Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

Caldesmon: a common actin-linked regulatory protein in the smooth muscle and nonmuscle contractile system

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.     

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin

Application of the myosin competition test (Lehman, W., and Szent-Gy?rgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.     

Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments.

Thin-filament preparations from four smooth muscle types (gizzard, stomach, trachea, aorta) all activate myosin MgATPase activity, are regulated by Ca2+, and contain actin, tropomyosin and a 120000-140000-Mr protein in the molar proportions 1:1/7:1/26. The 120000-140000-Mr protein from all sources is a potent inhibitor of actomyosin ATPase activity. Peptide-mapping and immunological evidence is presented showing that it is identical with caldesmon. Quantitative immunological data suggest that caldesmon is a component of all the thin filaments and that the thin-filament-bound caldesmon accounts for all the caldesmon in intact tissue. The myosin light-chain kinase content of thin-filament preparations was found to be negligible. We propose that caldesmon-based thin-filament Ca2+ regulation is a physiological mechanism in all smooth muscles.     

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound ⁴⁵Ca²⁺ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca²⁺ concentration. Crosslinking experiments with BS³ showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg²⁺-ATPase activity of myosin in the presence of Ca²⁺.     

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Phosphorylation of caldesmon by protein kinase C

Protein kinase C catalyzes phosphorylation of caldesmon, an F-actin binding protein of smooth muscle, in the presence of Ca2+ and phospholipid. Protein kinase C incorporates about 8 mol of phosphate/mol of chicken gizzard caldesmon. When calmodulin was added in the medium, there was an inhibition of phosphorylation. The fully phosphorylated, but not unphosphorylated, caldesmon inhibited myosin light chain kinase activity. The possibility that protein kinase C plays some role in smooth muscle contractile system through caldesmon, warrants further attention.     

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.     

Differences in phorbol-dependent phosphorylation of regulatory proteins and contraction of phasic and tonic smooth muscle

Smooth muscles are divided into slowly contracting tonic and relatively fast phasic muscles. In both cases Ca2+ is a key mediator of the contractile response. However, the appearance of a tonic component during sphincter or arterial muscle contraction and its absence in contracting visceral smooth muscle is characteristic of their difference. We have found that in chicken tissues phorbol 12,13-dibutyrate (PDBu) induces a sustained contraction in carotid arterial muscle, but provokes no contraction in phasic gizzard smooth muscle. Next we were aimed to find differences in PDBu-induced phosphorylation of the key proteins involved in regulation of smooth muscle contraction, i.e. caldesmon, myosin light chain kinase (MLCK), and the myosin light chain kinase-related protein (KRP, also known as telokin). Two correlative differences were observed. 1. PDBu stimulated phosphorylation of MLCK in tonic smooth muscle and had no effect on the level of MLCK phosphorylation in phasic muscle. Phosphopeptide mapping suggests the involvement of mitogen-activated protein (MAP) kinases in phosphorylation of MLCK in situ. 2. PDBu induced phosphorylation of MAP-kinase sites in caldesmon in both types of smooth muscle, but this phosphorylation had no significant effect on caldesmon functional activity in vitro. For the first time we have shown that in gizzard PDBu also stimulates a yet unknown transitory caldesmon-kinase different from protein kinase, C, Ca2+/calmodulin-dependent kinase II and casein kinase CK2. 3. No significant difference was found in the kinetics of PDBu-dependent phosphorylation of KRP in tonic and phasic smooth muscles. KRP was also demonstrated to be a major phosphoprotein in smooth muscle phosphorylated in vivo at several sites located within its N-terminal sequence. Protein kinases able to phosphorylate these sites were identified in vitro. Among them, MAP-kinase was suggested to phosphorylate a serine residue homologous to that phosphorylated in MLCK. 4. p42erk2 and p38 MAP-kinases were found in phasic and tonic smooth muscles. Both were responsive to PDBu in cultured chicken aortic smooth muscle cells, and their role in phosphorylation of MLCK and low molecular weight isoform of caldesmon was evaluated.     

