THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Smooth Muscle Calponin

Vol. 265, No. 17, Issue of June 15, pp. 1014%10155,199O Printed in U.S.A.

INHIBITION OF ACTOMYOSIN MgATPase AND REGULATION BY PHOSPHORYLATION*

(Received for publication, September 25, 1989)

Steven J. Winder+ and Michael P. Walsh8 From the Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Calponin isolated from chicken gizzard smooth mus- cle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system com- posed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phos- phorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phos- phorylation was unaffected. Furthermore, calponin in- hibited the actin-activated MgATPase of fully phos- phorylated or thiophosphorylated myosin. Although calponin is a Ca’+-binding protein, inhibition did not require Ca”. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not de- pendent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca*+/ calmodulin-dependent protein kinase II, but not by CAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein re- tained calmodulin and tropomyosin binding capabili- ties, but actin binding was greatly reduced. The cal- ponin-actin interaction, therefore, appears to be re- sponsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be in- volved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Cal- ponin function in turn is regulated by Ca’+-dependent phosphorylation.

Primary regulation of contraction in smooth muscle in- volves phosphorylation of the 20,000-dalton light chains of myosin by Ca’+/calmodulin-dependent myosin light chain kinase (1, 2). It is becoming increasingly clear, however, that other regulatory systems, having both direct and indirect calcium dependence, may have a role to play in the regulation of smooth muscle contraction (3). These mechanisms of reg- ulation include caldesmon/calmodulin (4,5), the calcium- and phospholipid-dependent protein kinase (protein kinase C) (6), and perhaps the direct binding of Ca2+ to myosin (7, 8). Recently, another smooth muscle protein has been described which may function to regulate the contractile state of the muscle (9, 10). This protein was named calponin for calcium-

* This work was support.ed by a grant from the Alberta Heart and Stroke Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

i A recinient of an Alberta Heritage Foundation for Medical Re- search Fellowship.

s A recipient of a Medical Research Council of Canada Scientist Award and Alberta Heritage Foundation for Medical Research Schol- arship. To whom all correspondence should be addressed.

and calmodulin-binding troponin T-like protein. Calponin, which has been purified from chicken gizzard (9)

and bovine aorta (lo), is a heat-stable, basic, 34-kDa protein which interacts with F-actin and tropomyosin in a Ca’+- independent manner and with calmodulin in a Ca’+-depend- ent manner. It is present in smooth muscle at the same molar concentration as tropomyosin (9). Electron microscopy sup- ports the idea that calponin is a bona fide thin filament protein: electron microscopy of smooth muscle tropomyosin paracrystals indicated that calponin binds to a site 16-17 nm from the C terminus of tropomyosin with 40 nm periodicity, i.e. identical to the binding pattern of skeletal muscle troponin T (11). The thin filament-bound form of calponin is degraded 500 times more slowly by calpain than is the free form of calponin, suggesting a very close association between calponin and the thin filament similar to the association of troponin T with the skeletal muscle thin filament (12). Calponin is clearly a distinct protein from caldesmon and myosin light chain kinase (lo), but it is antigenically related to the C-terminal half of rabbit skeletal and bovine cardiac troponin T (13). Recently, however, Lehman (14) has suggested that calponin may be a cytoskeletal or nuclear matrix protein rather than a thin filament component (see “Discussion”).

During characterization of native thin filaments prepared from chicken gizzard smooth muscle, we observed a 32-kDa protein in addition to actin, tropomyosin, and caldesmon (15). This protein was identified as calponin (13), a conclusion which we have confirmed using specific polyclonal antibodies to isolated gizzard calponin. The properties, including the binding to actin and tropomyosin, suggested that calponin may function in the regulation of smooth muscle contraction. To investigate this possibility, we carried out preliminary studies of the effects of purified calponin on the actin-acti- vated myosin MgATPase in vitro using purified smooth mus- cle proteins: actin, myosin, tropomyosin, calmodulin and my- osin light chain kinase (16). In the presence of 6 PM actin, 2 pM tropomyosin, and 1 FM myosin, calponin (2 pM) inhibited the actin-activated myosin MgATPase by 78%.

We report here further characterization of the inhibitory activity of calponin on the actin-activated myosin MgATPase of smooth muscle and demonstrate that phosphorylation of calponin reverses this inhibitory effect. Data presented indi- cate that the inhibitory action of calponin is due to its ability to bind actin which is lost upon phosphorylation.

EXPERIMENTAL PROCEDURES

Materiak-lIr-3’PIATP (20-40 Ci/mmol) was purchased from Amersham (Oykville, Ontaiio, Canada). Sephadex- G-75 was pur- chased from Pharmacia (Mississauga, Ontario, Canada) and CM- Sephadex, protein A-Sepharose, ribonuclease A, and chymotrypsin- ogen A from Sigma. Dithiothreitol and ATPrS’ were purchased from

1 The abbreviations used are: ATPrS, adenosine 5’-O-(3-thiotri- phosphate); EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- resis.

10148

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Boehringer Mannheim (Dorval, Quebec, Canada) and electrophoresis reagents from Bio-Rad. General laboratory reagents used were of analytical grade or better and were purchased from Fisher Scientific (Calgary, Alberta, Canada).

