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. Author manuscript; available in PMC: 2005 Nov 23.
Published in final edited form as: J Biol Chem. 1993 Jul 15;268(20):15305–15311.

Reversal of Caldesmon Binding to Myosin with Calcium-Calmodulin or by Phosphorylating Caldesmon*

Mark E Hemric *,‡, Frank W M Lu *, Richard Shrager *,§, Julie Carey *, Joseph M Chalovich *,
PMCID: PMC1289261  NIHMSID: NIHMS3601  PMID: 8325900

Abstract

Caldesmon, an actin-binding protein from smooth muscle and non-muscle cells, has previously been shown to bind stoichiometrically to smooth muscle myosin in an ATP-dependent manner. We now show quantitatively the effects of Ca2+-calmodulin and phosphorylation on the binding of caldesmon to myosin. Ca2+-calmodulin reduces the binding of caldesmon to myosin with the same effectiveness as it does the binding of caldesmon to actin. However, Ca2+-calmodulin is ineffective in antagonizing the binding of the purified myosin-binding region of caldesmon to myosin. These and other results suggest that Ca2+-calmodulin binding to the COOH-terminal region of caldesmon is responsible for reversal of binding to myosin. Phosphorylation of the NH2-terminal region of caldesmon by the co-purifying kinase, calmodulin-dependent protein kinase II, weakens but does not eliminate the binding of caldesmon to smooth muscle myosin. Finally, phosphorylation of smooth muscle myosin by smooth muscle myosin light chain kinase has no effect on the binding of caldesmon to myosin. Since Ca2+-calmodulin and phosphorylation of caldesmon weaken the binding of caldesmon to both actin and myosin, these events may be coordinately regulated.

Caldesmon inhibits the superprecipitation of actomyosin (Sobue et al., 1981), and activation of myosin-dependent ATP hydrolysis by actin (Dabrowska et al., 1985; Smith and Marston, 1985; Ngai and Walsh, 1985; Sobue et al., 1985; Lash et al., 1986). This activity, which resides in the COOH-terminal region of caldesmon (Fujii et al., 1987; Szpacenko and Dabrowska, 1986; Yazawa et al., 1987) is largely the focal point of caldesmon research. The addition of caldesmon or the actin binding polypeptide fragment of caldesmon to either skeletal (Brenner et al., 1991; Chalovich et al., 1991) or smooth (Pfitzer et al., 1992; Katsuyama et al., 1992) muscle inhibits force production.

The NH2-terminal region of caldesmon is also interesting because it binds to myosin (Hemric and Chalovich, 1988; Ikebe and Reardon, 1988; Sutherland and Walsh, 1989; Katayama et al., 1989; Marston et al., 1992); a bacterially expressed COOH-terminal fragment of caldesmon has also been reported to bind to myosin (Yamakita et al., 1992). The binding of caldesmon to myosin is about 20-fold stronger in the presence of ATP (Hemric and Chalovich, 1990). Caldesmon interacts primarily with myosin subfragment-2 (Hemric and Chalovich, 1988, 1990; Ikebe and Reardon, 1988), although weaker binding also occurs to myosin subfragment-1 (S-1)1 (Hemric and Chalovich, 1988, 1990). Several studies have indicated that, under some conditions, caldesmon can actually enhance contractile activity (Hemric and Haeberle, 1992; Walker et al., 1989; Hegmann et al., 1991; Lin et al., 1991). This enhancement occurs when the actin filament has low levels of bound caldesmon, so that the binding of myosin to actin is not greatly inhibited. The caldesmon, in effect, increases the binding of myosin to actin as a result of the simultaneous binding of caldesmon to both actin and to myosin (Lash et al., 1986; Hemric and Chalovich, 1988).

Earlier studies have shown that both Ca2+-calmodulin (Ikebe and Reardon, 1988) and caldesmon phosphorylation (Sutherland and Walsh, 1989) alter the caldesmon-myosin interaction. However, these studies did not quantitate the effect of these modifications, nor did they localize the site of the effects. We now show that Ca2+-calmodulin binding to the COOH region of caldesmon reverses the binding of myosin to the amino-terminal region of caldesmon. Phosphorylation of sites within the amino-terminal region of caldesmon weakens the binding of caldesmon to myosin, and the effect is graded with the number of phosphates incorporated into that region of caldesmon.

