Ca2+, calmodulin and cyclic AMP-dependent modulation of actin-myosin interactions in aorta

Incubation of bovine aortic native actomyosin with cyclic AMP and bovine aortic cyclic AMP-dependent protein kinase produced a rightward shift in the relation between free Ca2+ and both superprecipitation and actomyosin ATPase activity. The relation between free Ca2+ and phosphorylation of myosin light chains was also shifted to the right. The concentration of free Ca2+ required for half-maximal activation of both ATPase activity and myosin light chain phosphorylation was approximately 1.0 microM for control actomyosin and 2.5 microM for actomyosin incubated with cyclic AMP-protein kinase. Neither basal nor maximal activities were significantly affected by incubation with cyclic AMP-protein kinase. Addition of e microM calmodulin to cyclic AMP-protein kinase-treated actomyosin relieved inhibition of both superprecipitation and myosin light chain phosphorylation. These findings suggest that cyclic AMP-protein kinase-mediated inhibition of actin-myosin interactions in vascular smooth muscle involve a shift in the Ca2+ sensitivity of the system. This shift probably involves Ca2+-calmodulin interactions and the control of phosphorylation of the myosin light chains.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Interaction of flavonoid compounds with contractile proteins of skeletal muscle

Flavonoids (quercetin, rutin) influence ATPase activity and actomyosin superprecipitation. Low concentrations (below 20 mumol/l) of flavonoids were found to cause conformational changes in the myosin structure accompanied by an increase in ATPase activity. At higher concentrations an inhibitory action of flavonoids on both ATPase activity and actomyosin superprecipitation occurred. Conformational changes are likely to be due to flavonoids binding to regulatory site near the active centre of the myosin head. The effect of quercetin was stronger than that of rutin.     

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.     

Ca2+-independent gizzard myosin light chain kinase produced by cross-linking of the enzyme with calmodulin using glutaraldehyde

Cross-linked complex of gizzard myosin light chain kinase (MLCK) and calmodulin (CM) was produced by glutaraldehyde treatment of a mixture of these proteins in a high Ca2+ (0.1 mM) solution. Although the specific activity was reduced, this complex showed MLCK activity in a Ca2+-independent manner, different from the original MLCK whose activity was Ca2+-dependent. Chlorpromazine, one of the CM antagonists, was no longer able to inhibit the MLCK activity of this complex. These observations support the previously proposed hypothesis on the regulatory mechanism of MLCK activity via Ca2+. This complex could be regarded as another kind of Ca2+-independent MLCK different from that obtained by chymotryptic digestion of MLCK (Walsh, M.P., Dabrowska, R., Hinkins, S., & Hartshorne, D.J. (1982) Biochemistry 21, 1919-1925). This complex caused superprecipitation of gizzard actomyosin and enhanced actin-activated ATPase of myosin Ca2+-independently.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Actomyosin isolated from bovine tracheal smooth muscle.

A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of "classical" procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP, and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.     

Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon

Caldesmon, a major calmodulin- and actin-binding protein of smooth muscle (Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5652-5655), has been obtained in highly purified form from chicken gizzard by a modification of a previously published procedure (Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870) and was found to cause a significant inhibition of both superprecipitation and actin-activated myosin Mg2+-ATPase activity in a system reconstituted from the purified contractile and regulatory proteins without influencing the phosphorylation state of myosin. This inhibitory effect was seen both in the presence and absence of tropomyosin. A Ca2+-and calmodulin-dependent kinase which catalyzed phosphorylation of caldesmon was identified in chicken gizzard; this kinase is distinct from myosin light-chain kinase. Caldesmon prepared by calmodulin-Sepharose affinity chromatography was contaminated with caldesmon kinase activity and was unable to inhibit actomyosin ATPase activity or superprecipitation. Phosphatase activity capable of dephosphorylating caldesmon was also identified in smooth muscle. These results indicate that caldesmon can inhibit smooth muscle actomyosin ATPase activity in vitro, and this function may itself be subject to regulation by reversible phosphorylation of caldesmon.     

Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin

Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed.     