Protein Purifications-Calmodulin was purified from frozen bovine brains by a modification of the procedure of Gopalakrishna and Anderson (17) as described in detail by Walsh et al. (18). The following proteins were purified by previously described methods: chicken gizzard actin (19), tropomyosin (20), myosin (21) and myosin light chain kinase (22), and the bovine cardiac catalytic subunit of type II CAMP-dependent protein kinase (23). Chicken gizzard caldes- mon containing endogenous Ca’+/calmodulin-dependent kinase ac- tivity, and rat brain protein kinase C were purified as previously described (24,25) and generously provided by Drs. Gisele Scott-Woo and Janice Parente, respectively, of this laboratory. Cyclic GMP- dependent protein kinase (bovine lung) was generously provided by Dr. Tom Lincoln, University of South Alabama and Ca*+/calmodulin- dependent protein kinase II (bovine brain) by Dr. R. K. Sharma, University of Calgary. Calponin was purified as described by Taka- hashi et al. (9) with the modifications that CM-Sephadex was used for the ion-exchange chromatography step and Sephadex G-75 was used in place of Ultrogel AcA 44 for the gel filtration step.

Actin-actiuated Myosin MgATPase Assay-ATPase activities were measured as described previously (26) under the following conditions: 25 mM Tris-HCl (pH 7.5), 10 mM MgC12, 60 mM KCl, 0.1 mM CaCl* or 1 mM EGTA, 1 mM [r-“*P]ATP (-10 cpm/pmol), 1 pM myosin, 6 FM actin, 2 FM tropomyosin, 1 pM calmodulin, 74 nM myosin light chain kinase in reaction volumes of 1.0 ml at 30 “C. Other additions are indicated in the figure legends. Reactions were started by addition of ATP. For the determination of ATPase rates, aliquots (0.1 ml) of reaction mixtures were withdrawn at l-min intervals following the addition of ATP up to 9 min. Rates were calculated by linear regres- sion analysis of the time-course data. In one case, ATPase rates were determined at various actin and tropomyosin concentrations, main- taining a 3:l molar ratio of actin:tropomyosin, under otherwise stand- ard conditions.

Myosin Phosphorylation Assay-Levels of myosin phosphorylation were quantified on the same samples used for actomyosin ATPase measurements as described previously (27). Wherever necessary, the remainder of each reaction mixture was added to an equal volume of SDS gel sample buffer and boiled. Samples (50 ~1) were subjected to SDS-PAGE and autoradiography.

Superprecipitation Assay-Reaction conditions were exactly as de- scribed for the actomyosin ATPase assay with the exception that nonradiolabeled ATP was used. AGeO nm was recorded in an LKB Ultrospec.

Phosphorylation of Calponin-Phosphorylation by Ca’+/calmodu- lin-dependent protein kinase II was carried out under the following optimal conditions (24): 20 mM Tris-HCl (pH 7.5), 5 mM MgC12, 0.1 mM CaC12, 1 mM [r-“‘PIATP, 5.9 pM calponin, 4.6 pM calmodulin, 0.2 mg/ml caldesmon containing endogenous kinase activity or 5 pg/ ml bovine brain Ca’+/calmodulin-dependent protein kinase II at 30 “C. Samples (0.1 ml) of reaction mixtures were withdrawn at selected times for quantification of protein-bound [32P]phosphate. Wherever necessary, the remainder of each reaction mixture was added to an equal volume of SDS gel sample buffer and boiled prior to SDS-PAGE and autoradiography. Phosphorylation by protein kinase C was carried out under the following optimal conditions using the liposomal assay (28): 20 mM Tris-HCl (pH 7.5), 5 mM MgC12, 0.1 mM CaCl*, 40 fig/ml L-cu-phosphatidyl-L-serine, 0.8 pg/ml 1,3-diolein, 0.1 mM [y-“*P]ATP (-100 cpm/pmol), 5.9 pM calponin, and 4 kg/ml rat brain protein kinase C at 30 “C. In experiments designed to determine the effects of these phosphorylated forms of calponin on the actomyosin ATPase, reactions were quenched by addition of EGTA (8-9 mM), and phosphorylated calponins were purified by DEAE-Sephacel ion-exchange chromatography. Calponin did not bind to the resin whereas calmodulin, Ca’+/calmodulin-dependent protein kinase II and protein kinase C bound.

Phosphopeptide Mapping-Limit peptides of phosphorylated forms of calponin were generated by trypsin digestion and analysed by two- dimensional peptide mapping as described by Colburn et al. (29).

Phosphoamino Acid Analysis-Identification of phosphorylated amino acid residues was determined by one-dimensional thin layer electrophoresis (pH 3.5) as described previously (30).

Sedimentation Assay-Actin (11 PM) and calponin (4 FM) were incubated with or without tropomyosin (2.6 pM) at 25 “C for 30 min with gentle mixing in 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 2 mM MgCl,, 1 mM ATP, 1 mM dithiothreitol, 0.1 mM CaClz (9). Samples

(0.2 ml) were then centrifuged at 109,000 X g in a Beckman TL 100 centrifuge for 1 h at 2 “C in order to sediment actin and proteins bound to actin. Pellets and supernatants were subjected to SDS- PAGE. Sedimentation assays performed using the whole reconsti- tuted in vitro smooth muscle system were carried out in the presence of 2 pM phosphorylated or unphosphorylated calponin and nonradi- olabeled ATP as described for the MgATPase assay above. Following incubation at 30 “C for 7 min the samples were centrifuged at 109,000 x g in a Beckman TL 100 centrifuge for 1 h at 2 “C and pellets and supernatants were subjected to SDS-PAGE.