MATERIALS AND METHODS

Proteins

Smooth muscle myosin was prepared from chicken gizzards as described by Persechini and Hartshorne (1983) except that 0.1% Triton X-100 was included in the first wash and myosin was extracted with 40 mm imidazole-HCl (pH 7.2), 8 mm ATP, 60 mm KCl, 1 mm EDTA, 100 mg/liter streptomycin sulfate, and 0.5 mm dithiothreitol. Caldesmon was isolated from turkey gizzards by a modification of the method of Bretscher (1984) described previously (Velaz et al., 1989). Calmodulin was purified from porcine brains by the method of Yazawa et al. 1980. Smooth muscle myosin light chain kinase was purified by the method of Walsh et al. 1983. Phosphorylation of smooth muscle myosin was performed as described by Adelstein and Klee (1982), and quantitation of light chain phosphorylation was done by scanning isoelectric focusing gels (Wells and Bagshaw, 1984). The [14C]iodoacetamide-labeled caldesmon was prepared as previously described (Velaz et al., 1989) but without cellulose phosphate chromatography. The myosin-binding fragment of caldesmon was produced as previously described (Velaz et al., 1990) with some modifications. Caldesmon was cleaved with a 1:600 weight ratio of chymotrypsin for 5 min at 25 °C in a buffer composed of 0.1 m KCl, 20 mm Tris-HCl (pH 8), 1 mm EDTA, and 1 mm dithiothreitol. The digest was applied to a 1.5 × 95-cm Spectra/Gel AcA 54 gel filtration column equilibrated with digestion buffer brought to 1 m KCl. The myosin- and several actin-binding fragments in the 20–30-kDa molecular mass range were contained in the second peak. These myosin- and actin-binding fragments were subsequently dialyzed against 60 mm NaCl, 10 mm Tris-Mes (pH 7), 1 mm dithiothreitol and applied to a Waters Protein-Pak SP R (1 × 10 cm) cation exchange HPLC column equilibrated with dialysis buffer (Chalovich et al., 1992). The fragments were eluted with a gradient of NaCl to 500 mm. A larger amino-terminal caldesmon fragment was prepared by digestion of caldesmon with submaxillary Arg protease (1:500, w/w) for 90 min (Mornet et al., 1988). This fragment, which migrates at 90 kDa on SDS gels, was purified by gel filtration on Spectra/Gel AcA 54 followed by HPLC ion exchange chromatography on a Pharmacia Mono Q column.

Protein concentrations were determined by absorbance at 280 nm except for caldesmon and caldesmon fragments, whose concentrations were determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. The molecular weights used for calculations of protein concentrations are: smooth muscle myosin, 532,000 (Yanagisawa et al., 1987); calmodulin, 16,500; caldesmon, 87,000 (Bryan et al., 1989); and chymotryptic caldesmon myosin-binding fragment, 19,050 (Velaz et al., 1990).

Caldesmon Phosphorylation

Caldesmon and the co-purifying calmodulin-dependent protein kinase II were purified from turkey gizzards as previously described (Velaz et al., 1989) except, prior to heat treatment, extracted caldesmon was applied to a Cibricon Blue agarose (Bio-Rad) equilibrated with 0.5 m KCl, 25 mm Tris-HCl (pH 8), 1 mm EGTA, 1 mm EDTA, and 1 mm dithiothreitol. The caldesmon-kinase complex was eluted when the equilibration buffer was raised to 1 m KCl. Caldesmon was phosphorylated in 0.1 m NaCl, 20 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mm dithiothreitol, 0.125 mm CaCl2, 0.6 μm calmodulin, and 1 mm [32P]ATP (1 mCi) at 25 °C for 90 min and then boiled for 5 min to terminate phosphorylation and inactivate any phosphatases. The phosphorylation was dependent on the presence of Ca2+-calmodulin. Under conditions giving 1.2 mol of Pi/mol of caldesmon, in the presence of Ca2+-calmodulin, only 0.18 mol of Pi/mol of caldesmon was observed in the presence of EGTA and absence of added calmodulin. Phosphorylated caldesmon was dialyzed and applied to Sepharose G-50 gel filtration chromatography for final purification.

