©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Caltropin on Caldesmon-Actin Interaction (*)

(Received for publication, July 26, 1994; and in revised form, December 27, 1994)

Rajam S. Mani Cyril M. Kay (§)

From the Medical Research Council Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent probe acrylodan (6-acryloyl-2-(dimethylamino)napththalene). In co-sedimentation studies most of the caldesmon pelleted along with actin. However, when caldesmon in the presence of caltropin was mixed with actin, caldesmon did not pellet along with actin following high speed centrifugation, suggesting that caltropin has significantly weakened its binding to actin. The caltropin effect was noticed even when tropomyosin was included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 ± 2 nm. The addition of actin produced a nearly 70% increase in fluorescent intensity, accompanied by a blue shift in the emission maximum (i.e. = 505 ± 2 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K = 6 times 10^7M). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca medium, its affinity for caldesmon was lowered (K = 2 times 10^7M). Caltropin, which is very effective in reversing caldesmon's inhibition of the actin-activated myosin ATPase (Mani, R. S., McCubbin, W. D., and Kay, C. M.(1992) Biochemistry 31, 11896-11901), is shown in the present study to have a pronounced effect on its binding to actin, suggesting a major role for caltropin in regulating caldesmon in smooth muscle.


INTRODUCTION

In smooth muscle, calcium exerts its control via mechanisms based on both the thick (myosin) filaments (Sobieszek and Bremel, 1975) and the thin (actin-based) filaments (Chalovich, 1993; Sobue and Sellers, 1991). The thin filament-linked pathway involves actin-mediated regulation by caldesmon, a protein that is found bound to the thin filaments (Furst et al., 1986; Lehman et al., 1987; Walsh and Sutherland, 1989). When purified caldesmon binds to actin-tropomyosin, it inhibits the superprecipitation of actomyosin (Sobue et al., 1981) and the actin-activated ATPase activity of both myosin (Dabrowska et al., 1985; Moody et al., 1985; Sobue et al., 1985) and its subfragments (Lash et al., 1986; Chalovich et al., 1987). Recent studies with caldesmon fragments and smooth muscle fibers (Pfitzer et al., 1994; Katsuyama et al., 1992) demonstrate the physiological feasibility of the regulation of smooth muscle contraction by caldesmon. However, defining the precise role of caldesmon in cells requires an understanding of its interactions with several key proteins, namely actin, myosin, calmodulin, and caltropin.

Caldesmon binds to actin and myosin with strong affinity (Velaz et al., 1989; Hemric and Chalovich, 1990). Recent studies suggest that caldesmon inhibits the binding of myosin-ATP to actin, thereby inhibiting ATP hydrolysis (Velaz et al., 1989). In an earlier study (Mani and Kay, 1993), we have shown that caldesmon binds to smooth muscle heavy meromyosin with high affinity and that this interaction was weakened in the presence of caltropin (Ca), a high affinity smooth muscle calcium-binding protein. Caltropin, which is very potent in reversing the inhibitory effect of caldesmon in the presence of calcium (Mani et al., 1992), modulates the interaction between caldesmon and heavy meromyosin in a calcium-dependent manner. The caltropin effect on caldesmon may be summarized as follows. Caldesmon, by virtue of its ability to interact with both myosin and actin, is able to inhibit the actin-activated myosin ATPase activity by interfering in the actin-myosin interaction. In the presence of Ca caltropin, the affinity of caldesmon for myosin is lowered; as a consequence myosin is able to interact effectively with actin, resulting in restoring the actin-activated myosin ATPase activity. However, any proposed mechanism to account for the effect of caltropin on caldesmon should include not only the caldesmon-myosin interaction but also the caldesmon-actin interaction. For this reason, in the present study the effect of caltropin on the interaction between caldesmon and actin was investigated by the co-sedimentation method and fluorescence measurements using the fluorescent probe acrylodan (^1)to label both the sulfhydryl groups in caldesmon. Using these techniques we have shown that caltropin in the presence of calcium was effective in weakening the interaction between caldesmon and actin.


MATERIALS AND METHODS

Protein Purification

Caltropin and caldesmon were isolated from chicken gizzard using the procedure described in our earlier papers (Mani and Kay, 1990; Mani et al., 1992). F-actin was purified from rabbit skeletal muscle according to Pardee and Spudich(1982).

