The Small Heat Shock-related Protein, HSP20, Is Phosphorylated on Serine 16 during Cyclic Nucleotide-dependent Relaxation*

The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser16 modulates cAMP-dependent vasorelaxation.

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, HSP20 1 (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 Ser 16 is the site phosphorylated on HSP20 by PKA or PKG. We then prepared peptides in which the Ser 16 site was altered and determined whether these peptides altered the contractile responses of transiently permeabilized vascular smooth muscle.

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). [␥-32 P]ATP and [ 32 P]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 * This work was supported in part by a Veterans Affairs Merit Review award and National Institutes of Health Grant RO1 HL58027-01 (to C. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  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 Situ Phosphorylation of HSP20 -Strips of bovine carotid artery smooth muscle were incubated in 150 Ci/ml [ 32 P]orthophosphate in 10 mM Hepes (pH 7.4), 150 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO 4 , 1.0 mM NaH 2 PO 4 , 10 mM glucose, 1.5 mM CaCl 2 , and 25 mM Na 2 HCO 3 and oxygenated with with 95% O 2 , 5% CO 2 at 37°C for 4 h. The muscle strips were then stimulated with serotonin (1 M) for 10 min and then IBMX (1 mM) and forskolin (10 M) for 10 min, phorbol dibutyrate (1 nM-1 M) for 45 min, or phorbol dibutyrate (1 M) for 45 min followed by forskolin (10 M) 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 ϫ 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.
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 g 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 l/min. with the Smart System (Pharmacia Biotech, Uppsala, Sweden). 50-l 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 accord-ing 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 g 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 l 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(ϩ) cloning vector and transformed into Escherichia coli supercompetent cells according to the manufacturer's directions (pCR-Script SK(ϩ) 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 ϫ 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 125 I-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 Na 2 B 4 O 7 , 75 mM NaCl (pH 8.5)), and 100 l 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 antirabbit 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 ϫ 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)(16)(17)(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 Data Analysis-Values are reported as mean Ϯ 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).

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 Ser 16 on the HSP20 molecule. The fragment from peak 2 had a less suitable PKA consensus sequence (RAPS) corresponding to Ser 59 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.
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 sig-naling pathways, strips of bovine carotid artery smooth muscle were incubated with [ 32 P]orthophosphate and stimulated with forskolin (10 M) 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 Ser 16 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 Ser 16 representing the phosphorylation site. The proteolytic fragment from fractions 27/28 had a sequence of LPPGVD-PAAVTSALSPEG (100% homology with human HSP20), cor-

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 ϫ 10 Ϫ3 ). 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).
responding to the carboxyl terminus of the HSP20 molecule, and contained no consensus sites for PKA or PKG (Fig. 3).
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 [ 32 P]orthophosphate and stimulated with the phorbol ester, phorbol dibutyrate (PDBu, 1 M, 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 RYRLP-PGVPPAAVTSAL (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).
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.
To determine the sensitivity of the phosphorylation statespecific 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 g of phosphorylated HSP20 in a linear fashion (Fig. 6).
To determine the specificity of the phosphorylation state- 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.

FIG. 2.
In situ phosphorylation of HSP20 by forskolin. Radiolabeled strips of vascular smooth muscle were treated with the adenylate cyclase activator, forskolin (10 M), and the phosphodiesterase inhibitor, isobutylmethylxanthine (1 mM, for 10 min) (A), or with phorbol dibutyrate (1 M, 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 ϫ 10 Ϫ3 ). 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 LPPGVDPAAVTSAL-SPEG. 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. specific antibodies for HSP20 in intact tissues, homogenates of bovine carotid artery smooth muscles (30 g of protein) were treated with buffer alone (control), sodium nitroprusside (10 M, 10 min), or forskolin (10 M, 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).
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 M, 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 RRAS 16 site (19).

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 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 g 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 .   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).
cyclic nucleotide-dependent vasorelaxation, strips of human aortic smooth muscle were labeled with [ 32 P]orthophosphate and treated with IBMX (1 mM) and forskolin (10 M). 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.
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, WLRRAS p PLPGLK, in which Ser 16 was phosphorylated, significantly attenuated both KCl (110 mM)-and serotonin (5HT, 1 M)-induced contractions (Fig.  10). In addition, the synthetic peptide, WLRRAAPLPGLK, in 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 g 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 .   FIG. 7. Specificity of the phosphorylation state-specific antibodies. Bovine carotid artery smooth muscle was treated with buffer alone (control, C), sodium nitroprusside (10 M, 10 min, S), or forskolin (10 M, 10 min, F). The strips were homogenized, and 30 g 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).
FIG. 8. Immunoreactive small heat shock proteins. Strips of carotid artery smooth muscle were incubated in the presence of [ 32 P]orthophosphate and treated with phorbol dibutyrate (100 nM, for 45 min) followed by forskolin (10 M, 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.
FIG. 9. HSP20 isoforms in human aorta. Strips of human aortic vascular smooth muscle were labeled with [ 32 P]orthophosphate, treated with 3-isobutyl-1-methylxanthine (1 mM) and forskolin (10 M, 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.
which Ser 16 was replaced with an alanine, thus rendering the peptide "nonphosphorylatable" augmented both KCl (110 mM)and serotonin (5HT, 1 M)-induced contractions (Fig. 10). The synthetic peptides, WLRRASPLPGLK, in which Ser 16 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 M)-induced contractions (p Ͼ 0.05 compared with control (no peptide added) contractions).
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 Ϯ 214 base line versus 3379 Ϯ 315, n ϭ 3, p Ͼ 0.05) was observed. However, a larger peptide, EIPVPVQPSWLRRASAPLPGLK, was phosphorylated in vitro by PKA (1258 Ϯ 153 versus 4580 Ϯ 408, n ϭ 3, p Ͻ 0.05).

DISCUSSION
The present experiments demonstrate that the small heat shock-related protein, HSP20 is phosphorylated on Ser 16 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 Ser 16 during cAMP-dependent vasorelaxation. Antibodies generated against a peptide containing the Ser 16 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 RRAS 16 site (19). Taken together, these data suggest that there are species differences in HSP20 isoform expression and that Ser 16 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 twodimensional 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 Ser 16 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 Ser 16 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 mol-  16 was replaced with an alanine (n ϭ 5, * p Ͻ 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 M)-induced contractions (p Ͼ 0.05 compared with control, data not shown). ecule, 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 in-teraction 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 Ser 16 . Phosphorylation of HSP20 at Ser 16 is not only associated with cyclic nucleotide-dependent vasorelaxation but also inhibits agonist-induced contractile responses.