Immunoprecipitation of Calponin-Polyclonal antibodies to chicken gizzard calponin (which had been cut out of SDS-polyacryl- amide gels) were raised in rabbits using standard procedures. The IgG fraction of anti-calponin was purified as described previously (31). Calponin (0.25 mg) and anti-calponin (1.25 mg) were incubated for 4 h at 22 “C in a reaction volume of 0.455 ml. Protein A-Sepharose (0.5 ml) was added and centrifuged in a bench-top Eppendorf centri- fuge for 2 min. The effect of the supernatant on actomyosin ATPase activity was measured under standard conditions. A calponin concen- tration of 3.9 pM would be present in the ATPase reaction mixture if the protein was not immunoprecipitated. Two control immunoprecip- itation reactions were also carried out. In one case calponin was omitted (buffer control) and in the other anti-calponin was omitted (antibody control).

Other Procedures-The following enzymatic activities were assayed using published methods: CAMP-dependent protein kinase (23), cGMP-dependent protein kinase (32), myosin light chain kinase (27), protein kinase C (28), and Ca’+/calmodulin-dependent protein kinase II (24). Protein concentrations were determined by the Coomassie Brilliant Blue dye-binding assay (33) using dye reagent purchased from Pierce Chemical Co. or by spectrophotometric measurements, using the following absorption coefficients: calmodulin e$i., = 1.9 (34) ,m yosin & -= “In 4.5 (35), tropomyosin cRnrn = 2.9 (36), caldes- mon c&Z,, = 3.3 (37). and caluonin &., = 11.3. The absorption coefficient of calponinwas determined by recording the protein’s UV absorption spectrum after dialysis overnight against two changes (5 liters each) of 20 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol. The spectrum was recorded in a Beckman DU-8B UV-visible spectropho- tometer. The calponin preparation was determined to be 97.6% pure by scanning laser densitometry following SDS-PAGE. The calponin concentration was determined, by amino acid analysis (38), to be 0.49 mg/ml. Electrophoresis was performed in 7.5-20% polyacrylamide gradient slab gels (1.5 mm thick) with a 5% acrylamide stacking gel in the presence of 0.1% SDS at 36 mA using the discontinuous buffer svstem of Laemmli (39). Gels were stained in 45% (v/v) ethanol. 10% (v/v) acetic acid containing 0.14% (w/v) Coomassie Brilliant Blue R- 250 and diffusion destained in 10% (v/v) acetic acid. Destained gels were sealed in plastic bags and autoradiographed, if necessary, using Kodak X-Omat AR film in DuPont-Cronex cassettes fitted with DuPont Quanta III intensifying screens. Films were allowed to de- velop for 3 days or less at room temperature. Densitometry of de- stained gels was carried out in an LKB model 2202 Ultroscan laser densitometer equipped with a Hewlett-Packard model 3390A integra- tor.

RESULTS

Inhibition of Actomyosin ATPase by Calponin-We have shown previously that calponin has an inhibitory effect on smooth muscle actomyosin MgATPase activity (16). This effect was examined in greater detail using a reconstituted system comprising the purified contractile and regulatory proteins of chicken gizzard smooth muscle: myosin, actin, tropomyosin, calmodulin, and myosin light chain kinase. Fig. 1 shows that calponin produced a concentration-dependent inhibition of actomyosin ATPase with maximal (79%) inhi- bition being reached at concentrations of calponin >2 pM.

Inhibition was shown to be due to calponin since inhibitory activity could be removed by immunoprecipitation with spe- cific polyclonal antibodies to calponin (Table I). The inhibi- tory effect of calponin is not a nonspecific effect due to its basic nature (p1 = 8.4-9.1 (13)). Two other basic proteins, ribonuclease A (p1 = 9.6) and chymotrypsinogen (p1 = 9.5), exhibited no inhibitory effects on the actomyosin MgATPase, even at concentrations as high as 10 PM: ATPase rates of

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Smooth Muscle Calponin

,i,,,,, 0 1 2 3 4 5 s 7 B 8 10 CALPONIN +M)

FIG. 1. Inhibition of the actin-activated myosin MgATPase by calponin. Actomyosin ATPase rates were measured as described under “Experimental Procedures” at the indicated concentrations of calponin in the presence (0) and absence (W) of Ca*+. Myosin phos- phorylation levels were quantified on the same samples as described under “Experimental Procedures” in the presence (0) and absence (0) of Ca*+. Myosin phosphorylation data are presented as mean + SE. of 5-10 observations at each calponin concentration, and in the case of MgATPase rates data are the mean k S.E. of four to six observations, except at 7.5 and 10 pM calponin where these were three and two observations, respectively.

TABLE I

Zmmunoprecipitation of calponin and actomyosin ATPuse inhibitory activity

Immunoprecipitation was carried out as described under “Experi- mental Procedures” prior to assay of actomyosin ATPase.

