(Received for publication, November 20, 1996, and in revised form, February 3, 1997)
From the Departments of Surgery and Medicine and the Institute of
Molecular Medicine and Genetics, Medical College of Georgia, Augusta,
Georgia 30912, the Augusta Veterans Administration Medical Center,
Augusta, Georgia 30901, and the Department of
Biochemistry, Institute for Developmental Research, Human Service
Center, Kasugai, Aichi 480-03, Japan
Activation of cyclic nucleotide-dependent signaling pathways leads to the relaxation of various smooth muscles. One of the major phosphorylation events associated with cyclic nucleotide-dependent vasorelaxation in bovine trachealis and carotid artery smooth muscle is the phosphorylation of two 20-kDa phosphoproteins with pI values of 5.7 and 5.9 (previously designated pp8 and pp3, respectively). The present studies sought to determine the identities of pp3 and pp8 in vascular smooth muscle. The phosphopeptide maps for the pp8 and pp3 proteins were similar. Preparative two-dimensional gel electrophoresis and amino acid sequencing of a peptide fragment of the pp3 protein revealed a sequence identical to a 20-kDa heat shock-related protein (HSP20) previously purified from skeletal muscle. Western blot and immunoprecipitation analysis with anti-HSP20 antibodies demonstrated that the pp3 and pp8 proteins are phosphorylated forms of HSP20. In addition, HSP20 could be phosphorylated in vitro by both cAMP-dependent protein kinase and cGMP-dependent protein kinase. These data suggest that the phosphorylation of the heat shock-related protein HSP20 is associated with cyclic nucleotide-dependent relaxation of vascular smooth muscle.
Activation of cyclic nucleotide-dependent signaling pathways leads to the relaxation of vascular smooth muscle. Isoproterenol, prostacyclin, and forskolin stimulate the adenylate cyclase/cAMP pathway and activate cAMP-dependent protein kinase (PKA)1 (1). Nitric oxide, atrial natriuretic peptide, sodium nitroprusside, and nitroglycerin stimulate the guanylate cyclase/cGMP pathway and activate cGMP-dependent protein kinase (PKG) (2).
A number of investigators have sought to identify the protein targets for PKA and PKG that are involved in smooth muscle relaxation. Two sites of particular interest have emerged. First, the phosphorylation of myosin light chain kinase by PKA decreases its sensitivity to activation by Ca2+-calmodulin, leading to a decrease in the phosphorylation of the myosin light chains (3). Second, PKA and/or PKG activation leads to changes in the activities of one or more Ca2+ channels and/or Ca2+ pumps, thereby reducing the intracellular Ca2+ concentrations (4, 5). However, there is no simple correlation between the extent of myosin light chain phosphorylation and the state of contraction of vascular smooth muscle (6, 7). On the other hand, cyclic nucleotide-dependent relaxation of intact muscle strips can occur under conditions where the Ca2+ concentration is fixed at either a low or a high concentration (8-12).
The relaxation of several different types of smooth muscles by forskolin or sodium nitroprusside is associated with an increase in the extent of phosphorylation of two 20-kDa proteins with pI values of 5.9 and 5.7, previously described as proteins 3 (pp3) and 8 (pp8), respectively (13, 14). In addition, the extent of phosphorylation of these two proteins increases during forskolin or sodium nitroprusside-induced vasorelaxation under circumstances where the intracellular Ca2+ concentrations are low and fixed (12). Finally, there is no increase in the extent of the phosphorylation of these two proteins in human umbilical artery smooth muscle, a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation (15). Taken together, these data suggest that an increase in the phosphorylation of these two 20-kDa phosphoproteins plays a role in cyclic nucleotide-dependent vasorelaxation.
In the present study, preparative gel electrophoresis was employed to isolate one of these 20-kDa proteins (pp3) from bovine carotid smooth muscle. Amino acid sequencing of a peptide fragment of the pp3 protein revealed a sequence identical to a small heat shock-related protein (HSP20) recently isolated from rat and human skeletal muscle (16). Using highly specific antibodies to HSP20, in situ and in vitro phosphorylation studies of HSP20 show that the two 20-kDa phosphoproteins are phosphorylated forms of HSP20 and that HSP20 is a substrate for both PKA and PKG. These data indicate that there is a correlation between the ability of an intact vascular smooth muscle to undergo relaxation and an increase in the extent of the phosphorylation of a small heat shock-related protein, HSP20.