Affinity Chromatography

Smooth muscle myosin and calmodulin were covalently linked to CNBr-activated Sepharose 4B (10 mg of protein/ml of resin) according to the manufacturer’s directions (Pharmacia LKB Biotechnology Inc.). Unreacted groups on the column were blocked with ethanolamine to minimize the introduction of charged groups.

Electrophoresis and Autoradiography

SDS-polyacrylamide gels were run by the procedure of Laemmli (1970) and stained with Coomassie Blue. Dried gels were exposed to Kodak BB Blue film at −70 °C using Lightning Plus intensifying screens (Du Font-New England Nuclear).

Binding Studies

The binding of radioactively labeled caldesmon to smooth muscle myosin was determined by a low speed sedimentation assay described previously (Hemric and Chalovich, 1990). During modification, a fraction of caldesmon lost its ability to bind myosin and was determined to be 26% for [14C]iodoacetamide-labeled caldesmon and 22.5% for [32P]caldesmon. The fraction of protein that sedimented in the absence of smooth muscle myosin was 3% for [14C]iodoacetamide-labeled caldesmon and 4.5% for [32P]caldesmon. Finally, the fraction of smooth muscle myosin that did not sediment was 1%.

Mathematical Modeling

Two models of the binding of caldesmon with myosin and calmodulin were considered. In the first model, calmodulin binds to a single site on caldesmon (K2) which is located in the COOH-terminal region of caldesmon and which precludes myosin subfragment binding (K1). In the second model, calmodulin binds to a site at the NH2-terminal region of caldesmon (K2), which precludes myosin binding at K1, and to a second site at the COOH-terminal region (K3), which does not prevent the binding of myosin (see Table I). The observable reactants in the system are the bound forms of myosin (i.e. myosin with bound caldesmon with or without a maximum of 2 molecules of calmodulin). Theoretical values of the bound myosin with respect to changes in concentration of calmodulin (or caldesmon and calmodulin) were calculated using an iterative numerical procedure. Data fitting was done using the MATLAB mathematical modeling program (The Mathworks, Inc.).2

Table I. Estimated association constants for binding of caldesmon to both Ca2+-calmodulin and myosin.

Several data sets were analyzed by two models. Sets 1–4 were measured with varied caldesmon and calmodulin concentrations at a constant ratio of calmodulin to caldesmon. The molar ratios of calmodulin to caldesmon were 0, 0.25, 0.75, and 1.3 for sets 1 through 4. Set 6 was measured as the concentration of calmodulin was increased at constant caldesmon and myosin concentrations. Association constants have units of m−1 and are defined by the following scheme where C is caldesmon, M is myosin, and X is Ca2+-calmodulin, K2 and K3 are calmodulin binding to the competitive and noncompetitive sites, respectively, as shown below.

graphic file with name nihms3601f8.jpg

Sets fitteda K1 K2 K3
Single calmodulin binding site model
 1 5.1 × 105
 2, 3, 4, 6 1.5 × 105 2.1 × 105
 2, 6 2.0 × 105 2.6 × 105
 3, 4 9.6 × 104 4.9 × 105
Two calmodulin binding site model
 2, 3, 4, 6 1.5 × 105 2.8 × 105 3.8 × 104
 2, 6 1.9 × 105 3.9 × 105 7.3 × 104
 3, 4 9.6 × 104 4.9 × 105 1.1 × 100
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a

Conditions were the same as those described in Fig. 1.