Assay conditions Actomvosin ATPase nmol P,/mg myosin. nin

Actomyosin” - Ca*+ 6.6 Actomyosin + Caz+ 141.7 Actomyosin + Ca*+ + supernatantb 125.0 Actomyosin + Ca2+ + buffer control’ 133.5 Actomyosin + Ca*+ + antibody controk’ 37.6 ’ Actomyosin refers to the standard actomyosin ATPase reaction

system. b Supernatant refers to the supernatant remaining after immuno-

precipitation of calponin. ‘Buffer control refers to the supernatant from a control immuno-

precipitation reaction from which the antigen (calponin) was omitted. dAntibody control refers to the supernetant from a control im-

munoprecipitation reaction from which the antibody (anti-calponin) was omitted.

122.2 and 120.0 nmol of Pi/mg myosin.min were observed in the presence of 2 and 10 JLM ribonuclease A, respectively, and of 118.8 and 114.9 nmol of P,/mg myosin. min in the presence of 2 and 10 PM chymotrypsinogen, respectively. The control ATPase rate in these experiments was 120.7 nmol of PJmg myosin f min, which was reduced to 26.9 nmol Pi/mg myosin ’ min in the presence of 2 PM calponin. Calponin also inhibited superprecipitation of actomyosin in the reconstituted system (data not shown). On the other hand, myosin light chain phosphorylation was unaffected (Fig. l), even at calponin concentrations as high as 17.5 pM, in which case the phos- phorylation level was determined to be 1.82 2 0.05 mol of Pi/ mol myosin (n = 9). SDS-PAGE followed by autoradiography verified that specific phosphorylation of the 20,000-dalton myosin light chain occurred in all cases (data not shown).

These results indicate that inhibition of the actomyosin ATPase is not due to inhibition of myosin phosphorylation as a consequence, for example, of removal of calmodulin by binding to calponin. The mechanism of inhibition was further investigated by examining the effect of calponin on the actin- activated MgATPase activity of prephosphorylated myosin (Fig. 2). Myosin was first. phosphorylated in the presence of myosin light chain kinase and Ca’+-calmodulin to a level of 1.7 mol of Pi/m01 myosin. Actin was then added in order to activate the myosin MgATPase and the incubation continued in the presence or absence of calponin. In a third experiment, calponin was added 5 min after the addition of actin rather than with actin. As expected, the MgATPase of phosphoryl- ated myosin was strongly activated by actin, to 181.7 nmol of Pi/mg myosin + min (Fig. 2, 0); in a separate experiment the ATPase rate of unphosphorylated myosin in the presence of actin was determined to be only 1.1 nmol of Pi/mg myosin. min. Calponin added with actin caused a 78% decrease in the rate of ATP hydrolysis, to 39.7 nmol of Pi/mg myosin.min (Fig. 2, A), i.e. comparable to the inhibition seen in Fig. 1 where ATP hydrolysis and myosin phosphorylation occurred concomitantly. Calponin is capable not only of inhibiting actin activation of the MgATPase of prephosphorylated my- osin, but also inhibits the actin-activated MgATPase when added to the fully activated system: addition of calponin 5 min after actin resulted in 96% inhibition of the maximal rate of ATP hydrolysis, i.e. from 173 to 7 nmol of Pi/mg myosin. min (Fig. 2, A).

Inhibition of the actomyosin MgATPase by calponin is reversible by increasing the concentration of actin and tro- pomyosin. Under standard conditions, 2 PM calponin resulted in 74% inhibition (Fig. 3). This inhibition was progressively lost as the actin and tropomyosin concentrations were raised (maintaining a 3:l molar ratio of actin:tropomyosin) until, at 60 pM actin and 20 PM tropomyosin, inhibition almost com- pletely disappeared. On the other hand, if the calponin con- centration was also increased (e.g. 20 PM calponin, 20 FM

1

1 2 3 4 5 6 7 8 9 TIME (mid

FIG. 2. Inhibition of the actin-activated myosin MgATPase by calponin following prior phospborylation of myosin. Myo- sin was maximally phosphorylated by incubation for 8 min under the ATPase assay conditions described under “Experimental Procedures” with the exception that actin was omitted (tropomyosin was in- cluded). The following additions were then made simultaneously: 6 pM actin (0, A) and 6 pM actin + 5 pM calponin (A). Samples of reaction mixtures were withdrawn at the indicated times following actin addition for quantification of ATP hydrolysis. In one case (A) calponin (5 pM) was added 5 min after the addition of actin.

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I I I I I I I I I I

0 2 4 6 8 10

MOLAR RATIO TROPOMYOSIN:CALPONIN

FIG. 3. Reversal of calponin inhibition of the actomyosin MgATPase by actin/tropomyosin. Actomyosin MgATPase rates were measured as described under “Experimental Procedures” in the presence and absence of 2 pM calponin and at the indicated concen- trations of tropomyosin. The actin concentration was also varied so as to maintain a 3:l molar ratio of actin:tropomyosin. Control ATPase rates were 114 nmol of PJmg myosin.min in the presence of Ca2+ and 7 nmol of P,/mg myosin. min in the absence of Ca*+.

TABLE II Calponin inhibition of the actin-activated myosin MgATPase does not

require Ca” Myosin was prephosphorylated as described in the legend to Fig. 3

prior to the addition of 6 pM actin with or without 5 pM calponin in the presence of 0.1 mM CaCl* or 1 mM EGTA. ATPases and myosin phosphorylation levels were quantified as described under “Experi- mental Procedures.”

