Dissociation of the effect of caldesmon on the ATPase activity and on the binding of smooth heavy meromyosin to actin by partial digestion of caldesmon

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin

The binding of 125I- and 14C-caldesmon to actin and actin-tropomyosin was studied using a cosedimentation technique and was analyzed by the method of McGhee and von Hippel [1974) J. Mol. Biol. 86, 469-489) for the binding of large ligands to a homogeneous lattice. The binding was adequately described by a single class of binding sites with a stoichiometry between 1:7 and 1:10. The binding exhibited a small degree of positive cooperativity (omega = 5-6) which was the same in the presence and absence of tropomyosin. The association constant for the binding of caldesmon to an isolated binding site was enhanced, from about 6 X 10(5) to about 1.4 X 10(6) M-1, by the presence of smooth muscle tropomyosin. Caldesmon inhibited the actin-activated ATPase activity of skeletal myosin subfragment 1 in both the absence and presence of tropomyosin. Maximum inhibition of ATPase activity occurred when one caldesmon molecule bound to seven actin monomers. A greater degree of inhibition was observed in the presence of tropomyosin than in the absence. This greater inhibition cannot be explained totally by the increased strength of binding of caldesmon to actin in the presence of tropomyosin. Finally, Ca2+-calmodulin completely reversed the binding of caldesmon to actin.     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

Characterization of a caldesmon fragment that competes with myosin-ATP binding to actin.

The protein caldesmon inhibits actin-activated ATP hydrolysis of myosin and inhibits the binding of myosin.ATP to actin. A fragment isolated from a chymotryptic digest of caldesmon contains features of the intact molecule that make it useful as a selective inhibitor of the binding of myosin.ATP complexes to actin without having the complexity of binding to myosin. The COOH-terminal 20 kDa region of caldesmon binds to actin with one-sixth the affinity of caldesmon with a stoichiometry of binding of one fragment per two actin monomers. This contrasts with the 1:6-9 stoichiometry of intact caldesmon. The binding of the 20 kDa fragments to actin is totally reversed by Ca(2+)-calmodulin and, like intact caldesmon, the 20 kDa fragments are competitive with the binding of myosin subfragments to actin. This competition with myosin binding is largely responsible for the inhibition of ATP hydrolysis, although both the fragments and intact caldesmon also reverse the potentiation of ATPase activity caused by tropomyosin. These polypeptides are useful both in defining the function of caldesmon and in studying the role of weakly bound cross-bridges in muscle.     

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.     

Regulation of molluscan actomyosin ATPase activity

The interaction of myosin and actin in many invertebrate muscles is mediated by the direct binding of Ca2+ to myosin, in contrast to modes of regulation in vertebrate skeletal and smooth muscles. Earlier work showed that the binding of skeletal muscle myosin subfragment 1 to the actin-troponin-tropomyosin complex in the presence of ATP is weakened by less than a factor of 2 by removal of Ca2+ although the maximum rate of ATP hydrolysis decreases by 96%. We have now studied the invertebrate type of regulation using heavy meromyosin (HMM) prepared from both the scallop Aequipecten irradians and the squid Loligo pealii. Binding of these HMMs to rabbit skeletal actin was determined by measuring the ATPase activity present in the supernatant after sedimenting acto-HMM in an ultracentrifuge. The HMM of both species bound to actin in the presence of ATP, even in the absence of Ca2+, although the binding constant in the absence of Ca2+ (4.3 X 10(3) M-1) was about 20% of that in the presence of Ca+ (2.2 X 10(4) M-1). Studies of the steady state ATPase activity of these HMMs as a function of actin concentration revealed that the major effect of removing Ca2+ was to decrease the maximum velocity, extrapolated to infinite actin concentration, by 80-85%. Furthermore, at high actin concentrations where most of the HMM was bound to actin, the rate of ATP hydrolysis remained inhibited in the absence of Ca+. Therefore, inhibition of the ATPase rate in the absence of Ca2+ cannot be due simply to an inhibition of the binding of HMM to actin; rather, Ca2+ must also directly alter the kinetics of ATP hydrolysis.     

Theoretical studies on competitive binding of caldesmon and myosin S1 to actin: prediction of apparent cooperativity in equilibrium and slow-down in kinetics of S1 binding by caldesmon

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S1 to regulated actins in which the binding affinity of S1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that S1 binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both S1 and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of S1 binding to actin. This model successfully predicts an apparent cooperativity for the binding of S1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon.     

Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal alpha-actin. Fab fragments of these antibodies (S alpha N Fab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 degrees C. Binding experiments carried out in the presence of ATP at 5 degrees C revealed that the catalytic inhibition was related to the inhibition of S-1 binding to actin by Fab. At equimolar ratios of Fab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of Fab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.     

A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin.

Binding of caldesmon to actin causes a decrease in the quantity of bound myosin and results in a reduction in the rate of actin-activated adenosine triphosphate hydrolysis. It is generally assumed that the binding of caldesmon and myosin to actin is a pure competitive interaction. However, recent binding studies of enzyme digested caldesmon subfragments directed at mapping the actin binding site of caldesmon have shown that a small 8-kD fragment around the COOH-terminal can compete directly with the myosin subfragment 1 (S-1) binding to actin; at least one other fragment that binds to actin does not inhibit the actin-activated adenosine triphosphate activity of myosin. That is, only a part of the caldesmon sequence may be responsible for directly blocking the binding of S-1 to actin. This prompts us to question the actual mode of binding of intact caldesmon and myosin S-1 to actin: whether the entire intact caldesmon molecule is competing with S-1 binding (pure competitive model) or just a small part of it (mosaic multiple-binding model). To answer this question, we measured the amount of myosin S-1 and caldesmon bound per actin monomer as a function of the total concentration of S-1 added to the system at constant concentrations of actin and caldesmon. A formalism for calculating the titration data based on the pure competitive model and a mosaic multiple-binding model was then developed. When compared with theoretical calculations, it is found that the binding of caldesmon and S-1 to actin cannot be pure competitive if no cooperativity exists between S-1 and caldesmon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Caldesmon and thin-filament regulation of muscle contraction

Smooth muscle contraction is regulated by phosphorylation of myosin and also possibly by the actin associated protein, caldesmon. The properties of caldesmon are discussed and compared with those of tropomyosin-troponin, the well characterized actin-based regulatory system of striated muscle. Caldesmon functions quite differently from tropomyosin-troponin. Under relaxing conditions tropomyosin-troponin does not affect the binding of myosin subfragment-1 to actin. In contrast, caldesmon strongly inhibits the binding of subfragment-1 to actin in the presence of ATP. This inhibition of binding parallels the decrease in ATPase activity that occurs as the caldesmon concentration is increased. Caldesmon has the opposite effect on the two headed myosin subfragment, heavy meromyosin. The apparent binding of skeletal heavy meromyosin increases slightly as the caldesmon concentration is increased, although the rate of ATP hydrolysis is inhibited. It is suggested that in the presence of caldesmon, myosin·ATP does not bind to the productive actin binding site but interacts with a distinct site on actin-caldesmon. This could lead to both an inhibition of ATP hydrolysis and an inrease in resting stiffness of relaxed smooth muscle.     

Localization and characterization of a 7.3-kDa region of caldesmon which reversibly inhibits actomyosin ATPase activity.

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.     

Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.     

Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction

We have previously shown that p21-activated kinase, PAK, induces Ca(2+)-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J. E., Arrell, D. K., Foster, D. B., Strauss, J. D., Heinonen, T. Y., Furmaniak-Kazmierczak, E., Cote, G. P., and Mak, A. S. (1998) J. Biol. Chem. 273, 23433-23439). In this study, we provide biochemical evidence implicating a role for PAK in Ca(2+)-independent contraction of smooth muscle via phosphorylation of caldesmon. Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon. PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation. Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin. We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca(2+) sensitivity of smooth muscle contraction.     

Tension development in skinned glycerinated rabbit psoas fiber segments irrigated with soluble myosin fragments.

Single glycerinated rabbit psoas muscle fibers were skinned by splitting them lengthwise. The fiber segments thus obtained were more easily accessible to solutes in the surrounding medium than the intact fibers. Using such segments, active tension could be fully abolished by adding N-ethylmaleimide under conditions which lead to inhibition of actin activation of the ATPase activity of myosin. Such muscles could, however, develop tension after irrigation with myosin or with the water-soluble active myosin fragments heavy meromyosin (HMM) or its subfragment 1 (HMM-S1). The induced tensions increased with increasing protein concentration in the irrigating solution. At any given protein concentration, the tension generated by myosin was larger than that produced by HMM which was, in turn, greater than that induced by HMM-S1 e.g. at 15 mg/ml protein the tensions produced by these three myosin moieties were 44.0, 14.0 and 2.8 g/cm2, respectively. The tension was found to be intimately associated with ATP splitting; thus, HMM and HMM-S1 which have been treated with reagents abolishing actin-activated ATPase failed to induce tension development. A contractile force may thus be generated through the interaction with actin of the water-soluble, enzymatically active, myosin subfragments involving the splitting of ATP.