RESULTS

The effect of calmodulin on the binding of caldesmon to myosin was determined by two experimental protocols. In the first, shown in Fig. 1A, the amount of caldesmon bound to myosin was measured as a function of the total added calmodulin concentration at saturating Ca2+. In this type of experiment, the amount of caldesmon bound to myosin decreased to less than 4% of the initial value at high Ca2+-calmodulin concentrations. In the second protocol, a series of curves were generated where the caldesmon bound was measured as a function of the caldesmon concentration at constant ratios of calmodulin to caldesmon. Thus, the calmodulin and caldesmon were increased simultaneously. An example of this type of experiment is shown in Fig. 1B. Both types of experiments were analyzed to two models of calmodulin binding (see “Materials and Methods” and Table I). The solid line shows the best fit to the simple competitive model in which a single Ca2+-calmodulin site is assumed. The broken line is the fit of the model with two calmodulin binding sites, one competitive and the other noncompetitive for myosin binding. Both models can be fitted well to the data and give almost indistinguishable curves. More complex models, which incorporated a cooperative interaction between the presumptive noncompetitive calmodulin binding site and myosin, did not improve the fit (not shown).

Fig. 1. Effect of calmodulin on the binding of caldesmon to smooth muscle myosin.

Fig. 1

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A, binding was measured in the presence of 10 μm smooth muscle myosin, 18 μm [14C]iodoacetamide-labeled caldesmon, and varied concentrations of calmodulin (set 6 of Table I). B, binding was measured with 10 μm myosin and varied concentrations of labeled caldesmon at a constant ratio of 0.25 mol of calmodulin/mol of caldesmon (set 2 of Table I). The conditions were: 1 mm ATP, 30 mm NaCl, 5 mm MgCl2, 10 mm imidazole-HCl (pH 7), 1 mm CaCl2, and 1 mm dithiothreitol, at 25 °C. Theoretical curves were drawn assuming one (solid line) or two (broken line) calmodulin binding sites on caldesmon.

Both models allow estimations to be made for the association constants of myosin to caldesmon (K1) calmodulin to the competitive site of caldesmon (K2), and calmodulin to the noncompetitive site of caldesmon (K3). Values obtained from fitting the different data sets are listed in Table I. In the single calmodulin binding site model, the affinity of caldesmon for both calmodulin and myosin is about the same (1–5 × 105 m−1). In this model caldesmon can bind to either myosin or calmodulin but not to both simultaneously. If, on the other hand, we assume that there are two calmodulin binding sites then the affinity of calmodulin to the site which is competitive for myosin binding (the NH2-site) is about 6-fold greater than for the site which is noncompetitive for myosin (the COOH-site) binding if we exclude data sets 3 and 4, which are not consistent with model 2.

It is not likely that binding of Ca2+-calmodulin is nearly as strong at the NH2-site as at the COOH-site. We observed that only small amounts of the NH2-terminal, chymotryptic, myosin-binding fragment of caldesmon bound to calmodulin-Sepharose affinity columns in the presence of calcium. In contrast, all of the actin-binding fragments in the 20-kDa molecular mass range bound to the column (data not shown). We confirmed this result using the larger NH2-terminal fragments prepared from a submaxillary arginine-C protease digestion of caldesmon. This fragment begins at the NH2 terminus and ends around residue 482, where the 35-kDa CNBr fragment begins (Mornet et al., 1988). We observed, as did Mornet et al. 1988, that this fragment binds very weakly, if at all, to a calmodulin affinity column (data not shown). Therefore, if the NH2-region binds to calmodulin, it does so with an affinity much less than that of the COOH-terminal fragments. In contrast to the prediction of model 2, it is likely that Ca2+-calmodulin binding to the COOH-region of caldesmon controls binding of myosin to the NH2-terminal region of caldesmon.

We also examined this question more directly by measuring the effect of calmodulin on the binding of the purified myosin-binding fragment of caldesmon to myosin. While the purified myosin-binding fragment bound to smooth muscle myosin, as shown in Fig. 2A, the binding was rather insensitive to calmodulin. Interestingly, however, if the NH2-terminal fragment was not separated from the other caldesmon fragments in the digest, its binding was reversed upon the addition of Ca2+-calmodulin as shown in Fig. 2B. A component of the caldesmon molecule distinct from the NH2-region is required for reversal by Ca2+-calmodulin, and it need not be covalently linked to the NH2-region. Adding the purified 35-kDa actin-and calmodulin-binding, COOH-terminal fragment to the purified myosin-binding fragment was not sufficient for restoring Ca2+-calmodulin sensitivity (data not shown).