The catalytic subunit of PKA and endoproteinase
Lys-C were purchased from Promega (Madison, WI). The
[32P]orthophosphate and [-32P]ATP were
from Amersham Corp. The cAMP-dependent protein kinase inhibitor peptide was from Peninsula Labs (Belmont, CA). Serotonin and
protein A-Sepharose beads were from Sigma. Forskolin, leupeptin, and
aprotinin were from Calbiochem. Electrophoresis reagents and the DC
protein assay kit were from Bio-Rad. Rabbit anti-HSP20 antibody was
produced against purified HSP20 as described previously (16). Rabbit
anti-
B-crystallin was from Chemicon (Temecula, CA), and mouse
anti-HSP27 was a generous gift from Dr. Michael Welsh (University of
Michigan, Ann Arbor, MI). All other reagents were of analytical
grade.
Intact bovine carotid arteries were obtained from an abattoir. The adventitia was dissected free, and the endothelial layer was gently denuded. The arteries were opened longitudinally, and thin transverse strips were cut. The contractile viability of the vessels was confirmed by parallel muscle-bath experiments as described previously (17).
Whole Cell PhosphorylationStrips of bovine carotid artery smooth muscle were equilibrated in a bicarbonate buffer (120 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) bubbled with 95% O2, 5% CO2 for 1 h at 37 °C. The strips were then rinsed and incubated in a low phosphate buffer consisting of 10 mM Hepes, pH 7.4, 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 0.3 mM NaH2PO4 for 15-30 min. 250 µCi/ml [32P]orthophosphate was added 3 h before the addition of a vasorelaxant (10 µM forskolin or 10 µM sodium nitroprusside). The incubation was terminated by immersing the muscle strips in a dry ice/acetone slurry and then crushing the tissue with mortar and pestle under liquid N2. The powder was resuspended in homogenization buffer (20 mM Hepes, pH 7.4, 20 mM sucrose, 100 mM NaF, 15 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1.3% SDS) and boiled for 5 min. Protein concentrations were normalized using the Bio-Rad DC protein assay kit.
Two-dimensional Gel ElectrophoresisTwo-dimensional electrophoresis was performed using vertical slab isoelectric focusing gels with the modification described by Hochstrasser et al. (18). Briefly, the proteins in the samples were acetone-precipitated and reconstituted in 9 M urea and 2% CHAPS. The samples were protein-normalized, and 100 µg of protein was adjusted to a final concentration of 9 M urea, 2% CHAPS, 100 mM dithiothreitol, 15% glycerol, and 5% Ampholine (5 parts pH 6-8, 3 parts pH 5-7, 2 parts pH 3-10). The first dimensions were focused for 10,000 V-h and then run on a 12% SDS-PAGE second dimension (19). The gels were stained with Coomassie Brilliant Blue, and the dried gels were exposed to Kodak XAR-5 film.
For peptide sequencing, the second dimension gels were transferred to Immobilon-PSQ (Millipore, Bedford, MA) and stained with Ponceau Red to visualize the spots for isolation and sequencing. Eight to ten gels were transferred, and the pp3 protein was submitted to the Microchemical Facility, Winship Cancer Center, Emory University for amino acid sequencing. The protein was digested using endoproteinase Lys-C and separated by reversed phase microbore HPLC, and selected fragments were sequenced.
ImmunoblottingSecond dimension PAGE gels were transferred
to Immobilon-P (Millipore) and blocked with 5% milk in TBS (10 mM Tris, 150 mM NaCl, pH 7.4) for 1 h. The
blots were incubated with rabbit anti-HSP20 antibody (1:20,000), rabbit
anti-B-crystallin (1:2,000), or mouse anti-HSP27 (1:4,000) for
3 h at 4 °C. The blots were then washed in TBS, 0.5% Tween 20 (3 washes of 5 min each). Immunoreactive spots were detected using
horseradish peroxidase-conjugated goat anti-rabbit or donkey anti-mouse
for 1 h at 25 °C, and after washing (8 washes of TBS, 0.5%
Tween 20, 5 min each), Western blot chemiluminescence reagent was
applied (Dupont NEN), and the blots were exposed to Kodak XAR-5
film.
