolin treatment of human aortic smooth muscle.

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Fig. 9.
HSP20 isoforms in human aorta. Strips of
human aortic vascular smooth muscle were labeled with
[32P]orthophosphate, treated with
3-isobutyl-1-methylxanthine (1 mM) and forskolin (10 µM, for 10 min), homogenized, and separated by
two-dimensional electrophoresis. The proteins were transferred to
Immobilon and exposed to x-ray film (A). The blots were then
probed with phosphorylation state-specific antibodies (B).
The relative mobility of molecular weight markers is indicated on the
left and the isoelectric focusing gradient on the
top of A. The myosin light chains are indicated
with an m and isoform 3 of HSP20 with 3.
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The Effect of Synthetic Peptides on Contractile Responses in
Permeabilized Bovine Carotid Artery Smooth Muscle--
To determine
the effect of phosphorylation of HSP20 on smooth muscle physiology,
strips of bovine carotid artery smooth muscle were transiently
permeabilized and synthetic peptides introduced. The synthetic peptide,
WLRRASpPLPGLK, in which Ser16 was
phosphorylated, significantly attenuated both KCl (110 mM)- and serotonin (5HT, 1 µM)-induced contractions
(Fig. 10). In addition, the synthetic
peptide, WLRRAAPLPGLK, in which Ser16 was replaced with an
alanine, thus rendering the peptide "nonphosphorylatable" augmented
both KCl (110 mM)- and serotonin (5HT, 1 µM)-induced contractions (Fig. 10). The synthetic
peptides, WLRRASPLPGLK, in which Ser16 was not
phosphorylated, and PRKALWLGRPLA, a peptide containing a random
distribution of the amino acids, had no effect on KCl (110 mM)- or serotonin (5HT, 1 µM)-induced
contractions (p > 0.05 compared with control (no
peptide added) contractions).

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Fig. 10.
The effect of synthetic peptides on
contractile responses of transiently permeabilized vascular smooth
muscles. Strips of bovine carotid artery smooth muscles were
transiently permeabilized and incubated in the presence of synthetic
peptides. The strips were then treated with high potassium (110 mM, KCl), re-equilibrated in bicarbonate buffer, and
treated again with serotonin (5HT, 1 µM). A
representative tracing of the responses after incubation with the
synthetic peptide, WLRRASpPLPGLK (in which
Ser16 was phosphorylated, dotted line),
WLRRAAPLPGLK (in which Ser16 was replaced with an alanine,
dashed line), or with WLRRASPLPGLK (in which
Ser16 was not phosphorylated, solid line) is
depicted in A. Aggregate data, normalized to stress (where
stress (105 N/m2) was calculated as force
(gms) × 0.0987/area, where area = wet weight
(mg)/length (mm at Lmax)/1.055) are depicted in
B for KCl responses and C for serotonin
responses; lane 1 is the non-phosphorylated peptide;
lane 2 is the phosphorylated peptide; and lane 3 is the peptide in which Ser16 was replaced with an alanine
(n = 5, * p < 0.05 compared with
control). PRKALWLGRPLA, a peptide containing a random distribution of
the amino acids, also had no effect on KCl- (110 mM) or
serotonin (5HT, 1 µM)-induced contractions
(p > 0.05 compared with control, data not
shown).
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To determine if the phosphorylation state of the peptides could be
modified in vitro, the WLRRASPLPGLK peptide was
phosphorylated by the catalytic subunit of PKA. No increase in
phosphorylation of the WLRRASPLPGLK peptide (2518 ± 214 base line
versus 3379 ± 315, n = 3, p > 0.05) was observed. However, a larger peptide, EIPVPVQPSWLRRASAPLPGLK, was phosphorylated in vitro by PKA
(1258 ± 153 versus 4580 ± 408, n = 3, p < 0.05).
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DISCUSSION |
The present experiments demonstrate that the small heat
shock-related protein, HSP20 is phosphorylated on Ser16
during cAMP-dependent vasorelaxation (Fig. 1 and Fig. 2).
This serine is contained in a region with a consensus sequence for both
PKA and PKG (RRAS). There are three phosphorylated isoforms of HSP20 in
bovine carotid artery. Isoforms 3 and 8 are phosphorylated on
Ser16 during cAMP-dependent vasorelaxation.