Calcium-dependent control of caldesmon-actin interaction by S100 protein

Caldesmon from chicken gizzard muscle has been examined for ability to interact with S100 protein using sedimentation, low-shear viscosity, and affinity chromatography. Ca2+/S100 protein, like Ca2+/calmodulin, inhibited the binding of caldesmon to F-actin in a concentration-dependent manner and the inhibition was not observed in the absence of Ca2+. Caldesmon was bound to S100 protein-Sepharose in the presence of Ca2+ and released with EGTA, indicating that there is a direct interaction between caldesmon and S100 protein. The binding of S100 protein to caldesmon also relieved actomyosin Mg2(+)-ATPase inhibition by caldesmon. The molar ratio of S100 protein to caldesmon required for half-maximal restoration was about 0.3, a value less than that in the case of calmodulin. S100 protein, however, was less effective in terms of the maximal extent of the restoration. With respect to Ca2(+)-sensitivity, the restoration profiles were monophasic with a midpoint at 3 x 10(-5) M for S100 protein and 8 x 10(-6) M for calmodulin. The restoration by S100 protein was almost wholly inhibited by TFP, but not by W-7. Taken together, our results suggest that a Ca2(+)-binding protein other than calmodulin may regulate caldesmon-dependent cellular functions.     

Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: comparisons with gizzard caldesmon and MLC kinase

We have developed a simple and conventional purification method for caldesmon and MLC kinase from bovine arterial smooth muscle, and compared the arterial and gizzard proteins. Arterial caldesmon shares the alternative binding to calmodulin or F-actin in a Ca2+-dependent manner and the antigenic determinants with the gizzard protein. Both caldesmons have the same association constant with F-actin (1.3-1.7 X 10(7) M-1) and the same maximum binding (1 caldesmon per 12-14 actins). However, the molecular weight of arterial caldesmon (dimer of a 148 kDa polypeptides) was slightly different from that of gizzard caldesmon (heterodimer of 150/147 kDa polypeptides). The molecular weight of arterial MLC kinase (160 kDa) was much larger than that of the gizzard enzyme (135 kDa). The enzyme activities of both MLC kinases were comparable (Km = 9.5 microM, Vmax = 12.5 mumol/min X mg). The association constant of the arterial enzyme to F-actin (5.1 X 10(6) M-1) was much larger than that of the gizzard enzyme (9.0 X 10(5) M-1) but the maximum binding was the same (1 enzyme per 12-13 actins). Immunocytochemical examinations showed that caldesmon and MLC kinase in cultured arterial cells have a restricted localization along the stress fibers, suggesting functional linkages between both proteins and actin filaments in vivo.     

The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments

Chicken gizzard caldesmon causes up to 40% inhibition of Mg2+-ATPase activity of rabbit skeletal muscle actomyosin. In the presence of chicken gizzard tropomyosin this inhibition is significantly increased, reaching a maximum (around 80%) at a molar ratio of caldesmon to actin monomer of 1 to 10-13. The inhibition of actomyosin ATPase takes place over a wide pH range (from 6.0 to 8.0) but is decreased with an increase in KCl and MgCl2 concentrations. Caldesmon, in the range of caldesmon/ actin ratios within which it inhibits actomyosin ATPase, forms bundles of parallelly aligned actin filaments. Calmodulin in the presence of Ca2+ dissociates these bundles and restrains the inhibition of actomyosin ATPase, provided that it is used at a high molar excess over caldesmon.     

Caldesmon, calmodulin and tropomyosin interactions

Binary complex interactions between caldesmon and tropomyosin, and calmodulin and tropomyosin, and ternary complex interaction involving the three proteins were studied using viscosity, electron microscopy, fluorescence and affinity chromatography techniques. In 10 mM NaCl, caldesmon decreased the viscosity of chicken gizzard tropomyosin by 7-8 fold with a concomitant increase in turbidity (A330nm). Electron micrographs showed spindle-shaped particles in the tropomyosin-caldesmon samples. These results suggest side-by-side aggregation of tropomyosin polymers induced by caldesmon. Binding studies in 10 mM NaCl between caldesmon and chicken gizzard tropomyosin labelled with the fluorescent probe N-(1-anilinonaphthyl-4)maleimide (ANM) gave association constants from 5.3.10(6) to 7.9.10(6) M-1 and stoichiometry from 1.0 to 1.4 tropomyosin per caldesmon. Similar binding was observed for rabbit cardiac tropomyosin and caldesmon. Removal of 18 and 11 residues from the COOH ends of the gizzard and cardiac tropomyosin by carboxypeptidase A, respectively, had no significant effect on their binding to caldesmon. In the presence of Ca2+, chicken gizzard tropomyosin bound to a calmodulin-Sepharose-4B column and was eluted with a salt concentration of 140 mM. This interaction was weakened in the absence of Ca2+, and the bound tropomyosin was eluted by 65 mM KCl. ANM-labelled tropomyosin bound calmodulin in the presence of Ca2+ with a binding constant of 3.5.10(6) M-1 and a binding stoichiometry of 1 to 1.4 tropomyosin per calmodulin. In 10 mM NaCl, calmodulin reduced the specific viscosity of chicken gizzard tropomyosin in the presence of Ca2+ by 5 fold, while a 1.5-fold reduction in viscosity was observed in the absence of Ca2+. In either case, no significant increase in turbidity was observed suggesting that calmodulin reduced head-to-tail polymerization of tropomyosin. The interaction of caldesmon with the calmodulin-ANM-tropomyosin complex in the presence and absence of Ca2+ was also examined. The result is consistent with a model that in the absence of Ca2+, calmodulin binds weakly to either caldesmon or tropomyosin and has little effect on the tropomyosin-caldesmon interaction; whereas, Ca2(+)-calmodulin interacts with caldesmon and reduces its affinity to tropomyosin.     