Peptide mapping was performed according to the method of Cleveland et al. (20). The spots corresponding to the 20-kDa proteins pp3 and pp8 were cut from two-dimensional gels and rehydrated with 125 mM Tris, pH 6.8, 0.1% SDS, for 1 h. The rehydrated gel pieces were placed in the wells of a 15% SDS-PAGE gel and overlaid with 10 mg of Staphylococcus aureus V8 protease in 125 mM Tris HCl, pH 6.8, 0.1% SDS, and 15% glycerol. The gel was run at 150 V until the dye front reached the end of the gel. The gels were dehydrated in graded methanol (to 100% methanol), dried, and exposed to Kodak XAR-5 film.
ImmunoprecipitationStrips of bovine carotid artery smooth muscle were homogenized in TBS (0.5 g of tissue/ml of buffer), and then the samples were centrifuged at 10,000 × g for 15 min. The soluble proteins were then diluted 10-fold with TBS. The anti-HSP20 antiserum was added to the supernatants (1:100 dilution). The samples were shaken gently for 14 h at 4 °C. Protein A-Sepharose beads (0.1 volume) were added, and the samples were incubated for an additional 3 h at 4 °C. The beads were washed 3 times with TBS, 0.5% Tween 20. A final wash of 10 mM Tris, pH 7.4, was then done. The immunoprecipitated samples were phosphorylated in vitro (see below) or reconstituted in 9 M urea, 2% CHAPS, 100 mM dithiothreitol, 15% glycerol, and 5% Ampholine (5 parts pH 6-8, 3 parts pH 5-7, 2 parts pH 3-10) and separated by two-dimensional mini-gel electrophoresis.
In Vitro PhosphorylationImmunoprecipitated proteins were
phosphorylated in vitro in a 200-ml reaction mixture
containing 20 mM Tris, pH 7.4, 10 mM magnesium
acetate, 5 mM K2PO4, 5 mM EDTA, 2 mM 2-mercaptoethanol, and 50 µM isobutylmethylxanthine. In addition, 100 nM PKG, 2 µM cGMP, 1 µM protein
kinase inhibitor peptide or 15 units (40 nM) of the
catalytic subunit of PKA, or buffer alone (control) was added to the
reaction mixture. The reactions were initiated with the addition of 200 µM [-32P]ATP (800 cpm/pmol), incubated
for 10 min at 30 °C, terminated by the addition of SDS (1.3% final
concentration), and boiled for 10 min.
Densitometric analysis of the phosphoproteins was performed using a CCD camera interfaced with a two-dimensional analyzer software package (Inovision Software, Bioimage Corp., Ann Arbor, MI). The results are depicted as the mean ± S.E.. Statistical analysis was by one-way analysis of variance followed by the Tukey test, and p < 0.05 was considered significant.
Treatment of strips of bovine carotid artery
smooth muscle with either the adenylate cyclase activator forskolin (10 µM) or the guanylate cyclase activator sodium
nitroprusside (10 µM) resulted in a significant increase
in the phosphorylation of two 20-kDa proteins with pI values of 5.7 and
5.9 (previously designated pp8 and pp3, respectively (Fig.
1)). Sodium nitroprusside elicited a 2.7-fold increase
in pp8 phosphorylation and a 1.8-fold increase in pp3 phosphorylation
(Table I). Forskolin stimulated a 5.2-fold increase in
pp8 phosphorylation and a 3.0-fold increase in pp3 phosphorylation
(Table I). Also, as described previously (14) the phosphorylation of a
third 20-kDa protein with a pI of 6.0 (designated pp4) decreased
following treatment with either forskolin or sodium nitroprusside (Fig.
1). The increases in the phosphorylation of pp3 and pp8 are the major
phosphorylation changes observed during cyclic
nucleotide-dependent vasorelaxation of carotid artery smooth muscle as determined with whole cell phosphorylation and two-dimensional gel electrophoresis (14).
|
To assess the relationship of proteins pp3 and pp8, we performed
S. aureus V8 limited proteolysis (20) of pp3 and pp8
phosphorylated in response to either forskolin or sodium nitroprusside.