Fig. 2. Effect of calmodulin on the binding of the myosin-binding chymotryptic fragment of caldesmon to smooth muscle myosin·ATP.

Fig. 2

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Binding was measured with 3.1 μm smooth muscle myosin and varied concentrations of both [14C]iodoacetamide-labeled caldesmon fragment and calmodulin. The conditions were the same as in Fig. 1. A, binding of purified myosin-binding fragment to smooth muscle myosin. The concentrations of fragment used were 0.77 μm (▴), 1.0 μm (•), and 3.7 μm (▪). B, binding of myosin-binding fragment, unpurified from the total caldesmon digest, to smooth muscle myosin. Two preparations of digested caldesmon are shown, 3 μm (▪) and 4 μm (○). Inset, an autoradiograph of a SDS-polyacrylamide gel showing the binding of unpurified myosin-binding fragment to smooth muscle myosin in the presence of increasing concentrations of calcium-calmodulin. Only the region of the gel containing the myosin-binding fragment (MBF) is shown for clarity. The samples shown are pellets of a mixture of 3.1 μm smooth muscle myosin and 1 μm digested 125I-labeled caldesmon in the absence (a) or presence of 7.5 μm (b), 15 μm (c), 30 μm (d), and 37.5 μm (e) calmodulin.

Phosphorylation of caldesmon by its co-purifying calmodulin-dependent protein kinase II has been shown to eliminate its binding to a smooth muscle myosin-Sepharose affinity column (Sutherland and Walsh, 1989). Fig. 3 shows a similar affinity column experiment using caldesmon phosphorylated with [32P]ATP. Both the absorbance and radioactivity of the effluent are shown. The first peak of phosphorylated caldesmon did not interact with the column even under low ionic strength conditions. This caldesmon contained an average of 3 mol of phosphate/mol of caldesmon. A second population of phosphorylated caldesmon was retained by the column and eluted at 0.4 m NaCl (unphosphorylated caldesmon elutes at 0.5 m NaCl). The caldesmon retained by the column contained an average of 2 mol of phosphate/mol of caldesmon. Therefore, the binding became very weak only with the incorporation of 3 mol of Pi/mol of caldesmon.

Fig. 3. Smooth muscle myosin-Sepharose affinity chromatography of phosphorylated caldesmon.

Fig. 3

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25 mg of phosphorylated caldesmon was loaded onto a 30-ml smooth muscle myosin-Sepharose column equilibrated with 30 mm NaCl, 5 mm MgCl2, 10 mm imidazole-HCl (pH 7), and 1 mm dithiothreitol at 4 °C. After washing with the buffer, a 150-ml gradient of 30–0.75 m NaCl in the same buffer was applied to elute bound phosphorylated caldesmon.

Direct binding of the phosphorylated caldesmon to smooth muscle myosin was measured in the absence of ATP and the results shown in Fig. 4. Under these conditions, the affinity of phosphorylated caldesmon for myosin is quite low and saturation of binding could not be achieved. However, a reasonable fit was obtained assuming that the stoichiometry of binding was unchanged by caldesmon phosphorylation (2 mol of caldesmon/mol of myosin). This is reasonable since the stoichiometry is apparently dependent only on the myosin conformation (Lu and Chalovich, 1993). The association constant was reduced to 34% (2 mol of Pi/mol of caldesmon) and 17% (3 mol of Pi/mol of caldesmon) compared to unphosphorylated caldesmon.

Fig. 4. Effect of caldesmon phosphorylation of the binding of caldesmon to smooth muscle myosin.

Fig. 4

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Binding was measured in the presence of 10 μm smooth muscle myosin and varied concentrations of caldesmon phosphorylated with [32P]ATP. The conditions were the same as in Fig. 1, except that ATP and CaCl2 were excluded. The theoretical curves are for a single class of binding sites with a stoichiometry of 2 caldesmon molecules/myosin molecule and association constants of 5.6 × 104 m−1, 1.7 × 104 m−1, and 8.3 × 103 m−1 for unphosphorylated caldesmon (▪) and caldesmon phosphorylated to 2 (•) and 3 (○) mol of phosphate/mol of caldesmon, respectively.