Airfuge Binding Experiments

Rabbit skeletal muscle actin (10 µM) was polymerized using a solvent system consisting of 2 mM MgCl(2), 42 mM NaCl, 20 mM Tris, pH 7.5, 0.5 mM dithiothreitol, and 0.2 mM CaCl(2). Subsequent to polymerization, F-actin was incubated with caldesmon (1.0-3.0 µM) resulting in a range of caldesmon to actin ratios of 0.1-0.3. Following incubation for 20 min, the protein mixtures were centrifuged at 100,000 times g for 30 min in an air-driven ultracentrifuge (Beckman Instruments). Under these conditions caldesmon was not pelleted when centrifuged in the absence of actin. The pelleted samples were solubilized in SDS-gel running buffers and were analyzed using 12% SDS-polyacrylamide gels.

Fluorescence Studies

Fluorescence spectra were obtained with a Perkin-Elmer model MPF-44 spectrofluorometer, and all measurements were made at 20 °C. Caldesmon was labeled with acrylodan following the procedure of Prendergast et al. (1983) as described in our earlier paper (Mani et al., 1992). The ratio of label to protein was determined to be 1.9, suggesting that both of the sulfhydryl groups in caldesmon were labeled under these conditions. Labeled caldesmon was titrated with F-actin by following the changes in fluorescence intensity at 470 nm.

Acrylamide Fluorescence Quenching Studies

The excitation wavelength used was 375 nm, at which the probe acrylodan has maximum absorption. The initial A of the labeled caldesmon was leq0.05. The fluorescence quenching was measured at the emission maximum (505 nm) for the labeled caldesmon-actin complex following the addition of 8 M acrylamide in small aliquots. The theory of acrylamide quenching and the mathematical treatment of the data are described by Lehrer and Leavis(1978).


RESULTS

Binding Experiments

The binding of caldesmon to actin was studied by a co-sedimentation method. When caldesmon/actin mixtures of varying ratios (see ``Materials and Methods'') were subjected to high speed centrifugation, most of the caldesmon co-sedimented with F-actin whether Ca was present or not (Fig. 1, lane b). The addition of Ca/caltropin to actin prior to the addition of caldesmon had a pronounced effect on the ability of caldesmon to interact with actin. Under these conditions most of the caldesmon did not co-sediment with actin (Fig. 1, lane a), i.e. caldesmon was now present in the supernatant along with caltropin. However, when this experiment was repeated in the presence of 1 mM EGTA (i.e. in the absence of calcium) most of the caldesmon could be pelleted along with actin (Fig. 1, lane d), suggesting that caltropin has no significant effect on caldesmon-actin interaction in the absence of calcium. This finding is consistent with our earlier studies involving bioassays (Mani et al., 1992) where caltropin was shown to be effective in releasing the inhibition of caldesmon only in the presence of calcium. Caltropin was also effective in releasing caldesmon from actin filaments even in the presence of tropomyosin. Control experiments indicated that neither caldesmon nor caltropin sedimented by itself, and we also found no interaction between caltropin and actin.


Figure 1: Interaction between caldesmon and actin. The binding of caldesmon to actin was examined in the presence and absence of Ca and caltropin by the high speed centrifugation method described under ``Materials and Methods.'' The proteins that were pelleted were analyzed by 12% SDS-gels. Lanea, 1.5 µM caldesmon (CaD), 1 µM caltropin, and 10 µM actin in 0.2 mM Ca; laneb, 1.5 µM caldesmon and 10 µM actin in 0.2 mM Ca; lanec, 1.5 µM caldesmon and 10 µM actin in 1 mM EGTA; laned, 1.5 µM caldesmon, 1 µM caltropin, and 10 µM actin in 1 mM EGTA.