Digests of the two 20-kDa proteins gave similar phosphopeptide maps
(Fig. 2). These data suggested that the pp3 and pp8
proteins are structurally related and are phosphorylated within closely
related peptide sequences in response to both forskolin and sodium
nitroprusside.
Identification of 20-kDa Phosphoproteins
To identify the
20-kDa proteins, the pp3 phosphoprotein was isolated from
preparative two-dimensional gels. A Lys-C peptide digest was
resolved on HPLC, and a single major peptide peak was isolated and
submitted for sequencing. Amino acid analysis of the Lys-C peptide
fragment revealed a sequence of HFSPEEIAVK. This sequence contained an
80% sequence identity with the small heat shock protein
B-crystallin (HFSPEELKVK) and was completely identical to another
protein recently purified from skeletal muscle (HSP20 (16)).
To confirm that HSP20 was phosphorylated during cyclic
nucleotide-dependent relaxation, immunoblots with
anti-HSP20 antibodies were performed. Intact strips of bovine carotid
artery smooth muscle were labeled with
[32P]orthophosphate and then stimulated with 10 µM forskolin for 10 min. The strips were homogenized, and
the 10,000 × g supernatant proteins were separated by
two-dimensional gel electrophoresis. The proteins were transferred to
Immobilon, and an autoradiograph was developed. The membranes were
subsequently probed with rabbit anti-HSP20 antibodies. Both pp8 and
pp3, which demonstrated increases in phosphorylation with
cAMP-dependent vasorelaxation, were immunoreactive with
anti-HSP20 antibodies (Fig. 3). A more basic
non-phosphorylated protein was also immunoreactive with anti-HSP20
antibody. The non-phosphorylated immunoreactivity comigrated with
purified rat HSP20 (data not shown). Immunoreactivity for the
non-phosphorylated protein decreased with forskolin stimulation. In
addition, the pp4 protein was recognized by the HSP20 antiserum and,
similar to pp4 phosphorylation, immunoreactivity decreased with
stimulation by forskolin. Antibodies against closely related
B-crystallin and another small heat shock protein (HSP27)
did not cross-react with any of the HSP20 immunoreactive proteins (data
not shown).
Finally, to confirm the identities of pp3 and pp8 intact strips of
bovine carotid arteries were stimulated with 10 µM
forskolin for 10 min, and muscle proteins were immunoprecipitated with
anti-HSP20 antibodies (Fig. 4). The anti-HSP antibodies
immunoprecipitated two proteins with similar molecular masses and
isoelectric points to the pp3 and pp8 proteins phosphorylated in
response to forskolin stimulation (Fig. 4).
In Vitro Phosphorylation of HSP20
The phosphorylation of
HSP20 increased under conditions where intracellular cAMP and/or cGMP
concentrations were elevated. Increases in cAMP and cGMP are thought to
effect cellular responses via the activation of PKA and PKG,
respectively (1, 2). To determine if PKA and PKG phosphorylate HSP20
in vitro, HSP20 was immunoprecipitated from cytosolic
protein fractions of carotid artery smooth muscle and phosphorylated
in vitro with purified PKG or the catalytic subunit of PKA
(Fig. 5). HSP20 was phosphorylated in vitro
by both PKG and PKA and produced two-dimensional patterns similar to
the in situ phosphorylation observed after sodium
nitroprusside or forskolin treatment of intact strips of carotid
artery. In vitro phosphorylation with PKA also variably led
to an additional minor, more acidic isoform. No phosphorylation of
HSP20 was observed in immunoprecipitated fractions in which PKG or PKA
was not added. Thus, while another small HSP (B-crystallin) has been
shown to contain "autokinase" activity (21), these data suggest
that HSP20 does not co-immunoprecipitate with autokinase activity.