Interaction between chicken gizzard caldesmon and tropomyosin

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.     

Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin

Caldesmon is an F-actin cross-linking protein of chicken gizzard smooth muscle whose F-actin binding activity can be regulated in vitro by Ca2+-calmodulin (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, 1981, Proc. Natl. Acad. Sci. USA, 78:5652-5655). It is a rod-shaped, heat-stable, F-actin bundling protein and is the most abundant F-actin cross-linking protein of chicken gizzard smooth muscle presently known (Bretscher, A., 1984, J. Biol. Chem., 259:12873-12880). We report the use of polyclonal antibodies to caldesmon to investigate its distribution and localization in other cells. Using immune blotting procedures, we have detected immunoreactive, heat-stable forms of caldesmon in cultured cells having either approximately the same apparent polypeptide molecular weight as gizzard caldesmon (120,000-140,000) or a substantially lower molecular weight (71,000-77,000). Through use of affinity-purified antibodies in indirect immunofluorescence microscopy, we have localized the immunoreactive forms to the terminal web of the brush border of intestinal epithelial cells and to the stress fibers and ruffling membranes of cultured cells. At the light microscope level caldesmon is distributed in a periodic fashion along stress fibers that is coincident with the distribution of tropomyosin and complementary to the distribution of alpha-actinin.     

Calcium-dependent regulation of caldesmon by an 11-kDa smooth muscle calcium-binding protein, caltropin.

Caldesmon from chicken gizzard muscle has been examined for its ability to interact with caltropin using affinity chromatography and the fluorescent probe acrylodan. The action of caltropin on the inhibitory effect of caldesmon on actomyosin ATPase was also studied. Like calmodulin, caltropin could release the inhibitory effect of caldesmon in the presence of Ca2+. Complete reversal was obtained when 1 mol of caltropin was added per mol of caldesmon. When caldesmon was applied to caltropin-Sepharose in the presence of Ca2+, most of the caldesmon was bound to the column and could be eluted with EGTA, indicating that there is a direct interaction between caldesmon and caltropin. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 504 nm. Addition of caltropin in the presence of Ca2+ resulted in a nearly 50% increase in fluorescence intensity, and this was accompanied by a blue shift in the emission maximum (i.e., lambda em,max 492 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of caltropin with labeled caldesmon indicated a strong affinity for this protein (Kd was in the order of 8 x 10(-8)-2 x 10(-7) M). However, when caltropin was added to labeled caldesmon in the presence of EGTA, there was no indication of any interaction. Caltropin was at least as potent as calmodulin, if not better, in reversing the inhibitory effect of caldesmon in the presence of calcium, making it a potential Ca2+ factor in regulating caldesmon in smooth muscle.     

Isolation of the native form of chicken gizzard myosin light-chain kinase.

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.     

Caldesmon association with smooth muscle thin filaments isolated in the presence and absence of calcium

Thin filaments isolated from chicken gizzard smooth muscle in either the presence or the absence of Ca2+ possess identical caldesmon contents. Hence, a 'flip-flop' mechanism, involving Ca2+-dependent association and dissociation of caldesmon and thin filaments, does not appear to operate in vivo and is an unlikely model for caldesmon function.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.