Antibodies generated against a peptide containing the Ser16
phosphorylation site were both sensitive and specific for
phosphorylated HSP20 (Figs. 5-7). These antibodies recognized isoforms
3 and 8 but did not recognize isoform 4 (Fig. 8). Only one
phosphorylated isoform (isoform 3) was recognized in human vascular
tissue with the phosphorylation state-specific antibody (Fig. 9). In
addition, another small heat shock protein in muscle, MKBP, is not
phosphorylated when muscles are stimulated with PDBu or with
IBMX/forskolin (Fig. 8). Although this protein has considerable
sequence homology with HSP20, it does not contain the
RRAS16 site (19). Taken together, these data suggest that
there are species differences in HSP20 isoform expression and that
Ser16 is the physiologically relevant phosphorylation site
for cAMP-dependent vasorelaxation.
The isoforms that are phosphorylated after activation of adenylate
cyclase with forskolin or guanylate cyclase with sodium nitroprusside
stimulation have similar mobilities on two-dimensional gels (4). The
peptide maps after proteolytic digestion of the two phosphorylated
isoforms of HSP20 with S. aureus V8 protease are similar
(4). The phosphorylation of HSP20 by PKG in vitro resulted
in a similar mobility of the phosphorylated peptide on the SMART system
as the phosphorylation of HSP20 by PKA in vitro (Fig.
1). Finally, the phosphorylation state-specific antibodies recognize the same phosphorylated isoforms in vessels treated with
sodium nitroprusside as with forskolin (Fig. 7). These data suggest
that HSP20 is phosphorylated on Ser16 after activation of
cGMP-dependent signaling pathways.
Whereas there are three phosphorylated isoforms of HSP20 in bovine
carotid artery smooth muscles, two of the isoforms, 4 and 8, have
peptide maps that differ from that of isoform 3 (Fig. 4) suggesting
that they may represent proteins that contain different amino acid
sequences. The simplest explanation of our present results is that
isoform 4 is phosphorylated on a carboxyl-terminal site when agonists
induce contraction, and then Ser16 is phosphorylated when
PKA is activated. This leads to a shift from isoform 4 to isoform 8.
Using the identified phosphorylation site on the HSP20 molecule,
peptides corresponding to this site were synthesized. The effects of
these peptides on contractile physiology was determined by transiently
permeabilizing strips of bovine carotid artery smooth muscle (15, 16,
17, 18). The introduction of a phosphorylated peptide into carotid
artery smooth muscle inhibited both high extracellular potassium and
serotonin-induced contractions (Fig. 10). This response is similar to
the effect of phosphorylated HSP20 on vascular smooth muscle, it
inhibits agonist-induced smooth muscle contraction. This peptide may be
inhibiting a HSP20 phosphatase or the peptide may act on the same
target as the HSP20 protein. The introduction of a peptide in which the
phosphorylated serine was replaced with an alanine enhanced contractile
responses (Fig. 10). This peptide may inhibit the phosphorylation of
endogenous HSP20 or inhibit the effects of the HSP20 molecule. Peptides
that were not phosphorylated or contained a scrambled sequence had no
effect on contractile responses. The synthetic peptide could not be
phosphorylated by PKA in vitro, suggesting that contractile responses were not altered by phosphorylation of the peptides in the
strips of muscle. These data supply direct but incomplete evidence that
the phosphorylation of HSP20 may be a critical event in the relaxation
of tonic vascular smooth muscle.
Heat shock proteins are a group of proteins whose synthesis is induced
by heat or other stressors. These proteins are divided into several
groups based on molecular weights. The small heat shock proteins
(15-30 kDa),
B-crystallin,
A-crystallin, HSP20, HSP27, and the
MKBP all share considerable sequence homology (approximately 50%)
(19). HSP20 and HSP27 are highly expressed in muscle cells (7), and
both exist in phosphorylated and non-phosphorylated forms (4, 20). The
specific physiologic functions of the small heat shock proteins are not
known. However, increases in the phosphorylation of HSP27 have been
associated with vascular smooth muscle contraction (21, 22) and
increases in the phosphorylation of HSP20 with vascular smooth muscle
relaxation (4). HSP27 has also been implicated in stabilizing the actin
cytoskeleton (23). HSP20 has also been shown to be an actin-binding
protein, and the association of HSP20 with actin in vitro is
dependent on the phosphorylation state of HSP20 (24). Thus, the small heat shock proteins may be late phase signaling molecules that modulate
smooth muscle contractile responses via a direct interaction with
specific cytoskeletal and/or contractile elements.
In sum, these data suggest that the cyclic
nucleotide-dependent vasorelaxation is associated with
increases in the phosphorylation of HSP20 at Ser16.
Phosphorylation of HSP20 at Ser16 is not only associated
with cyclic nucleotide-dependent vasorelaxation but
also inhibits agonist-induced contractile responses.