Cyclic nucleotide-dependent vasorelaxation is associated with an increase in the phosphorylation of two 20-kDa phosphoproteins, pp8 and pp3 (14, 15). These two phosphoproteins share partial sequence identity and are immunoreactive with a recently identified small heat shock-related protein (HSP20). They appear to represent phosphorylated forms of the same protein. The phosphorylation of HSP20 increases with cyclic nucleotide-dependent vasorelaxation under physiologic Ca2+ conditions and under conditions where the intracellular Ca2+ is low and fixed (12, 14). However, the phosphorylation of HSP20 does not increase in a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle (15). These data suggest that the phosphorylation of HSP20 is an important event in cyclic nucleotide-dependent relaxation of vascular smooth muscle.
The phosphopeptide maps of the two isoforms of HSP20 are similar for muscles that were stimulated to relax with either forskolin or sodium nitroprusside. These results indicate that HSP20 is phosphorylated on similar sites by both PKA and PKG. HSP20 can also be phosphorylated in vitro by the purified cyclic nucleotide-dependent protein kinases PKA and PKG. The in vitro phosphorylation of HSP20 by PKA and PKG leads to phosphoproteins of similar mobility on two-dimensional gels, again suggesting that HSP20 represents a common substrate for both PKA and PKG. Indeed, the protein sequence for HSP20 (16) contains a consensus sequence (RRAS) for both PKA and PKG phosphorylation.
An additional 20-kDa phosphoprotein (pI 6.0) previously referred to as phosphoprotein 4 (pp4 (14)) was also immunoreactive with anti-HSP20 antibodies. However, the phosphorylation of this protein increases with stimuli that induce contraction of the vascular smooth muscle and decreases with stimuli that induce relaxation (14). Treatment of carotid artery smooth muscle with phorbol esters also elicits an increase in pp4 phosphorylation (14). Thus, pp4 may represent a population of HSP20 that is phosphorylated by protein kinase C or another unidentified kinase. Alternatively, it is possible that pp4 represents a different protein that shares homology with HSP20.
Heat shock proteins represent a family of phylogenetically well
conserved proteins whose expression is induced by cellular stress (for
review, see Ref. 22). Many HSPs (including HSP20) are also expressed
constitutively and thus appear to play a role in normal cellular
behavior. B-crystallin, HSP27, and HSP20 are all members of the low
molecular weight HSP family ("small HSPs"). The small HSPs share
considerable sequence homology and often copurify in large
macromolecular aggregates (16, 23). HSP20 was originally identified as
a by-product of the purification of HSP27 (16). While HSP20 is
ubiquitously distributed, it is found in much higher levels in
skeletal, smooth, and heart muscle (16). The prevalence of HSP20 in
muscle tissue supports a role for HSP20 in contractile physiology.
Unlike
B-crystallin and HSP27, the amount of HSP20 does not increase
after heat shock in rat skeletal muscle (16). However, HSP20 does
redistribute from a cytosolic to an insoluble fraction and dissociates
from an aggregated form after cellular stress (16). Thus, HSP20 does share some of the functional properties of the other small HSPs.
While the precise functions of the HSPs are not known, many HSPs act as "molecular chaperones" and assist in the assembly, disassembly, stabilization, and internal transport of intracellular proteins. Recent studies suggest that small HSPs are important regulatory components of the actin-based cytoskeleton (24), and phosphorylation of HSP27 has been implicated in regulating the contraction of rectal sphincter smooth muscle (25). Other investigations have suggested that small HSPs interact with intermediate filaments (26), which in turn may play a regulatory role in vascular smooth muscle contraction and relaxation (27).
Although the specific role that the phosphorylation of HSP20 plays in
vasorelaxation is not known, the phosphorylation of HSP20 may alter its
ability to associate either with components of either the actin-myosin
contractile domain or with the intermediate filament domain of smooth
muscle cells (28, 29). The localization of another small HSP
(B-crystallin) with the Z band of cardiac muscle along with the
evidence that the dense bodies are the structural counterpart of the Z
band in smooth muscle raises the possibility that small HSPs act at the
level of the dense bodies (30). Since the dense bodies are the sites at
which both the actin-myosin contractile filament and the intermediate
filament domains are anchored, cyclic nucleotide-dependent
relaxation may lead to a simultaneous reorganization of each fibrillar
domain and release the muscle from the so-called "latch" state
(31).
We thank Shannon Lamb for technical assistance.