Additions at t = 0 ATPase rate t = 1 min t = 9 min

Actin Calmnin C2’

nmol P,/mg myosin. min mol PJmol my&n

+ - + 114.7 1.71 1.64 + - - 91.4 1.86 1.23 + + + 40.4 1.89 1.78 + + - 25.5 1.72 1.32

tropomyosin, 60 pM actin), 63.6% inhibition of the MgATPase was observed (data not shown).

Having established the inhibitory effect of calponin on the actomyosin ATPase in the presence of Ca*‘, we investigated the possibility that calponin inhibition of the ATPase may be calcium-dependent. This was prompted by the demonstration by Takahashi et al. (40) using UV difference spectroscopy that calponin binds Ca2+ with an affinity in the micromolar range. We have also found that calponin immobilized on nitrocellulose binds Ca2+ when incubated with 45CaC12 as described by Maruyama et al. (41) (data not shown). Previ- ously (15), we had not detected Ca” binding to calponin in thin filament preparations using this technique in which 0.3 pg of calponin was transblotted. However, a clear signal was observed after transblotting 10 kg of purified calponin.

Myosin was prephosphorylated as before prior to addition of actin with or without calponin in the presence or absence of Ca*+ (Table II). Inhibition by calponin was observed both in the presence of Ca*+ (65% inhibition) and in the absence of Ca*+ (78% inhibition). The lower ATPase rates observed in the absence of Ca*+ in each case were due to a low level of dephosphorylation of myosin by Ca’+-independent phospha- tase activity once Ca2+ was removed from the assay system (Table II). However, to overcome this problem of dephospho-

rylation during reactions in the absence of Ca2+, we repeated the experiments of Table II using thiophosphorylated myosin which is resistant to the action of myosin phosphatase (42). Experimental conditions were exactly as described in Table II except that ATPyS replaced ATP in the prephosphoryla- tion step. ATPase rates were then measured after addition of ATP and actin in the presence and absence of calponin and in the presence and absence of Ca’+. In the presence of Ca*‘, calponin inhibited the ATPase rate by 63.1% and in the absence of Ca2+ by 63.6%. We conclude, therefore, that the inhibitory effect of calponin on the actin-activated myosin MgATPase is Ca2+-independent.

Phosphorylation of Culponin-Calponin inhibition of the actomyosin ATPase is not, therefore, regulated directly by Ca*+ or by Ca”-calmodulin (calponin binds calmodulin in a Ca*+-dependent manner (9)). We considered the possibility, however, that calponin may be regulated by phosphorylation. Calponin was found to be phosphorylated by protein kinase C (to 1.0 mol of PJmol), by Ca*+/calmodulin-dependent pro- tein kinase II (to 1.0 mol of Pi/mol) and by a Ca*+/calmodulin- dependent protein kinase copurifying with smooth muscle caldesmon (to 1.9 mol of PJmol), but not by CAMP- or cGMP- dependent protein kinases or myosin light chain kinase (~0.01 mol of PJmol). In each case, specific phosphorylation of calponin was verified by SDS-PAGE and autoradiography (data not shown). We previously published evidence suggest- ing that the Ca’+/calmodulin-dependent protein kinase activ- ity copurifying with caldesmon actually resides within the caldesmon molecule itself (24). However, this conclusion is not consistent with the recently deduced amono acid sequence of caldesmon (43). The properties of this kinase most closely resemble those of Ca’+/calmodulin-dependent protein kinase II (44). We, therefore, examined the site specificity of phos- phorylation of calponin by this kinase activity and by purified bovine brain Ca*+/calmodulin-dependent protein kinase II using two-dimensional phosphopeptide mapping of limit tryp- tic peptides (Fig. 4). Identical phosphopeptide maps were obtained (compare Fig. 4, A and B), so we conclude that the kinase activity copurifying with caldesmon is a smooth muscle form of Ca2’/calmodulin-dependent protein kinase II. Phos- phopeptide mapping of calponin phosphorylated by protein kinase C revealed a simpler pattern of phosphopeptides (Fig. 4C), only three major phosphopeptides being detected. If tryptic digests of calponin phosphorylated by protein kinase C and Ca”/calmodulin-dependent protein kinase II were combined prior to two-dimensional phosphopeptide mapping, the three phosphopeptides (labeled 1-3) comigrated (data not shown) suggesting that the two major sites of phosphorylation by Ca*+/calmodulin-dependent protein kinase II (sites 1 and 2) are also phosphorylated by protein kinase C. Site 3 which is strongly phosphorylated by protein kinase C is poorly phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Additional sites of phosphorylation by Ca2+/calmodulin- dependent protein kinase II are apparent (Fig. 4, A and B), but are minor relative to sites 1 and 2.

Thin layer electrophoresis of acid hydrolysates of phos- phorylated calponin followed by autoradiography indicated that Ca*+/calmodulin-dependent protein kinase II and protein kinase C phosphorylated both serine and threonine residues. Neither kinase incorporated radiolabeled phosphate onto ty- rosine residues.