Higher levels of saturation of binding could be obtained in the presence of ATP where the binding of caldesmon to myosin is enhanced. Fig. 5 shows that incorporation of 1.4 mol of Pi reduces the association constant to 24%, whereas the presence of 3.1 mol of Pi reduces the association constant to 4% of the initial value. In both cases, the best fit to the data occurs with a stoichiometry within experimental error of that measured with zero phosphorylation (1:1). Thus, the association constant of caldesmon for myosin decreases with increasing levels of caldesmon phosphorylation.

Fig. 5. Effect of caldesmon phosphorylation on the binding of caldesmon to smooth muscle myosin·ATP.

Fig. 5

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Smooth muscle myosin was 2.5 μm for experiments with unphosphorylated caldesmon and 10 μm for experiments with phosphorylated caldesmon. The conditions were the same as in Fig. 1 except for the exclusion of 1 mm CaCl2. The theoretical curves were calculated for a single class of binding sites. Unphosphorylated caldesmon (dashed line) and caldesmon phosphorylated with [32P]ATP to 1.4 (•) and 3.1 (○) mol of phosphate/mol of caldesmon are shown. The association constants for the curves were 9.9 × 105 m−1, 2 × 105 m−1, and 4 × 104 m−1, respectively. The corresponding stoichiometries are 0.92, 1.2, and 1.0 mol of caldesmon/mol of myosin.

Even though calmodulin-dependent protein kinase II phosphorylates the NH2 terminus of caldesmon preferentially, some COOH-terminal sites are also phosphorylated. Ikebe and Reardon (1990) showed that with a total phosphorylation of 2.8, the distribution of 32Pi was 28.6% in Ser-26, 36.4% in Ser-73, 12.5% in Ser-726, and the rest in other sites. Thus, even with 3 total phosphates in caldesmon, the two NH2- terminal sites are not totally phosphorylated. To demonstrate that phosphorylation of the NH2-terminal sites of caldesmon is responsible for weakening of its binding to myosin, the binding of cleaved caldesmon to myosin was studied. Fig. 6 shows overloaded SDS-polyacrylamide gels of the supernatants and pellets formed after sedimenting cleaved 32Pi-labeled caldesmon with myosin. By comparing the Coomassie Blue-stained gels with the autoradiographs, it can be seen that the actin-binding fragments (ABF) were not significantly phosphorylated in this experiment. With only 2 mol of 32P incorporated, only a small amount of phosphorylation in the actin binding regions is expected. In addition, phosphorylation at Ser-726 may be underestimated due to the production of small polypeptides during chymotrypsin digestion. In contrast, the upper band of the myosin-binding fragments (MBF) was heavily phosphorylated (an unidentified component of intact caldesmon is highly phosphorylated and its digestion products are visible in lane b′). It is noteworthy that, even in this heavily loaded gel, none of the phosphorylated myosin-binding fragment is observed to co-sediment with myosin. Quantitation of this result was done by measuring the radioactivity remaining in the supernatant following sedimentation with myosin. While 14% of the nonphosphorylated myosin-binding fragment, in caldesmon digests, bound to myosin, only 2% of the phosphorylated myosin-binding fragment bound to myosin. Therefore, phosphorylation at the NH2-terminal region by calmodulin-dependent protein kinase II is responsible for the reversal of caldesmon binding to myosin.

Fig. 6. Effect of phosphorylation on the binding of chymotryptically cleaved, phosphorylated caldesmon to smooth muscle myosin·ATP.