Superprecipitation of an actomyosin-like complex isolated from Naegleria gruberi amoebae

A protein complex similar to muscle actomyosin and plasmodial myosin B has been isolated from Naegleria gruberi amoebae. This extract, which comprises approximately 0.7% of the total cell protein, has the solubility properties of actomyosin, displays Ca²⁺-activated, Mg²⁺-inhibited ATPase activity, forms microfilaments, and undergoes a strong superprecipitation reaction. Superprecipitation is initiated by ATP and is preceded by a very brief clearing phase. Although added Mg²⁺ is not essential for superprecipitation of the extract, the reaction proceeds maximally when 7 mM Mg²⁺ is provided. This extract does not appear to have a Ca²⁺ requirement, and superprecipitation is in fact inhibited by added Ca²⁺ ion at all concentrations greater than 0.1 mM. Both ATPase activity and superprecipitation of the actomyosin-like complex are inhibited by the sulfhydryl inhibitor salyrgan.     

pH-dependence of kinetic parameters in the superprecipitation reaction and ATPase activity of myometrial actomyosin

The investigation of pH-dependence of superprecipitation reaction and ATPase activity of myometrium actomyosin in the interval of pH 5.5-8.0 has detected cupola-shaped curves with maximal activity of both processes by pH 6.5. On the basis of calculating the constants of ionization it was supposed that in the case of actomyosin ATPase imidazole groups of two histidins had an essential role in reaction of ATP hydrolysis and in superprecipitation process--imidazol group of histidine and carboxyl group of asparagin acid. The investigation of [ATP]- and [Mg2+]-dependence of superprecipitation reaction by pH 6.0, 6.5 and 7.0 has demonstrated different pH-sensitiveness of Michaelis constants and maximal speeds relatively Mg2+ and ATP for both processes. It was shown that pH-optimum of ATPase activity of myometrium actomyosin coincided with maximal affinity of actomyosin with ATP and Mg2+ while as for superprecipitation reaction the correlation between value of process by certain pH and affinity with ATP and Mg2+ was not detected.     

Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.     

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Calpain abolishes the effect of filamin on the actomyosin system in platelets

Platelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of filamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-actin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for contraction of the activated platelets.     

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

Effects of K-252a, (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, purified from the culture broth of Nocardiopsis sp., on the activity of myosin light chain kinase were investigated. 1) K-252a (1 x 10(-5) M) affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca2+-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca2+-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10(-6) M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10(-4) M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lower in the presence of 100 microM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [gama-32P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP (Ki = 20 nM). These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase. The direct action of the compound on the enzyme would explain the multivarious inhibition of myosin ATPase, of superprecipitation, and of the contractile response of smooth muscle.     

The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin

The dependence of the onset and course of turbidity changes ( superprecipitation) induced by ATP were studied in a natural actomyosin suspension with the dephosphorylated and phosphorylated forms of light chains (LC2) of myosin. It was found that the onset and time course of the changes in turbidity of the natural actomyosin suspension are strongly dependent on the (phosphorylated and dephosphorylated) form of these chains of myosin. The ATPase activity of actomyosin with phosphorylated LC2 was lower and the half-time for achieving maximal turbidity of actomyosin suspension after addition of ATP was higher than that of actomyosin with dephosphorylated LC2. Natural actomyosin preparations contain endogenous light-chain kinase and phosphatase. The changes of turbidity induced by ATP in the natural actomyosin suspension are greatly diminished in the presence of phosphate. Thiophosphorylation of LC2 of myosin leads to a decrease of the extent of superprecipitation of natural actomyosin. The release of [32P]phosphate from actomyosin containing [32P]ATP-phosphorylated LC2 of myosin increases with increased turbidity of actomyosin suspension. The change of the form LC2 as a kind of additional myosin-linked regulation of superprecipitation is discussed.     

Calcium-sensitivity of pig-carotid-actomyosin ATPase in relation to phosphorylation of the regulatory light chain

Ca2+-dependent phosphorylation of the 20000-Mr regulatory light chain was found to be a necessary condition for the Ca2+-sensitivity of the Mg2+-dependent ATPase activity and superprecipitation of pig carotid actomyosin. Actin-myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.