The effect of phosphorylation of calponin on its ability to inhibit the actomyosin MgATPase was then investigated. Calponin was phosphorylated to 1.11 mol of Pi/m01 calponin by Ca’+/calmodulin-dependent protein kinase II and to 1.20 mol of Pi/m01 calponin by protein kinase C. The effects of

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10152 Smooth Muscle Calponin

c

,3 t-‘;

y, * O*-TLE

0 0 FIG. 4. Peptide mapping of phosphorylated forms of cal-

ponin. Approximately 1 rg of each limit tryptic digest of phosphoryl- ated calponin was subjected to two-dimensional phosphopeptide map- ping as described by Colburn et al. (29). Thin layer electrophoresis (TLE) was performed from anode @ to cathode 8 followed by ascend- ing thin layer chromatography (TLC). Autoradiographs presented are: A, calponin phosphorylated by Ca’+/calmodulin-dependent pro- tein kinase II (1.0 mol of P,/mol calponin); B, calponin phosphoryl- ated by caldesmon with copurifying kinase activity (1.9 mol of P,/mol calponin); C, calponin phosphorylated by protein kinase C (1.0 mol of P,/mol calponin). 0, represents the origin and 1, 2, and 3 represent the major phosphopeptides referred to in the text.

FIG. 5. Inhibition of the actomyosin MgATPase by calponin is blocked by phosphorylation. ATPase rates were measured under standard conditions in the presence of the indicated concen- trations of unphosphorylated calponin (0) or calponin phosphoryl- ated by either Ca’+/calmodulin-dependent protein kinase II (0) or protein kinase C (A).

varidus concentrations of phosphorylated and unphosphory- lated calponins on the actomyosin ATPase were examined (Fig. 5). At concentrations as high as 5 FM, calponin phos- phorylated by Ca’+/calmodulin-dependent protein kinase II or protein kinase C had little or no effect on the actin- activated myosin MgATPase when compared with unphos- phorylated calponin. Partial inhibition (from 117 to 89 nmol of Pi/mg myosin. min) was observed in other experiments in the presence of 8 j.tM phosphorylated calponin. The same concentration of unphosphorylated calponin reduced the ATPase rate to 6 nmol of Pi/mg myosin.min. Therefore,

calponin loses its ability to inhibit the actomyosin ATPase upon phosphorylation.

Although regulation of actomyosin MgATPase by calponin is not mediated directly by calcium, but via calcium-dependent kinases, the possibility exists that calponin could be acting in a troponin-like manner by immobilizing tropomyosin on the actin filament, i.e. not permitting tropomyosin to move rela- tive to the actin filament in such a way as to permit normal actin-myosin interaction. In the presence of actin (7 PM) and presence or absence of 1 pM tropomyosin, calponin produced a concentration-dependent inhibition of the actin-activated myosin MgATPase (Fig. 6A) reaching maximum (70-80%) inhibition in both cases at or above ‘2 pM calponin. This effect can be seen more clearly in Fig. 6B where the data have been normalized in order to compensate for the lower ATPase rates seen in the absence of tropomyosin. Calponin inhibition of the actin-activated MgATPase of myosin is not, therefore, dependent on the presence of tropomyosin.

It is known from the work of Takahashi et al. (9, 13) that calponin binds to calmodulin, tropomyosin, and actin. Ex- amination of the effects of phosphorylation on these binding properties of calponin may help to shed light on the mecha- nism whereby calponin inhibits the actin-activated myosin MgATPase. We found that phosphorylation of calponin did not affect its interaction with either calmodulin-Sepharose or tropomyosin-Sepharose (data not shown) but, as shown in Fig. 7A, did affect its binding to actin. Unphosphorylated calponin bound to actin and to actin-tropomyosin as deter- mined in a sedimentation assay (Fig. 7A, lanes 1 and 2, 5 and 6). Upon phosphorylation by either protein kinase C or Ca*+/ calmodulin-dependent protein kinase II, most of the calponin did not bind to actin or actin-tropomyosin (Fig. 7A, lanes 3 and 4, 7 and 8). Similarly in sedimentation experiments performed in the presence of actin, tropomyosin, myosin, calmodulin and myosin light chain kinase, i.e. the MgATPase assay system, unphosphorylated calponin bound to actin-

0. 0 2 4 6 8

CALPONIN (PM)

FIG. 6. Inhibition of actin-activated myosin MgATPase by calponin in the presence and absence of tropomyosin. Acto- myosin ATPase rates were measured as described under “Experimen - tal Procedures” at the indicated concentrations of calponin in the presence of actin (7 FM) and in the presence (0, n ) or absence (0) of 1 pM tropomyosin and in the presence (0,O) or absence (m) of Ca’+. A, data presented as ATPase rate in nanomoles of Pi/mg myosin. min. B, data normalized by expressing ATPase rate in the presence or absence of tropomyosin as a percentage of the maximum.

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Smooth Muscle Calponin 10153

SPSPSPSP A.

