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.     

Properties of phosphorylated myofibrils from gizzard smooth muscle

Phosphorylation of chicken gizzard myosin light chain in myofibril and its effect on myofibrillar ATPase activity were investigated in the contracted state of myofibrils. When myofibrils were incubated for two hours at 30 degreeds C with ATP, magnesium and calcium, the myosin light chain was phosphorylated by endogenous light-chain kinase. Standing overnight, the phosphorylated light chain was dephosphorylated by endogenous light-chain phosphatase. Control myofibril had much higher ATPase activity than phosphorylated and phosphorylated-dephosphorylated myofibrils. It was very interesting that the phosphorylated and phosphorylated-dephosphorylated myofibrils were quite similar in ATPase activity. However, phosphorylated myofibril differed from phosphorylated-dephosphorylated myofibril in Ca2+ dependency of Mg2+-ATPase activity. The phosphorylated-dephosphorylated myofibril was not affected by the presence or absence of Ca2+. In contrast, phosphorylated myofibril apparently showed a negative Ca2+-sensitivity. On the other hand, the results indicating that the superprecipitation gel formed by phosphorylated-dephosphorylated myosin could not be dissolved in 0.6 M NaCl, suggest that the phosphorylation-dephosphorylation process of the actomyosin system in gizzard myofibril results in stronger actin-myosin interaction.     

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.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin.

Ca2+-dependent phosphorylation of the myosin light chains in bovine aortic native actomyosin is markedly depressed in the presence of cyclic AMP and its dependent protein kinase. This inhibition occurs with either cardiac, skeletal, or aortic protein kinase plus cyclic AMP, while little or no inhibition occurs with either cyclic AMP or protein kinase alone. The extent of inhibition is related to the concentration of protein kinase and approaches a maximum of approximately 50%. Concomitant with the inhibition of myosin light chain phosphorylation is (a) an increased phosphorylation of a 100,000-dalton moiety which possibly corresponds to the myosin light chain kinase present in the native actomyosin preparation and (b) a decrease in the actomyosin Mg2+-ATPase activity. These findings suggest that modulation of actin-myosin interactions by the cAMP system directly at the level of the contractile proteins may represent a mechanism by which beta adrenergic relaxation occurs in mammalian vascular smooth muscle.     

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.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.     

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.     

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.     

Myosin light-chain phosphatase.

1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.     

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.     

Caldesmon150 regulates the tropomyosin-enhanced actin-myosin interaction in gizzard smooth muscle

Using a reconstituted system in which myosin was preferentially phosphorylated, we examined the regulatory action of caldesmon150 on the smooth muscle actin-myosin interaction. Caldesmon150 inhibited the tropomyosin-enhanced actomyosin ATPase activity in a Ca2+-independent manner. This inhibitory effect of caldesmon150 was observed to be overcome by the addition of calmodulin in a Ca2+-dependent manner. In accordance with the observations of ATPase activity, we demonstrated evidence that the regulatory action of caldesmon150 on the actin site was mainly through control of the tropomyosin-enhanced actin-myosin interaction and calmodulin confers the Ca2+-sensitivity upon the caldesmon150 action determined by the cosedimentation method.     

A protein-bound polysaccharide from Basidiomycetes enhances myosin phosphorylation but inhibits myosin ATPase activity: studies with a crude actomyosin preparation of chicken gizzard smooth muscle.

Chicken gizzard actomyosin, containing the calmodulin-myosin light chain kinase (MLCK) system, was incubated in the presence of various concentrations of PSK, a protein-bound polysaccharide from Basidiomycetes, together with Ca2+ and Mg-ATP. The phosphorylation of myosin was enhanced half-maximally by 10-4 g/ml of PSK. However, a similar concentration of PSK reduced the Mg-ATPase activity of the actomyosin. The former was brought about through stimulation of the MLCK activity and the latter through inhibition of the myosin ATPase activity.     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

An effect of trypsin on the actin-myosin interaction

Three different lines of evidence were obtained to show that trypsin modifies the actin-myosin interaction: (I) At trypsin to actomyosin or myosin ratios between 1 to 300 and 1 to 500, 30 min of trypsin treatment causes an 8-fold increase in the Ca²⁺-modified ITPase activity of actomyosin but has no effect on the Ca²⁺-modified ITPase activity of myosin alone. At these same trypsin to actomyosin ratios, the Mg²⁺ + Ca²⁺-modified ATPase activity increases by 10–30% during the first 1–2 min of trypsin digestion, and then decreases rapidly to less than 20% of its original activity after 60 min of digestion. Trypsin has no effect on the Mg²⁺ + Ca²⁺-modified ATPase activity of pure myosin. (2) The rate of turbidity response of reconstituted actomyosin suspensions is first increased and then decreased by trypsin treatment. At trypsin to actomyosin ratios of 1 to 3000, rate of turbidity response is maximal after 5 min of trypsin digestion and then decreases; after 60 min, the turbidity response is much slower than the response of the control actomyosin. (3) Supercontracted sarcomeres, shortened to less than 50% of their initial length, are lengthened to 70% of their initial length by 4 min of trypsin treatment. Myosin B from such lengthened sarcomeres has less than 35% of its myosin converted to light meromyosin and heavy meromyosin.

These results show that trypsin modifies the actin-myosin interaction in at least two ways: (1) a very rapid initial modification that increases the Mg²⁺ + Ca²⁺-modified ATPase activity and the rate of turbidity increase, and (2) a slower modification that decreases the Mg²⁺ + Ca²⁺-modified ATPase activity and rate of turbidity response, and that lengthens contracted sarcomeres. Tryptic modification is not due to cleavage of myosin to light and heavy meromyosin. Since tryptic modification occurs more rapidly than conversion of myosin to light and heavy meromyosin, all heavy meromyosin preparations will be modified.     

Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts.

Myosin purified from a murine myeloid leukaemia cell line (M1) that had been incubated with [32P]orthophosphate incorporated 32P into the heavy, but not the light, chain. When the heavy chain was dephosphorylated by bacterial alkaline phosphatase, myosin that had low actin-activated ATPase activity gained higher activity only in the presence of the light-chain kinase. In the absence of the light-chain kinase, however, the Mg2+-stimulated ATPase activity of myosin was not activated by actin, regardless of phosphatase treatment. These results indicate that the activity of M1 myosin ATPase is regulated by phosphorylation of both the light and heavy chains. A scheme for this regulation by phosphorylation is presented and discussed.     

Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.     

Calcium ion-regulated thin filaments from vascular smooth muscle.

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 x 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.