Fig. 6

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Caldesmon was phosphorylated to 2 mol of phosphate/mol of caldesmon and digested with chymotrypsin. 4 μm cleaved caldesmon was co-sedimented with 3.1 μm smooth muscle myosin under the same conditions as in Fig. 1 except for the omission of CaCl2. Samples from the supernatant and pellet and standards of caldesmon and cleaved caldesmon were applied to an SDS 7–20% polyacrylamide gradient gel as follows: intact caldesmon (a and a′), digested caldesmon (b and b′), supernatant (c and c′), and pellet (d and d′). The gel was stained with Coomassie Blue (a–d) and exposed to x-ray film (a′–d′). The positions of intact caldesmon (CaD), myosin-binding fragment (MBF), and the actin-binding fragments (ABF) are shown. Note the presence of a low molecular weight polypeptide, which is highly labeled (lane a′) but not visible in the Coomassie Blue-stained gel (lane a). Digestion of this polypeptide may contribute to the radioactivity seen in lanes b′ and c′ below the myosin-binding fragment.

Phosphorylation of smooth muscle myosin regulatory light chains does not have an effect on the affinity of myosin for actin (Sellers et al., 1982). We investigated whether light chain phosphorylation affected myosin binding to caldesmon. The binding of caldesmon to smooth muscle myosin phosphorylated to about 0.66 mol of phosphate/mol of subfragment-1 is shown in Fig. 7. The binding to phosphorylated myosin is fit reasonably well by the curve obtained from our earlier experiments of caldesmon binding to unphosphorylated smooth muscle myosin (Hemric and Chalovich, 1990). Thus, phosphorylation of smooth muscle myosin has no effect on its binding to caldesmon.

Fig. 7. Effect of myosin light chain phosphorylation on the binding of caldesmon to smooth muscle myosin·ATP.

Fig. 7

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Binding was measured in the presence of 2.5 μm smooth muscle myosin and varied concentrations of [14C]iodoacetamide-labeled caldesmon. The conditions were the same as in Fig. 1 except for the omission of CaCl2. The theoretical curve is the same as that calculated for the binding of caldesmon to unphosphorylated smooth muscle myosin (Hemric and Chalovich, 1990). The curve is calculated for a stoichiometry of 0.92 mol of caldesmon/mol of myosin and a binding constant of 9.9 × 105 m−1.

DISCUSSION

Calmodulin inhibits the direct binding of caldesmon to myosin in a concentration-dependent manner. It is likely that the reversal of binding of caldesmon to both myosin and actin are controlled by the binding of Ca2+-calmodulin to the same site on caldesmon. Thus, the value of the binding constant for binding of Ca2+-calmodulin to the inhibitory site, K2, is 3–5 × 105 m−1 from myosin competition data and 3.6 × 105 m−1 from actin competition data (estimated from Velaz et al. (1989)). In contrast, NH2-terminal myosin-binding fragments of caldesmon bind weakly, if at all, to calmodulin affinity columns (present study; Mornet et al., 1988). Actin-binding fragments, from the COOH-terminal region, bind tightly to such column under the same conditions. Furthermore, deletion mutants that encode for the myosin binding region (residues 1–239, see Hayashi et al. (1991)) or the myosin binding region plus the helical region (residues 1–458, see Hayashi et al. (1991); residues 1–578, see Redwood et al. 1990) do not bind to calmodulin affinity columns. Although our modeling cannot exclude two binding sites for Ca2+-calmodulin as suggested by others (Wang et al., 1989), it appears that the COOH-terminal Ca2+-calmodulin site regulates binding of caldesmon both to actin and to myosin.

If Ca2+-calmodulin binding to the COOH-terminal region of caldesmon affects myosin binding at the NH2-region of caldesmon, then it is likely that caldesmon is folded under some conditions. This is true because there is a large extended helical region separating these two domains (Wang et al., 1991) and direct communication between them is unlikely. Folding of caldesmon is known to occur since the thiol groups at the two ends of caldesmon can form a disulfide bond (Lynch et al., 1987).