Fluorescence Spectroscopy

Acrylodan, a sulfhydryl-specific fluorescent probe, is very useful for studying protein-protein interactions. Because sulfhydryl groups in chicken gizzard caldesmon can be modified without the protein losing its activity (Hemric and Chalovich, 1990), we decided to label both the sulfhydryl groups in caldesmon with acrylodan. When the labeled caldesmon was excited at 375 nm, the emission maximum occurred at 515 ± 2 nm in both the presence and the absence of calcium. The addition of actin resulted in a blue shift in the emission maximum, which was then centered at 505 ± 2 nm. Even though there was no significant change in fluorescence intensity around the emission maximum, a significant increase in relative fluorescence intensity (nearly 70%) was observed at 470 nm (Fig. 2). For this reason, during the titration of labeled caldesmon with actin, the fluorescence intensity at 470 nm was monitored. Analysis of the titration curve indicated that the maximum increase in fluorescence intensity was achieved by the time 7 mol of actin were added per mol of caldesmon (Fig. 3). Also shown in the figure is the titration of actin with the caldesmon-caltropin complex. Addition of caltropin to labeled caldesmon in the presence of calcium produced an increase in fluorescence intensity. The observed fluorescence intensity in the presence of caltropin was taken as the starting value for the titration with actin. In this instance also the maximum change was reached when 7 mol of actin were added. However, as can be seen from Fig. 3, actin binds to the caldesmon-caltropin complex with lower affinity. For example, for a 50% increase in fluorescence intensity, the amount of actin required for caldesmon and the caldesmon-caltropin complex corresponded to 0.15 and 0.25 µM, respectively. The shape of the titration curve suggests the existence of more than one class of binding site. If all seven actins were to bind with the same affinity, then the observed increase in fluorescence intensity would be linear until 7 mol of actin were added, at which stage the observed increase in fluorescence intensity would correspond to the maximum level (i.e. it will level off). If one were to draw tangents to the initial slope of the titration curve and to the asymptotic part of the curve (i.e. where the curve levels off), then the point of intersection should correspond to the number of high affinity binding sites. The stoichiometry obtained using this approach corresponds to a value of 2.8 and 3.5 mol of actin for the caldesmon and the caldesmon-caltropin complex, respectively. In other words, out of seven actins that are required for saturation, only three are binding with high affinity. If one assumes that the observed fluorescence change is the result of actin binding to caldesmon, then one can calculate the amount of actin bound at any given point in the titration curve. Knowing the amount of actin added, one can deduce the amount of free actin that is present. When Scatchard analysis was carried out, with the amounts of the bound and free actin and the caldesmon concentration used for the titration known, a best fit was also obtained when the ratio of actin to caldesmon was 3:1 (Fig. 4). From the slope term of the Scatchard plot, a K(a) value of (6.0 ± 0.5) times 10^7M was calculated from three sets of titration data, suggesting that caldesmon binds to actin with high affinity. Also shown in Fig. 3is the titration of actin with the caldesmon-caltropin complex. Actin binds to this binary complex with lower affinity, because the K(a) value obtained was only (2.5 ± 0.5) times 10^7M. The presence of caltropin has lowered the affinity of caldesmon for actin more than 2-fold.


Figure 2: Fluorescence emission spectra of acrylodan-caldesmon (solid line) and acrylodan-caldesmon-actin (dashed line) complexes in 25 mM Tris, pH 7.5, 42 mM NaCl, 2 mM MgCl(2), 0.2 mM CaCl(2), and 1 mM dithiothreitol at 20 °C. The excitation wavelength was 375 nm.




Figure 3: Influence of actin on acrylodan-caldesmon (bullet) and acrylodan caldesmon-caltropin fluorescence (circle). The initial caldesmon concentration was 1.0 times 10^7M. Measurements were carried out in 25 mM Tris, pH 7.5, 42 mM NaCl, 2 mM MgCl(2), 0.2 mM CaCl(2), and 1 mM dithiothreitol at 20 °C. Relative changes in fluorescence intensities (DeltaF/DeltaF(max)) at 470 nm are plotted as functions of actin concentration. The excitation wavelength was 375 nm. The data points shown on the titration curves are based on the results obtained from three sets of titrations. The size of the symbols on these curves takes into account the observed experimental errors. The inset shows the relative changes in fluorescence intensities (DeltaF/DeltaF(max)) at 470 nm as functions of molar ratios of actin added to caldesmon.




Figure 4: Scatchard plot of actin binding to acrylodan caldesmon (bullet) and acrylodan-caldesmon-caltropin (circle) with [actin] corrected to [free actin] using a 3:1 stoichiometry for actin to caldesmon. Bound actin was determined from the change in fluorescence intensity, which is directly proportional to binding. is the number of moles of ligand bound to 1 mol of macromolecules; it is defined as = [A]/[P]. The conditions were the same as described in the legend of Fig. 3.



The change in fluorescence intensity as a function of added actin was also analyzed using double-inverse plots. The affinities of actin for caldesmon and the caldesmon-caltropin complex determined from the abscissa intercepts of the double inverse plots were 1 times 10^6 and 5 times 10^5M, respectively (data not shown), and this probably represents the average K(a) values for the high and the low affinity binding sites. In earlier studies involving the interaction of caldesmon and actin, several other investigators have also suggested the existence of high and low affinity binding sites (Moody et al., 1985; Lash et al., 1986; Horiuchi et al., 1986; Smith et al., 1987). We also wanted to see if caltropin could influence caldesmon-actin interaction in the presence of tropomyosin. For this reason we carried out fluorescence titration experiments with labeled caldesmon containing tropomyosin with actin in the presence and the absence of caltropin. In this instance the affinity of caldesmon for actin was also lowered by more than 2-fold when caltropin was present, suggesting that caltropin could function even in the presence of tropomyosin.