- -- --.-- 0-A

1 2345618

SPSPSP a c - .. w-M

a- -

b- -

‘- ‘.--A - - - -Tm

d-v -- - -

-Cap

e- w 7 - f-r - -

- -LCzo - -Lc,l

1234561

FIG. 7. Binding of phosphorylated and unphosphorylated calponin to actin, actin-tropomyosin and the reconstituted actomyosin system. A, actin and phosphorylated or unphosphory- lated calponin were sedimented at high speed in the presence and absence of tropomyosin as described under “Experimental Proce- dures.” Supernatants (S) and pellets (P) were analyzed by SDS- PAGE. Lanes 1 and 2, actin + unphosphorylated calponin; lanes 3 and 4, actin + phosphorylated calponin; lanes 5 and 6, actin + tropomyosin + unphosphorylated calponin; lanes 7 and 8, actin + tropomyosin + phosphorylated calponin. In this experiment, calponin was phosphorylated to 1.0 mol of Pi/mol calponin by Ca’+/calmodu- lin-dependent protein kinase II. Identical results were obtained if calponin was phosphorylated to 1.0 mol of Pi/mol calponin by protein kinase C. In separate experiments, calponin alone (phosphorylated or unphosphorylated) did not sediment under these conditions. B, phosphorylated or unphosphorylated calponin (2 pM) was sedimented at high speed in the presence of actin (6 j.~cM), myosin (1 PM), tropomyosin (2 PM), myosin light chain kinase (74 nti) and calmod- ulin (1 pM) as described under “Experimental Procedures.” Super- natants (S) and pellets (P) were analyzed by SDS-PAGE. Lane 1, molecular weight markers (a, phosphorylase b,-97,400, b, bovine serum albumin, 66,200; c, ovalbumin, 45,000; d, carbonic anhydrase, 29,000; e, soybean trypsin inhibitor, 20,100, /, lysozyme, 14,400); lanes 2 and 3, myosin, actin, tropomyosin, myosin light chain kinase, and cal- modulin alone; lanes 4 and 5, as lanes 2 and 3 but plus unphosphor- ylated calponin; lanes 6 and 7, as lanes 2 and 3 but plus phosphoryl- ated calponin. In this experiment calponin was phosphorylated to 2.3 mol of Pi/m01 calponin by Ca*+/calmodulin-dependent protein kinase II. M, myosin; A, actin; Tm, tropomyosin; Cap, calponin; L&, 20- kDa light chain of myosin; LC17, 17-kDa light chain of myosin.

tropomyosin (Fig. 7B, lanes 4 and 5) and was recovered predominantly in the pellet along with myosin. Phosphoryl- ated calponin clearly does not bind to actin-tropomyosin and was recovered in the supernatant along with some unsedi- mented actin (Fig. 7B, lanes 6 and 7). Quantitative data (Table III) were obtained by laser densitometry of the gels shown in Fig. 7.

DISCUSSION

In spite of being a major protein component of smooth muscle, calponin was only recently reported (9). Its properties, as determined by Takahashi and coworkers (g-13), suggest that it is a bona fide thin filament protein, although this has recently been challenged by Lehman (14) who suggested that a 35-kDa protein identified as calponin may be part of the insoluble cytoskeleton or possibly a component of the nuclear matrix. This suggestion was based on two observations: first, thin filaments immunoprecipitated with anti-caldesmon did

TABLE III Dissociation of calponin from actin induced by phosphorylation

A, actin; Cap, calponin; P-Cap, phosphorylated calponin; Tm, trooomvosin.

Conditions Calponin”

Supernatant Pellet

% A. A + CaP 2.5 97.5

A + P-Cap 70.8 29.2

A + Tm + CaP 10.0 90.0 A + Tm + P-Cap 81.1 18.9

B. ATPase system + CaP 24.7 75.3 ATPase system + P-Cap 100.0 0.0

’ Results were obtained by laser densitometry of the gels shown in Fig. 7, A and B.

not contain calponin and, secondly, gizzard “ghost” cells from which actin and myosin were extracted were enriched in the 35-kDa protein. However, the results of the immunoprecipi- tation experiments suggest that calponin may have been completely proteolyzed, a reasonable possibility given the prolonged incubation at 25 “C and its known susceptibility to proteolysis. Furthermore, if comparable amounts of the his- tones in washed muscle and ghost cells were loaded on the gels in Fig. 6, a and b, of Ref. 14, it would be apparent that the amount of calponin retained in the ghost cells is small relative to that extracted with actin and myosin. The weight of evidence, therefore, favors calponin as a thin filament- associated protein which, as shown by Takahashi et al. (lo), is specific to smooth muscle and is, therefore, of interest as a potential regulator of smooth muscle contraction. This pos- sibility is supported by the in vitro binding properties of calponin: it interacts with F-actin and tropomyosin in the presence or absence of Ca*+, and with calmodulin only in the presence of Ca*+ (9, 13). We initially encountered this protein in preparations of native thin filaments from chicken gizzard (15); it is clear that the 32-kDa protein observed in such preparations is identical to calponin (13).* Consequently, we have investigated the effects of purified calponin on the actin- activated myosin MgATPase in an in vitro system recon- structed from purified smooth muscle contractile and regula- tory proteins: actin, myosin, tropomyosin, calmodulin, and myosin light chain kinase.

Calponin inhibits the actomyosin ATPase maximally (-80%) when present in the assay system at a concentration equimolar to tropomyosin. Takahashi et al. (9) have shown that the molar concentrations of calponin and tropomyosin in chicken gizzard smooth muscle are equal. The inhibitory effect of calponin on the actomyosin ATPase is apparently due to its interaction with actin and is clearly not due to inhibition of myosin phosphorylation. Furthermore, although calponin can bind Ca*+ directly (40), its ability to inhibit the actomyosin ATPase is not Ca*+-dependent. This observation prompted us to search for an alternative mechanism of regu- lating calponin function. Phosphorylation was an obvious one to investigate. We found that calponin could be phosphoryl- ated by protein kinase C and Ca*+/calmodulin-dependent protein kinase II, but not by CAMP- or cGMP-dependent protein kinases or by myosin light chain kinase. Most inter- estingly, phosphorylation by either protein kinase C or Ca”+/ calmodulin-dependent protein kinase II abolished inhibition of actomyosin ATPase activity by calponin. Phosphorylation also blocked the actin-calponin interaction. However, phos- phorylation did not affect the interaction of calponin with

’ S. J. Winder and M. P. Walsh, unpublished observations.