Several kinases have been shown to phosphorylate caldesmon. Protein kinase C, p34cdc2 kinase, and p44mpk kinase phosphorylate sites located in the COOH-terminal region (Ikebe and Hornick, 1991; Wawrzynow et al., 1991; Childs et al., 1992), casein kinase II phosphorylates one site at the NH2-terminal region of caldesmon (Wawrzynow et al., 1991), and calmodulin-dependent protein kinase II phosphorylates sites located in the COOH- and NH2-terminal regions (Ikebe and Reardon, 1990). Caldesmon has been shown to become phosphorylated in several model systems (Adam et al., 1989; Marston, 1986; Barany, 1991; Litchfield and Ball, 1987; Yamashiro et al., 1991; Hettasch and Sellers, 1991). However, to date, the only kinases implicated in in vivo phosphorylation are p34cdc2 and p44mpk kinases (Yamashiro et al., 1991; Adam et al., 1992), but it would not be surprising to find that caldesmon can be phosphorylated by multiple kinases since this occurs in other regulatory systems such as with glycogen synthase kinase (Fiol et al., 1990).

Ca2+-calmodulin-dependent protein kinase II, which co-purifies with caldesmon (Abougou et al., 1989; Scott-Woo et al., 1990), was used in the present study. This kinase apparently binds very tightly to caldesmon, since it is separated from caldesmon only with great difficulty. It is also interesting that phosphorylation by this kinase decreases the binding of caldesmon to both actin (Ngai and Walsh, 1987) and to myosin (Sutherland and Walsh, 1989). Interestingly, serines 73 and 26, within the myosin binding region of caldesmon, are phosphorylated preferentially to Ser-726 and Ser-587 in the actin binding region (Ikebe and Reardon, 1990). Likewise, we find that the NH2-region of caldesmon is predominantly phosphorylated (Fig. 6). Phosphorylation of the NH2-region of caldesmon is sufficient to reverse binding to myosin, since the phosphorylated myosin-binding fragment of caldesmon binds only weakly to myosin.

Phosphorylation of caldesmon with Ca2+-calmodulin-dependent protein kinase II does not totally eliminate binding to myosin but produces a graded inhibition depending on the level of phosphorylation. The incorporation of 3 phosphates per caldesmon reduces the binding to constant to 4% of its initial value. Such a reduction in the affinity of caldesmon to myosin could alter the cross-linking of actin to myosin, which presumably is responsible for the increased interaction between actin and myosin under some conditions (Hemric and Haeberle, 1992; Walker et al., 1989; Hegmann et al., 1991; Lin et al., 1991; Lash et al., 1986; Hemric and Chalovich, 1988).

Marston et al. 1992 have recently reported their results of the interaction of caldesmon with myosin. We are in agreement with their data in terms of the binding constant of caldesmon to smooth myosin and with the lack of effect of myosin phosphorylation on this interaction. However, differences exist between their observations with sheep aorta caldesmon and ours with turkey gizzard caldesmon. For example, they found that the stoichiometry of binding is 3 caldesmon molecules per myosin and that neither the stoichiometry nor the affinity are altered in the presence of ATP. Our present report (Figs. 4 and 5) supports our previous finding (Hemric and Chalovich, 1990) that the affinity of caldesmon for myosin increases in the presence of ATP. Furthermore, we observed the stoichiometry to be 1:1, in the presence of ATP, and 2 or 3 caldesmon molecules per myosin in the absence of ATP. The different binding observed could be due to different sources of caldesmon. Marston et al., 1992 also reported that caldesmon does not bind to skeletal myosin or to the S-1 region of smooth myosin. In contrast, we have observed binding to skeletal heavy meromyosin (Hemric and Chalovich, 1988, 1990) and a weak interaction with the S-1 region of smooth and skeletal S-1 (Hemric and Chalovich, 1988 and Fig. 6 of Hemrich and Chalovich, 1990). At present, we do not know the reason for these different observations.

Footnotes

*

This work was supported by Grant AR35216 from the National Institutes of Health (to J. M. C.) and Grant 1988-89-A16 from American Heart Association, North Carolina Affiliate (to M. E. H.). A preliminary report was presented at the Biophysical Society Meeting, February 24, 1991, San Francisco, CA (Hemric et al., 1991).

1

The abbreviations used are: S-1, myosin subfragment-1; HPLC, high performance liquid chromatography; Mes, 4-morpholineethane-sulfonic acid.

2

The program used to solve these equations, in the MATLAB language, may be obtained by writing to R. I. Shrager.

References

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