The observed increase in fluorescence intensity accompanied by a blue shift in the emission maximum upon actin binding could be due to a decrease in the exposure of the probe to solvent quenchers. Fig. 5shows the effect of the acrylamide quencher on the fluorescence intensity of caldesmon, the caldesmon-actin complex, the caldesmon-caltropin (Ca)-actin ternary complex, the caldesmon-tropomyosin-actin complex, and the caldesmon-tropomyosin-caltropin (Ca)-actin complex. The linear portion of the plots at low acrylamide concentration gave Stern-Volmer constants (K) of 2.1, 1.7, 1.2, 1.1, and 0.6 for the caldesmon, caldesmon-actin, caldesmon-caltropin-actin, caldesmon-tropomyosin-actin, and caldesmon-tropomyosin-caltropin-actin complexes, respectively. The value of the Stern-Volmer quenching constant obtained is higher for caldesmon than for the caldesmon-actin complex. Thus actin does shield the label on caldesmon from the solvent quencher. The addition of Ca caltropin to the caldesmon-actin complex results in further lowering of the quenching constant, suggesting that the label now moves to a more hydrophobic environment (i.e. the label is now more protected from the quencher). This effect of caltropin was also noticed for the caldesmon-tropomyosin-actin complex. In other words, caltropin was effective in shielding the label even when tropomyosin was present in the caldesmon-actin complex. Even though both the sulfhydryl groups in caldesmon (Cys-153 and Cys-580) are labeled, we believe the observed effect of caltropin and actin on acrylamide quenching is due to the binding of these proteins at or near the label Cys-580, because actin and caltropin are known to bind at the COOH-terminal end of caldesmon (Szpacenko and Dabrowska, 1986; Zhuang et al., 1994) and thus shield the label from collisions with quenchers in the medium.


Figure 5: Steady-state acrylamide quenching plots for the acrylodan-caldesmon (up triangle), acrylodan-caldesmon-actin (bullet), acrylodan-caldesmon-caltropin-actin (circle), acrylodan-caldesmon-tropomyosin B-actin (box), and the acrylodan-caldesmon-tropomyosin B-caltropin-actin () complexes. Excitation and emission wavelengths were 375 and 505 nm, respectively. The solvent system used was the same as described in the legend of Fig. 3.




DISCUSSION

Caldesmon binds tightly to myosin (K(a) = 10^6M), actin (K(a) = 10^6-10^7M), and tropomyosin (K(a) = 10^6M) and inhibits the actin-activated ATPase of smooth and skeletal myosins and their subfragments (Lash et al., 1986; Hemric and Chalovich, 1988; Velaz et al., 1989; Horiuchi and Chacko, 1988). These characteristics of caldesmon make it a potential candidate for the regulation of actomyosin interaction. Simultaneous binding of caldesmon to actin and myosin may provide an additional role for caldesmon in stabilizing the structure of the contractile apparatus or may even maintain tension in the latch state of smooth muscle.

The interaction between caldesmon and actin is important for several reasons. First, the binding of caldesmon to the NH(2)-terminal end of actin results in the inhibition of ATP hydrolysis by actomyosin (Crosbie et al., 1994), suggesting a regulatory role for caldesmon in the filament-linked regulation of smooth muscle contraction. Even though we know that the COOH-terminal region of caldesmon binds to the NH(2)-terminal end of actin, many questions regarding the interaction remain unanswered. Caldesmon appears to bind along the actin filament (Lehman et al., 1989) with each caldesmon monomer covering several actin monomers (Velaz et al., 1989). Unlike tropomyosin, caldesmon does not have repeating actin-binding structural units, and for this reason it is unclear how the binding of the COOH-terminal region of caldesmon stabilizes its binding along several actin monomers. In the presence of calcium, calcium-binding proteins such as caltropin and calmodulin can release caldesmon's inhibition of the actin-activated myosin ATPase. In order to get an insight into the mechanism by which these calcium-binding proteins release the inhibition of caldesmon, we studied the influence of caltropin on the caldesmon-actin interaction using co-sedimentation and fluorescence measurements using labeled caldesmon. Caltropin in the presence of Ca was very effective in weakening the interaction between caldesmon and actin.