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10154 Smooth Muscle Calponin

immobilized tropomyosin or calmodulin. In separate experi- ments (data not shown) we have found that calponin, whether phosphorylated or not, does not bind to an affinity column of smooth muscle myosin (phosphorylated or unphosphorylated) coupled to Sepharose 4B.

The electron microscopic observations made by Takahashi et al. (11) indicate that calponin is distributed regularly along tropomyosin paracrystals with ~40 nm periodicity and is located 16-17 nm from the C terminus of each tropomyosin molecule. This is remarkably similar to the distribution of troponin T along striated muscle thin filaments (45). Fur- thermore, calponin and troponin T exhibit immunological cross-reactivity (13). We have also observed that our poly- clonal antibodies to chicken gizzard calponin cross-react with striated muscle troponin T.2 Functionally, however, calponin resembles troponin I in that it is capable of inhibiting the MgATPase activity of desensitized actomyosin in the pres- ence or absence of tropomyosin. However, unlike troponin I with which maximal inhibition is observed at one troponin 1:one actin monomer in the absence of tropomyosin and at one troponin 1:three to four actin monomers in the presence of tropomyosin (46), maximal inhibition of calponin occurs at approximately one calponin:three actin monomers in both the presence and absence of tropomyosin. Since calponin binds to tropomyosin and inhibits the actomyosin ATPase, it may resemble the troponin T-troponin I complex of striated mus- cles. Further studies will be required to determine if calmod- ulin in smooth muscle plays a role analogous to troponin C in striated muscles through its Ca’+-dependent interaction with calponin. However, calponin phosphorylation (see below) may provide an alternative mechanism of regulation of calponin function.

While calponin binds to immobilized tropomyosin, it is the interaction with actin that confers its inhibitory properties. Our observations that phosphorylated calponin binds to im- mobilized tropomyosin but not to reconstituted thin filaments in a sedimentation assay suggest that calponin may not, in fact, interact with tropomyosin on the thin filament. However, we cannot rule out the possibility that calponin may bind to both actin and tropomyosin on the thin filament and, whereas phosphorylation prevents the actin interaction, it weakens the tropomyosin interaction. We may suggest, therefore, that calponin is bound to actin, and possibly also tropomyosin, when the sarcoplasmic free Ca2+ concentration is low, as in the resting smooth muscle cell. Stimulation of the cell, with activation of protein kinase C and/or Ca’+/calmodulin-de- pendent protein kinase II, leads to phosphorylation of cal- ponin preventing its interaction with actin, and thus facili- tating its release from the thin filament allowing actin to activate the MgATPase of phosphorylated myosin.

The functional relationship between calponin and caldes- mon is now a matter of great interest. It is clear that they are not related through proteolysis, for example, and the tissue content of calponin (9), equal to that of tropomyosin (150 FM) (47), is significantly higher than that of caldesmon (11 pM) (48), so it is unlikely that they function as components of a complex analogous to the troponin complex of striated muscles. The two proteins do, however, exhibit several simi- larities. Both proteins (i) inhibit the actomyosin ATPase, (ii) bind F-actin and tropomyosin in a Ca’+-independent manner and calmodulin in a Ca*+-dependent manner, (iii) are sub- strates of protein kinase C and Ca’+/calmodulin-dependent protein kinase II, and (iv) lose their ability to inhibit the actomyosin ATPase upon phosphorylation. Several investi- gators have suggested that caldesmon may be involved in latch-bridge formation (4,49-51), i.e. the formation of slowly

cycling or noncycling cross-bridges which are responsible for force maintenance at low levels of ATP consumption and in the presence of intermediate Ca2+ concentrations (52-56). A similar argument could be made for calponin but neither is, as yet, entirely satisfactory.

A great deal of evidence now exists supporting the central role of myosin phosphorylation in the regulation of smooth muscle contraction (3). It is, however, clear that at least one secondary or modulatory mechanism exists which can alter the contractile state of a smooth muscle cell or its response to extracellular signals. Some such mechanisms also utilize Ca2+ as the second messenger. Possibilities which involve Ca*+ include caldesmon (4,5), protein kinase C (6), and direct binding of Ca” to myosin (7, 8). Calponin may be added to this list.

Acknowledgments-We are grateful to Drs. Janice Parente, Gisele Scott-Woo, and R. K. Sharma of the University of Calgary for supplies of protein kinase C, caldesmon containing endogenous kinase activity, and Ca*+/calmodulin-dependent protein kinase II, respec- tively; to Dr. Tom Lincoln, University of South Alabama for cGMP- dependent protein kinase; to Dr. John Kendrick-Jones, Medical Re- search Council Laboratorv of Molecular Bioloav. Cambridee. United Kingdom for his helpful comments and suggestions during prepara- tion of this manuscript; to Elaine Fraser for help with antibody production; to Cindy Sutherland for purified smooth muscle myosin and myosin light chain kinase; and to Chris Horgan, Marla Farmer, and Gerry Garnett for word processing.

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S J Winder and M P Walshphosphorylation.

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by

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