The addition of actin to labeled caldesmon produced an increase in fluorescence intensity that leveled off when the mole ratio of actin to caldesmon was nearly 7. There is uncertainty in the in vivo and in vitro stoichiometric ratio of caldesmon to actin, raising questions as to the assembly and arrangement of caldesmon on the actin filament. According to Marston et al.(1992), the in vivo ratio is about one caldesmon molecule/14 actin monomers, whereas the in vitro values reported range from 3 to 7 actin monomers/caldesmon molecule for maximum inhibition of ATPase activity. The length of caldesmon has been estimated to be 740 Å. If caldesmon binds to both sides of an actin filament like tropomyosin, then a maximum of 14 actin monomers could be covered by a single caldesmon molecule, consistent with the in vivo determinations. Graceffa and Jancso(1991) studied cross-linking of caldesmon to actin as a function of caldesmon concentration and demonstrated that only 3-4 actin monomers could be cross-linked to one caldesmon molecule, hence reinforcing the difference in the in vivo and in vitro determinations. Native thin filaments also contain calponin (Nishida et al., 1990), which also binds actin. The presence of calponin might limit the amount of caldesmon bound to actin, and this could explain the observed difference between the in vivo and the in vitro estimations. From this study we conclude that caltropin in the presence of Ca is effective in weakening the interaction between caldesmon and actin. Caldesmon has been implicated to act as a competitive inhibitor of myosin binding to actin. Caldesmon can bind to actin with strong affinity at or near the myosin binding region (i.e. at the NH(2)-terminal end of actin), thus blocking myosin from binding to actin and resulting in lowered ATPase activity (Chalovich et al., 1987). Caldesmon, which binds caltropin in the presence of Ca with high affinity (Mani et al., 1992), undergoes a conformational change; as a consequence we believe it binds to actin with lower affinity and is no longer able to compete with myosin for binding to actin, resulting in the recovery of the ATPase activity. In this context Graceffa and Jansco(1991) have also evoked a similar mechanism to explain the effect of Ca/calmodulin on caldesmon-actin interaction.

A possible fine tuning mechanism for cooperative caldesmon regulation of the thin filament is shown in Fig. 6. The regulatory properties of caldesmon are similar to troponin in most details as indicated by Marston and Redwood(1993). The COOH-terminal region of caldesmon named domain 4b comprising amino acid residues 658-756 is closely analogous to the inhibitory peptide of troponin (troponin-I). Domain 3 of caldesmon (i.e. residues 508-565) is homologous to the CB(2) region of troponin-T (i.e. residues 71-151, which are implicated in tropomyosin binding). For this reason we have indicated in our model that region 4b and domain 3 of caldesmon bind to actin and tropomyosin, respectively. Tropronin-C (the Ca-binding subunit) is part of the troponin complex, whereas in smooth muscle the Ca factor caltropin is not a part of caldesmon.


Figure 6: A troponin-like model for cooperative caldesmon regulation of the thin filament caldesmon (CaD), tropomyosin (TM), actin (A), and myosin (M). Domain 1 corresponds to the NH(2)-terminal region of caldesmon; domains 3 and 4b represent COOH-terminal amino acid residues 508-565 and 658-756, respectively. CaT, caltropin.



Caldesmon can bind tightly to myosin as well as to actin, and caltropin has no effect on these interactions in the absence of calcium as shown in our model (Fig. 6). Caldesmon is known to compete with myosin and myosin fragments for the binding to actin in rigor (Hemric and Chalovich, 1988), and this indicates the existence of at least a partial overlap in their actin-binding sites and could form the basis of the regulatory function of caldesmon. According to Chalovich et al.(1987), caldesmon acts as a competitive inhibitor of the binding of myosin to actin, because the COOH-terminal end of caldesmon that does not bind to myosin is able to inhibit ATP hydrolysis. Calmodulin and caltropin in the presence of Ca can release the inhibition of caldesmon without displacing caldesmon from the actin filament (Smith et al., 1987; Marston and Smith, 1985; Mani et al., 1992). According to our proposed model, in the presence of Ca, caltropin binds to caldesmon with high affinity (Mani et al., 1992) and induces a conformational change in caldesmon with the result that caldesmon binds to actin with lower affinity (3-fold) and is no longer able to compete with myosin for binding to actin resulting in the release of the inhibition.

Current studies have clearly demonstrated the importance of this cooperative unit consisting of caldesmon-tropomyosinactin in regulating the smooth muscle thin filament. Even though in vitro studies involving the interaction of caldesmon with individual proteins, namely tropomyosin, actin, and myosin, and the calcium-binding proteins have been studied in detail, caution should be exercised in interpreting these results, because most of these studies involve interaction of caldesmon with either thick or thin filament proteins. It is necessary to relate these findings with the individual proteins to the integrated system consisting of both the thick and thin filament assembly, namely the actomyosin system.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviation used is: acrylodan, 6-acryloyl-2-(dimethylamino) napththalene.


ACKNOWLEDGEMENTS

We thank A. Keri, K. Oikawa, and L. Hicks for excellent technical assistance.


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